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https://openalex.org/W2909937546
https://elpub.vir.nw.ru/jour/article/download/169/119
Russian
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AGROBIOLOGICAL CHARACTERISTICS OF THE MALTING SPRING BARLEY CULTIVAR ‘OMSKY 100
Trudy po prikladnoj botanike, genetike i selekcii
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Ключевые слова: яровой многорядный ячмень, вегетационный период, по­ ражение головней, высота растений, форма колоса, за­ зубренность остей, цвет зерна, стабильность, пла­ стичность, гомеостатич­ ность АГРОБИОЛОГИЧЕСКАЯ ХАРАКТЕРИСТИКА ПИВОВАРЕННОГО СОРТА ЯРОВОГО ЯЧМЕНЯ ОМСКИЙ 100 П. Н. Николаев1, П. В. Попол- зухин1, Н. И. Аниськов2, О. А. Юсова1, И. В. Сафонова2 Федеральное государственное бюджетное научное учреждение Сибирский научно-исследова­ тельский институт сельского хо­ зяйства, 644012 г. Омск, пр. Коро­ лева 26, Актуальность. Селекция пивоваренного ячменя в России - перспективное направление растениеводства. Одним из главных факторов, влияющих на необходимость поиска новых сортов ячменя, являются климатические условия. Сорта пивоваренного ячменя, завозимые из стран Европы, зача­ стую не выдерживают специфических погодных условий Западной Си­ бири. Объектом исследований выступал новый перспективный сорт яро­ вого ячменя пивоваренного направления 'Омский 100' ('Медикум 4747'), переданный на ГСИ в 2015 г. Материалы и методы. Проведение исследо­ ваний сопровождалось постановкой полевых опытов на селекционном стационаре лаборатории селекции ячменя (третий селекционный севооб­ орот, четвертая культура после пара) на опытных полях Сибирского научно-исследовательского института сельского хозяйства, расположен­ ных в южной лесостепи Омской области. Селекционная проработка мате­ риал велась на основе методики ГСИ. Проведены оценка на устойчивость к болезням и биохимический анализ зерна. Математическая обработка проведена методом дисперсионного анализа, рассчитаны параметры ста­ бильности, пластичности и гомеостатичности. Результаты. Яровой ячмень 'Омский 100' выведен путем гибридизации (Медикум 4365 х Ме­ дикум 4549) в 1998 году с последующим индивидуальным отбором. Сорт 'Омский 100' относится к лесостепной экологической группе сортов, харак­ теризуется высокой устойчивостью к полеганию, слабой восприимчиво­ стью к черной головне, средней - к пыльной головне и сильной - к камен­ ной головне. В среднем за 2011-2015 гг., новый сорт 'Омский 100' имел пониженное содержание белка (12,8%, «-»0,5% st.), натура зерна соста­ вила 634 г/л, («+»44 г/л st.), пленчатость зерна 8,5% («-»0,2% st.). По дру­ гим биохимическим показателям - экстрактивности (80,6%), пленчатости (8,5%) и массе 1000 зерен (53,3 г) - сорт 'Омский 100' соответствовал тре­ бованиям ГОСТа на пивоваренный ячмень. По продуктивности сорт 'Ом­ ский 100' относится к высокоурожайным в условиях Западной Сибири (4,5 т/га, «+»0,4 т/га st.), отзывчив на улучшение условий выращивания (62d = 2,3, КМ = 2,6) и способен сочетать высокую потенциальную урожай­ ность с минимальным ее снижением в неблагоприятных условиях выра­ щивания (Hom = 0,4). Заключение. Сорт 'Омский 100' соответствует требо­ ваниям ГОСТа на пивоваренный ячмень и рекомендуется для использова­ ния в пивоваренной промышленности. e-mail: sibniish@bk.ru 2Федеральный исследователь­ ский центр Всероссийский инсти­ тут генетических ресурсов расте­ ний имени Н. И. Вавилова, 190000 г. Санкт-Петербург, ул. Большая Морская 42-44, e-mail: i.safonova@vir.nw.ru e-mail: i.safonova@vir.nw.ru УСПЕХИ ОТЕЧЕСТВЕННОЙ СЕЛЕКЦИИ НА СОВРЕМЕННОМ ЭТАПЕ УСПЕХИ ОТЕЧЕСТВЕННОЙ СЕЛЕКЦИИ НА СОВРЕМЕННОМ ЭТАПЕ DOI: 10.30901/2227-8834-2017-4-90-99 УДК 633.16:631.527:631. 526.32(527.1) DOI: 10.30901/2227-8834-2017-4-90-99 УДК 633.16:631.527:631. 526.32(527.1) ОРИГИНАЛЬНАЯ СТАТЬЯ ОРИГИНАЛЬНАЯ СТАТЬЯ DOI: 10.30901/2227-8834-2017-4-90-99 ORIGINAL ARTICLE Поступление: 20.09.2017 90 AGROBIOLOGICAL CHARACTERISTICS OF THE MALTING SPRING BARLEY CULTIVAR 'OMSKY 100 I. Vavilov All-Russian Institute of Plant Genetic Resources, 42-44. Bolshaya Morskaya St., St. Petersburg, 190000, Russia, e-mail: i.safonova@vir.nw.ru Key words: y common spring barley, growing season, smut incidence, plant height, spike shape, awn serra­ tion, kernel color, stability, flexi­ bility, homeostaticity AGROBIOLOGICAL CHARACTERISTICS OF THE MALTING SPRING BARLEY CULTIVAR 'OMSKY 100 P. N. Nikolaev1, P .V. Popolzu- khin1, N. I. Anisimov2, O. A. Yusova1, I. V. Safonova2 P. N. Nikolaev1, P .V. Popolzu- khin1, N. I. Anisimov2, O. A. Yusova1, I. V. Safonova2 1Siberian Research Institute of Ag­ riculture, RAAS, Background. Breeding of malting barley in Russia is a promising trend in plant production. One of the main factors urging the need for new barley varieties is climate. Malting barley cultivars imported from Europe often cannot withstand the specific weather conditions of Western Siberia. Object. The object of re­ search was the new and promising cultivar of malting spring barley 'Omsky 100' ('Medicum 4747') submitted for the State Trials in 2015. Materials and meth­ ods. The research was accompanied by field experiments at the stationary breeding nursery of the Barley Breeding Lab (third crop rotation, fourth crop after fallow) in the experimental fields of the Siberian Research Institute of Ag­ riculture located in the southern forest-steppe area of Omsk Province. Breed­ ing-oriented study of the material was based on the methods of the State Vari­ ety Trials. Disease resistance assessment and biochemical grain analysis were performed. Analysis of variance was used in mathematical processing, and the parameters of stability, plasticity and homeostaticity were calculated. Results. The spring barley cultivar 'Omsky 100' was developed in 1998 through hybridi­ zation ('Medicum 4365' x 'Medicum 4549'), followed by individual selection. The cultivar 'Omsky 100' belongs to the forest-steppe environmental group of varieties characterized by high resistance to lodging, low susceptibility to false loose smut, medium to loose smut, and high to covered smut. On average. in 2011-2015 'Omsky 100' demonstrated lower protein content (12.8%, "-"0.5% st.), grain-unit level of 634 g/l, ("+"44 g/l st.), and grain hull content of 8.5% ("­ "0.2% st.). In other biochemical parameters, such as extract efficiency (80.6%), hull percentage (8.5%) and 1000 grain weight (53.3 g), 'Omsky 100' complied with the State Standard's requirements for malting barley. In terms of produc­ tivity, 'Omsky 100' is among high-yielding cultivars in the environments of West Siberia (4.5 t/ha, "+"0.4 t/ha st.). It is responsive to improvement of cultivation conditions (regression coefficient = 2.3; multiplier coefficient = 2.6) and is able to combine high potential yield with its minimal decrease in adverse cultivation environments (homeostaticity = 0.4). Conclusions. The variety meets the re­ quirements of the State Standard for malting barley, and is recommended for use in brewing industry. e-mail: sibniish@bk.ru 2N. P. N. Nikolaev1, P .V. Popolzu- khin1, N. I. Anisimov2, O. A. Yusova1, I. V. Safonova2 1Siberian Research Institute of Ag­ riculture, RAAS, 26 Koroleva Ave., Omsk, 644012, Russia, e-mail: sibniish@bk.ru 2N. I. Vavilov All-Russian Institute of Plant Genetic Resources, 42-44. Bolshaya Morskaya St., St. Petersburg, 190000, Russia, e-mail: i.safonova@vir.nw.ru Введение ного ячменя, к которым относится и За­ падная Сибирь. Более полное использо­ вание гидротермических ресурсов таких зон может быть реализовано лишь на ос­ нове создания и возделывания пивова­ ренных сортов местной селекции. В Ячмень относится к наиболее важным зерновым культурам благодаря своим огромным приспособительным возмож­ ностям, высокой урожайности и разно­ стороннему использованию. В России лишь 8% производимого зерна ячменя расходуется на приготовление пива (Anisimov et al., 2010; Kalashnikov et al, 2005; guidelines, 2000; Nelevic et al., 1981; Surin et al., 1993). Основные пара­ метры пивоваренного ячменя изложены в ГОСТ 5060-86 (Anisimov et al., 2010) и ГОСТ 29294-92 (Instruction on process control..., 1967), в которых к сортам пи­ воваренного ячменя предъявляются жесткие требования. Зерна должно быть крупным и выравненным (масса 1000 зе­ рен 40 г и выше), иметь пленчатость не выше 9%. Слишком высокое содержание белка (свыше 13%) в зерне ячменя де­ лает его малопригодным для пивоваре­ ния: ухудшается вкус пива и уменьша­ ется его выход. Хороший пивоваренный ячмень содержит 9-11% белка. Выход пива тем больше, чем больше в зерне крахмала, от количества которого зави­ сит экстрактивность солода, т. е. способ­ ность отдавать в раствор сухое веще­ ство. Она должна составлять 78-84%. Более 80% пивоваренного ячменя выра­ щивается из семян сортов зарубежной селекции. Как правило, они обладают хорошими технологическими характе­ ристиками, отвечающими требованиям современного пивоваренного производ­ ства. Однако при выращивании ино­ странных сортов в условиях Западной Сибири показатели произведенного из них солода и пива зачастую не дости­ гают заявленных характеристик (Guidelines, 2000). Известно, что пивова­ ренный ячмень гарантированно можно получить лишь в зонах, где из года в год складываются благоприятные гидротер­ мические условия для формирования низкобелкового зерна. Но в отдельные годы благоприятная обстановка может сложиться также в зонах, не входящих в список районов заготовок пивоварен­ ренных сортов местной селекции. В 2016 г. Государственным Реестром се­ лекционных достижений в Западной Си­ бири допущено к использованию 33 сорта ячменя, из них 11 относятся к пи­ воваренным. Наибольший вклад в фор­ мирование сортовых ресурсов ярового ячменя внесли сибирские селекционеры. Они создали 23 сорта (70%), из них 7 пи­ воваренных. Received: 20.09.2017 91 91 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Введение В том числе в Сибирском НИИ сельского хозяйства создано 2 сорта ячменя: ‘Омский 90’ (медикум, пи­ воваренный, ценный, 2000 г.), ‘Ом­ ский 91’ (нутанс, пивоваренный, цен­ ный, 2004 г.); Алтайском НИИ земледе­ лия и селекции сельскохозяйственных культур - 2 сорта: ‘Ворсинский’ (дву­ рядный, пивоваренный, ценный, 2001 г.), ‘Сигнал’ (пивоваренный, ценный, 1997 г.); Сибирском НИИ растениевод­ ства и селекции: ‘Ача’ (пивоваренный, ценный, двурядный, 1997 г.); Кемеров­ ском НИИ сельского хозяйства: ‘Ни­ кита’ (двурядный, пивоваренный, цен­ ный, 2004 г.), Челябинский НИИ сель­ ского хозяйства - ‘Челябинский 99’ (пи­ воваренный, 2002 г.). Также Государ­ ственным Реестром допущено к исполь­ зованию 3 пивоваренных сорта инорай- онной селекции: Германия - ‘Беатрис’ (пивоваренный, ценный, 2008 г.); Укра­ ина - ‘Одесский 100’ (пивоваренный, ценный, 1984 г.); Ставропольский НИИ сельского хозяйства - ‘Гетьман’ (пиво­ варенный, 2005 г.); (Public register..., 2016). Пивоваренные сорта занимают достаточно большую площадь посева, но для приготовления пива используется зерно ячменя местного производства в недостаточном количестве. Большая часть по-прежнему завозится из евро­ пейской части России и других стран. В настоящее время необходимость создания местной сырьевой базы для пи­ воваренной промышленности определя В настоящее время необходимость создания местной сырьевой базы для пи­ воваренной промышленности определя­ ется экономическими предпосылками, поскольку огромные средства уходят за В настоящее время необходимость создания местной сырьевой базы для пи­ воваренной промышленности определя­ ется экономическими предпосылками, поскольку огромные средства уходят за 92 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 1985). Оценку на устойчивость к болез­ ням определяли в лаборатории иммуни­ тета, биохимический анализ зерна про­ водили в лаборатории генетики, биохи­ мии и физиологии растений. Объектом исследований, результаты которых представлены в данной статье, являлся новый перспективный пивоваренный сорт ярового ячменя ‘Омский 100’. Математическая обработка проведена методом дисперсионного анализа (Dospekhov, 1985), рассчитаны пара­ метры стабильности, пластичности и го­ меостатичности (Dragavcev, 1984; Nettevich, 1985; Hangil'din,1977; Eberhart et al., 1966). 1985). Оценку на устойчивость к болез­ ням определяли в лаборатории иммуни­ тета, биохимический анализ зерна про­ водили в лаборатории генетики, биохи­ мии и физиологии растений. Объектом исследований, результаты которых представлены в данной статье, являлся новый перспективный пивоваренный сорт ярового ячменя ‘Омский 100’. пределы Сибири. Цель настоящего ис­ следования - охарактеризовать новый пивоваренный сорт ярового ячменя ‘Ом­ ский 100’ по качеству зерна, урожайно­ сти и устойчивости к комплексу болез­ ней. Результаты и обсуждение Яровой ячмень ‘Омский 100’ (‘Медикум 4747’) выведен в ФГБНУ СибНИИСХ путем гибридизации сортов (Ме- дикум 4365 х Медикум 4549) с последу­ ющим индивидуальным отбором (рис. 2). Большое значение в селекционной ра­ боте имеют образцы мировой коллекции ВИР (Loskutov, 2012), которые и были использованы в качестве исходного ма­ териала, что отражено на рисунке 2. Скрещивание сортов проведено в 1998 году, в 1998 - размножение в теплице, в 1999 - размножение в СП-1. В гибрид­ ном питомнике в 2000 г. проведен отбор элитного растения, которое было высе­ яно в 2001 году в СП-1. В полевых усло­ виях эта линия изучалась в СП-II - 2002 г. и КП - 2003 г. Объектом исследований выступал но­ вый перспективный сорт ярового ячменя пивоваренного направления ‘Омский 100’ (‘Медикум 4747’), переданный на ГСИ в 2015 г. В качестве стандарта ис­ пользован сорт ‘Омский 95’ (Тогу- зак х Омский 88). Разновидность нутанс, относится к степной экологической группе, засухоустойчив, среднеспелый, вегетационный период 79-90 дней. Сорт также характеризуется высокой устой­ чивостью к полеганию, слабой воспри­ имчивостью к каменной и черной го­ ловне и средней восприимчивостью к пыльной головне. Рекомендуется к ис­ пользованию на кормовые цели, а также, благодаря крупности зерна, в крупяной промышленности. Сорт включен в Гос- реестр по Уральскому (9) и Западно-Си­ бирскому (10) регионам. Патент № 3102, зарегистрирован в Государственном ре­ естре селекционных достижений РФ 26.04.2006 г. Сорт рекомендован для возделывания во всех зонах Западной Сибири. В качестве примера для сравне­ ния приведены данные последнего пере­ данного на ГСИ (2014 г.) сорта ‘Подарок Сибири’ (‘Медикум 4712’). Сорт харак­ теризуется белковостью зерна на уровне 13,5% (+0,3% st.), а также содержанием С 2004 по 2015 г. ‘Медикум 4747’ проходил испытание в КСИ. Сорт яч­ меня ‘’Омский 100 относится к разно­ видности медикум. Куст полупрямосто- ячий. Толщина и прочность стебля сред­ ние. Лист средней ширины - промежу­ точный (табл. 1). Влагалища нижних ли­ стьев без опушения. Антоциановая окраска ушек имеется, очень слабая. Встречаемость растений с наклоненным флаговым листом низкая. Восковой налет на влагалище слабый. Окраска стеблевых узлов коричневая. Ушки сер­ повидные, светлые. Язычок обыкновен­ ный. Колос цилиндрический, двуряд­ ный, соломенно-желтый, рыхлый, сред­ ней длины, прямостоячий. Переход цве­ точной чешуи в ость постепенный. Нер­ вация цветочной чешуи слабо выражена. Ости длинные, расположены парал­ лельно колосу, гладкие, легко осыпаю­ щиеся при созревании, желтые. Первый сегмент колосового стержня со слабым изгибом. На среднем колоске длина ко­ лосовой чешуи и ости равна зерновке. Зерно желтое, пленчатое, полу-удлинен- ное, крупное. Материалы и методы Экспериментальная часть работы прово­ дилась на опытных полях Сибирского научно-исследовательского института сельского хозяйства (ФГБНУ Сиб- НИИСХ). Проведение исследований со­ провождалось постановкой полевых опытов на селекционном стационаре ла­ боратории селекции ячменя (третий се­ лекционный севооборот, четвертая куль­ тура после пара). Селекционная прора­ ботка материал велась на основе Мето­ дики государственного сортоиспытания сельскохозяйственных культур (Fedin, Математическая обработка проведена методом дисперсионного анализа (Dospekhov, 1985), рассчитаны пара­ метры стабильности, пластичности и го­ меостатичности (Dragavcev, 1984; Nettevich, 1985; Hangil'din,1977; Eberhart et al., 1966). По данным гидрометеорологического центра (ОГМС), в черте г. Омска в пе­ риод исследований с 2011 по 2015 гг. сложились контрастные условия, рису­ нок 1. Рис. 1 Характеристика периодов вегетации 2011-2015 гг., (Омская ГМОС) Fig. 1. Characteristics of growing seasons in 2011-2015, (Omsk Weather Station) Рис. 1 Характеристика периодов вегетации 2011-2015 гг., (Омская ГМОС) Fig. 1. Characteristics of growing seasons in 2011-2015, (Omsk Weather Station) Периоды вегетации 2011 и 2014 гг. ха­ рактеризуются засушливыми условиями (ГТК 0,90-0,92), очень сухими в период вегетации 2012 г. (ГТК 0,69), сухими и холодными в 2015 г. (0,70). Достаточ­ ным увлажнением отличался период ве­ гетации 2013 года (ГТК = 0,99). Средне­ многолетнее значение ГТК составляет 0,82, что означает засушливые условия. Западная Сибирь традиционно считается зоной рискованного земледелия. Ти­ пично континентальный климат южной 93 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 части Западной Сибири с коротким веге­ тационным периодом, поздним прекра­ щением заморозков весной и ранним наступлением их осенью, проявлением региональных типов засух и ливневых осадков обусловливают необходимость внедрения в производство сортов зерно­ вых, выносливых к экстремальным усло­ виям возделывания. Период формирова­ ния зерновки овса (третья декада июля - август) характеризовался недо­ бором количества осадков в 2011, 2012, 2014 гг., а также в июле 2015 г. (13 ^ 95 % к норме), что, несомненно, отрази­ лось на качестве зерна. На этом фоне наблюдается превышение средних тем­ ператур воздуха в июле 2011 г., июле - августе 2012 г., августе 2014 г. (+0,4 ^ +3,2°С) и недобор их в августе 2011 г., в июле 2013, 2014 гг. (-0,6 ^ - 3,4°С). крахмала и сырого жира на уровне стан­ дарта (55,2 и 2,2% соответственно). Результаты и обсуждение Масса 1000 зерен, в сред­ нем, составляет 52-54 г. Сыпучесть зерна при посеве хорошая. 94 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Табл. 1. Морфологическое описание пивоваренного сорта ярового ячменя ‘Омский 100’ Tabl. 1. Morphological description of the malting s pring barley cultivar ‘Omsky 100’ Признак Характеристика Растение: тип куста Полупрямостоячий Нижние листья: опушение листовых влагалищ Отсутствует Флаговый лист: антоциановая окраска уш ек Имеется Флаговый лист интенсивность антоциановой окраски уш ек Слабая Флаговый лист: встречаемость растений с наклоненным флаговым листом Низкая Флаговый лист: восковой налет на влагалище Слабый Время колошения Среднее Ости: антоциановая окраска кончиков Имеется Ости: интенсивность антоциановой окраски кончиков Средняя Колос: восковой налет Средний Колос: положение Прямостоячий Растение: длина Средняя Колос: количество рядков Два Колос: форма Цилиндрический Колос: плотность Рыхлый Колос: длина (исключая ости) Средняя Ости: длина по сравнению с колосом Длинные Ости: зазубренность краев Отсутствует Стержень колоса: длина первого сегмента Короткий Стержень колоса: изгиб первого сегмента Слабый Средний колосок: длина колосковой чешуи и ости по отноше­ нию к зерновке Равна Зерновка: тип опушения основной щетинки Волосистая, длинный Зерновка: пленчатость Имеется Зерновка: опушение брюшной бороздки Отсутствует Зерновка: расположение лодикул Охватывающее Зерновка: окраска алейронового слоя Белая Тип развития Яровой Зерновка: окраска алейронового слоя сравнении со стандартным сортом ‘Ом­ ский 95’. Биохимический анализ зерна ячменя образцов КСИ свидетельствует, что, в среднем, за 2011-2015 гг., новый сорт ‘Омский 100’ имел пониженное со­ держание белка - 12,8%, что на 0,5% ниже, чем у пивоваренного сорта ‘Ом­ ский 90’ (13,3%), и на 1,1% меньше, чем у пивоваренного сорта ‘Беатрис’ (13,9%). Натура зерна исследуемого сорта, в среднем, составила 634 г/л, («+»44 г/л st.), пленчатость зерна 8,5% («-»0,2% к стандарту и «-»0,9% к по­ следнему переданному в ГСИ сорту ‘По­ дарок Сибири’). Сорт ‘Омский 100’ относится к лесо­ степной экологической группе сортов, характеризуется высокой устойчиво­ стью к полеганию, среднерослый (64-80 см), соломина прочная, засухоустойчив. Сорт среднеспелый, период вегетации, в среднем, составила 81-89 дней, что на уровне стандарта ‘Омский 95’ и на 5 дней больше, чем у сорта ‘Подарок Си­ бири’. За годы испытания на искусствен­ ном инфекционном фоне сорт, в целом, характеризовался слабой восприимчиво­ стью к черной головне, средней - к пыльной головне и сильной - к каменной головне, но отличался более высокой устойчивостью к этим заболеваниям в 95 3 в era n к» к» м ^ » S & о ста « -! Результаты и обсуждение Ьа <т> о » 03 о а “*s as Нутанс 58 ТЗ » g ж чз - н9» в а ста -в О* О 1S И ■S о * 1 п н С " X 2 Л ее < I is а ■s " 3 % £. Я - £с о е ® о Паллиссер ТТанад^^“ Омский 13709 ф^ н^ Е н й й о Г Паллиссер Омский 13709 СибНИИС Х (Хайпроли х Мед.134) х Нут.244 I ДМ Украина Славутич Ткрайнг^ 3 в era n к» к» м ^ » S & о ста « -! Ьа <т> о » 03 о а “*s as ТЗ » g ж чз - н9» в а ста -в О* О 1S И ■S о * 1 п н С " X 2 Л ее < I is а ■s " 3 % £. Я - £с о е ® о 40 On Харьковский 70 ^украйн^ Медикум 4365 Ш1ЛЬН.Шх Донецкий 9 Окраин? Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 Максимальная урожайность была полу­ чена в КСИ СибНИИСХ в 2015 г. - 6,6 т/га, прибавка к стандартному сорту Омский 95 составила 0,8 т/га. В среднем, за 5 лет ис­ пытаний (2011-2015 гг.) при урожайности 4,5 т/га прибавка к стандартному сорту ‘Омский 95’ составила 0,4 т/га, к ранее пе­ реданному сорту ‘Подарок Сибири’ - 0,2 т/га (табл. 2). По другим биохимическим показателям - экстрактивности (80,6%), пленчатости (8,5%) и массе 1000 зерен (53,3 г) - сорт ‘Омский 100’ соответствует требованиям ГОСТа на пивоваренный ячмень и реко­ мендуется для использования в пивоварен­ ной промышленности. По продуктивности сорт ‘Омский 100’ относится к высокоуро­ жайным в условиях Западной Сибири. Табл. 2. Урожайность ярового среднеспелого пивоваренного сорта ‘Омский 100’, т/га Table 2. Yield of the middle-ripening malting spring barley cv. ‘Omsky 100’, t/ha С о р т 2011 г . 2012 г . 2 0 1 3 г . 2 0 1 4 г . 2 0 1 5 г . _ Х ± s t . О м с к и й 9 5 , s t . Результаты и обсуждение 5 , 3 2,2 3 , 4 3 , 6 5 , 8 4 , 1 - О м с к и й 1 0 0 5 , 8 2,8 3 , 5 3 , 9 6,6 4 , 5 + 0 , 4 П о д а р о к С и б и р и 5 , 8 1,8 3 , 4 4 , 3 6 , 3 4 , 3 + 0 , 2 Н С Р 0 5 0,11 0,12 0,10 0,11 0 , 1 3 - - _Х - средние значения st. - стандарт Табл. 2. Урожайность ярового среднеспелого пивоваренного сорта ‘Омский 100’, т/га Table 2. Yield of the middle-ripening malting spring barley cv. ‘O Табл. 2. Урожайность ярового среднеспелого пивоваренного сорта ‘Омский 100’, т/га Yield of the middle-ripening malting spring barley cv. ‘Omsky 100’, t/ha ники, так как только в этом случае они да­ дут максимум отдачи. В случае, когда ко­ эффициент регрессии меньше, сорта пока­ зывают лучшие результаты в неблагопри­ ятных условиях выращивания. Из изучае­ мых сортов наиболее отзывчивыми на улучшение условий оказались сорта ‘По­ дарок Сибири’, ‘Омский 100’, ‘Омский 95 ’ (при повышении среднего уровня уро­ жайности на 1,0 т/га они увеличивали свою на 1,5; 1,3; 1,2 т/га, соответственно, таблица 3). Наименее отзывчив на улуч­ шение условий выращивания ‘Омский 91’ (с повышением среднего уровня урожай­ ности на 1,0 т/га он увеличил свою только на 0,9 т/га). По уровню стабильности сорта расположились следующим обра­ зом: ‘Омский 91’, ‘Омский 95’, ‘Омский 100’, ‘Подарок Сибири’ (62d = 0,7; 1,9; 2,3; 3,3, соответственно). В резко-континентальных условиях За­ падной Сибири очень важно обращать внимание на создание сортов, наиболее адаптированных к условиям выращива­ ния. Вновь созданный сорт может быть допущен к использованию в производстве только при условии, если он способен формировать более высокие и стабильные урожаи, чем лучшие в этой зоне сорта дан­ ной культуры. Поэтому изучение и оценка экологической пластичности сортов, сферы их применения и адаптации к кон­ кретным природно-климатическим ситуа­ циям является важной задачей современ­ ного сельхозпроизводства. Оценку эколо­ гической пластичности сортов и гибридов проводят с использованием математиче­ ских методов, позволяющих получить ин­ дивидуальную характеристику по этому показателю в различные годы. Метод S. A. Eberhart, W. A. Russell (Eberhart et al., 1966) позволяет оценить сорта по их отзывчивости на условия вы­ ращивания путем определения коэффици­ ента регрессии (bi) и вариансы стабильно­ сти (62d). Результаты и обсуждение Считается, что чем выше еди­ ницы коэффициент регрессии, тем силь­ нее отзывчивость сорта на улучшение условий выращивания. Такие сорта требо­ вательны к высокому уровню агротех­ Коэффициент мультипликативности (КМ) характеризует приспособленность сортов к тем или иным условиям обитания (Dragavcev, 1984). Чем выше числовое значение этого коэффициента, тем выше их отзывчивость на улучшение условий среды. Таким требованиям удовлетво­ ряют сорта ‘Подарок Сибири’, ‘Омский 100’, ‘Омский 95’. Лимитирующим фак­ тором урожайности является не потенци­ альная продуктивность, а устойчивость к 97 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 неблагоприятным условиям внешней указывает на их способность сочетать вы- среды, т. е. гомеостатичность (Hangil'din, сокую потенциальную урожайность с ми- 1977). Чем выше значение этого пара- нимальным ее снижением в неблагопри- метра, тем стабильнее сорт. У изученных ятных условиях выращивания. сортов этот показатель был одинаков, что неблагоприятным условиям внешней указывает на их способность сочетать вы- среды, т. е. гомеостатичность (Hangil'din, сокую потенциальную урожайность с ми- 1977). Чем выше значение этого пара- нимальным ее снижением в неблагопри- метра, тем стабильнее сорт. У изученных ятных условиях выращивания. сортов этот показатель был одинаков, что неблагоприятным условиям внешней среды, т. е. гомеостатичность (Hangil'din, 1977). Чем выше значение этого пара- метра, тем стабильнее сорт. У изученных сортов этот показатель был одинаков, что Табл. 3. Оценка адаптивной способности, стабильности и гомеостатичности ярового пивоваренного сорта ячменя ‘Омский 100’ Tabl. 3. Assessment of adaptability, stability and homeostaticity in the malting spring barley cv. ‘Omsky 100’ Сорт bi 62d КМ Ш т ПУСС Омский 95, st. 1,2 1,9 2,4 0,4 165,2 Омский 100 1,3 2,3 2,6 0,4 225,0 Подарок Сибири 1,5 3,3 2,7 0,4 227,7 Омский 91 0,9 0,7 1,8 0,4 100,0 Sх 0,1 0,4 0,1 0,1 30,2 bi - коэффициент регрессии 6 2d - варианса стабильности КМ - коэффициент мультипликативности №т - гомеостатичность ПУСС - показатель уровня и стабильности сорта Табл. 3. Оценка адаптивной способности, стабильности и гомеостатичности ярового пивоваренного сорта ячменя ‘Омский 100’ Tabl. 3. Assessment of adaptability, stability and homeostaticity in the malting spring barley cv. ‘Omsky 100’ Из показателей стабильности более наглядную информацию дает показатель уровня и стабильности сорта (ПУСС), являющийся комплексным, поскольку позволяет одновременно учитывать уро­ вень и стабильность урожайности и ха­ рактеризует способность отзываться на улучшение условий выращивания, а при их ухудшении поддерживать достаточно высокий уровень продуктивности. Этот показатель рассчитывают по данным средней урожайности сортов за годы ис­ пытания, коэффициенту вариации уро­ жайности и относительной урожайности сорта, выраженной в процентах к стан­ дарту. Результаты и обсуждение В соответствии с этим подходом более урожайными и стабильными явля­ ются сорта ‘Подарок Сибири’, ‘Омский 100’, ‘Омский 95’ (ПУСС = 227; 225; 155, соответственно). гим биохимическим показателям - экс­ трактивности (80,6%), пленчатости (8,5%) и массе 1000 зерен (53,3 г) - сорт соответ­ ствует требованиям ГОСТа на пивоварен­ ный ячмень и рекомендуется для исполь­ зования в пивоваренной промышленно­ сти. 3. На искусственном инфекционном фоне сорт ячменя ‘Омский 100’, в целом, характеризуется слабой восприимчиво­ стью к черной головне и средней воспри­ имчивостью к пыльной головне. Отлича­ ется более высокой устойчивостью к этим заболеваниям в сравнении со стандарт­ ным сортом ‘Омский 95’. 4. Оценка хозяйственно-биологиче­ ских и морфологических признаков и свойств новых сортов ячменя с учетом их адаптивной реакции показала, что сорт ‘Омский 100’ имеет практический интерес для выращивания пивоваренного зерна в условиях Западно-Сибирского региона и рекомендуется для испытания в 10-м реги­ оне. На основе изучения эксперименталь­ ного материала из использованных мето­ дов оценки адаптивности следует обра­ тить особое внимание на показатель ста­ бильности сортов и коэффициент мульти­ пликативности. References/Литература [ i n R u s s i a n ] (Инструкция по технологиче­ скому к о н т р о л ю п и в о в а р е н н о г о п р о и з в о д ­ с т в а . М . : И з д - в о « П и щ е в а я п р о м ы ш л е н ­ н о с т ь » , 1 9 6 7 . 2 3 4 с . ) . DospekhovB. A. M e t h o d s o f f i e l d e x p e r i e n c e . M o s ­ c o w : A g r o p r o m i z d a t , 1 9 8 5 , 3 5 2 p . [ i n R u s s i a n ] (Доспехов Б.А. М е т о д и к а п о л е в о г о о п ы т а . М . : А г р о п р о м и з д а т , 1 9 8 5 . 3 5 2 с . ) . Dragavtsev V. A., Cil'ke V. A., Reiter B. G. t h e G e ­ n e t i c s o f p e r f o r m a n c e t r a i t s o f s p r i n g w h e a t i n W e s t e r n S i b e r i a . N o v o s i b i r s k : N a u k a , 1 9 8 4 , 2 2 9 p . [ i n R u s s i a n ] (ДрагавцевВ. А., Цильке В. А., Рейтер Б. Г. Г е н е т и к а п р и з н а к о в п р о д у к т и в ­ н о с т и я р о в о й п ш е н и ц ы в З а п а д н о й С и б и р и . References/Литература I n s t i t u t e o f a g r i c u l t u r e a n d c h e m i c a l i z a t i o n . A l t a i r e s e a r c h I n s t i t u t e o f a g r i c u l t u r e a n d c r o p b r e e d ­ i n g . N o v o s i b i r s k , 2 0 0 0 , 5 2 p . [ i n R u s s i a n ] (Пи­ воваренный ячмень в З а п а д н о й С и б и р и : м е ­ т о д . р е к о м е н д а ц и и / М С Х Р Ф А П К . С и б - Н И И з Х и м . А Н И И З и С . Н о в о с и б и р с к , 2 0 0 0 . 5 2 с . ) . Anis'kov N. I., Popolzukhin P. V. S p r i n g b a r l e y i n W e s t e r n S i b e r i a ( B r e e d i n g , s e e d p r o d u c t i o n , v a ­ r i e t i e s ) : M o n o g r a p h . O m s k : V a r i a n t - O m s k , 2 0 1 0 , 3 3 8 p . [ i n R u s s i a n ] (АниськовН. И., По- ползухин П. В. References/Литература , 1 9 8 5 . 2 5 0 с . ) . Hangil'din V. V., Asfondiyarova R R. M a n i f e s t a t i o n o f h o m e o s t a s i s i n h y b r i d s o f P i s u m s a t i v u m // B i ­ o l o g i c a l S c i e n c e s , 1 9 7 7 , n o . 1 , p p . 1 1 6 - 1 2 1 [ i n R u s s i a n ] (Хангильдин В. В., АсфондияроваР. Р. П р о я в л е н и е г о м е о с т а з а у г и б р и д о в г о р о х а п о с е в н о г о // Б и о л о г и ч е с к и е н а у к и . 1 9 7 7 . N ° 1 . С . 1 1 6 - 1 2 1 ) . Surin N. A. References/Литература I m p r o v e m e n t o f a d a p t i v e q u a l i t i e s o f b a r l e y , u s i n g t r a d i t i o n a l a n d m o d e r n m e t h o d s o f b r e e d i n g // B r e e d i n g o f c r o p s f o r h i g h g e n e t i c p o ­ t e n t i a l , y i e l d a n d q u a l i t y : m a t e r i a l s o f t h e i n t e r n a ­ t i o n a l s c i e n t i f i c - p r a c t i c a l c o n f e r e n c e . T y u m e n , 2 0 1 2 , p p . 3 0 - 4 0 [ i n R u s s i a n ] (Сурин Н. А. References/Литература С о ­ в е р ш е н с т в о в а н и е а д а п т и в н ы х с в о й с т в я ч ­ м е н я с и с п о л ь з о в а н и е м с т а р о д а в н и х и с о в р е ­ м е н н ы х м е т о д о в с е л е к ц и и // С е л е к ц и я с е л ь ­ с к о х о з я й с т в е н н ы х к у л ь т у р н а в ы с о к и й г е н е ­ т и ч е с к и й п о т е н ц и а л , у р о ж а й и к а ч е с т в о : М а ­ т е р и а л ы м е ж д у н а р о д н о й н а у ч н о - п р а к т и ч е ­ с к о й к о н ф е р е н ц и и . Т ю м е н ь , 2 0 1 2 . С . 3 0 - 4 0 ) . Kalashnikov N. A., Kozlov G. Y., Anisimov N. I. G e ­ n e t i c s o f p r o d u c t i v i t y a n d g r a i n q u a l i t y o f m a l t ­ i n g b a r l e y i n t h e c o n d i t i o n s o f t h e M i d d l e I r t y s h r e g i o n . N o v o s i b i r s k , 2 0 0 5 , 1 3 2 p . [ i n R u s s i a n ] (Калашников Н. А., Козлова Г. Я., Аниськов Н. References/Литература Н о в о с и б и р с к : Н а у к а , 1 9 8 4 . 2 2 9 c . ) . Nettevich E. D., Anikanova Z . F., Romanova, L M., t h e C u l t i v a t i o n o f m a l t i n g b a r l e y . M o s c o w : K o ­ l o s , 1 9 8 1 , 2 0 4 p . [ i n R u s s i a n ] (Неттевич Э. Д., Аниканова З. Ф., Романова Л. М. В ы р а щ и в а ­ н и е п и в о в а р е н н о г о я ч м е н я . М . : К о л о с , 1 9 8 1 . 2 0 4 с . Nettevich E. D., Morgunov A. I., Maksimenko M. I. i m p r o v i n g t h e e f f i c i e n c y o f s e l e c t i o n o f s p r i n g w h e a t o n t h e s t a b i l i t y , y i e l d a n d g r a i n q u a l i t y // B u l l e t i n o f a g r i c u l t u r a l s c i e n c e , 1 9 8 5 , n o . 1 , p p . 6 6 - 7 3 [ i n R u s s i a n ] (Неттевич Э. Д., Моргунов А. И., Максименко М. И. References/Литература П о в ы ш е н и е э ф ф е к ­ т и в н о с т и о т б о р а я р о в о й п ш е н и ц ы н а с т а ­ б и л ь н о с т ь , у р о ж а й н о с т ь и к а ч е с т в о з е р н а // В е с т н и к с е л ь с к о х о з я й с т в е н н о й н а у к и . 1 9 8 5 . № 1 . С . 6 6 - 7 3 ) . Eberhart S. A., Russell W. A. S t a b i l i t y p a r a m e t e r s f o r c o m p a r i n g v a t i i i e t i e s // C r o p . s c i . 1 9 6 6 , v o l . 6 , n o . 1 , p p . 3 6 - 4 0 . Fedin M. A. M e t h o d o l o g y o f s t a t e v a r i e t y t e s t i n g o f a g r i c u l t u r a l c r o p s . T h e c o m m o n p a r t . M o s c o w , 1 9 8 5 , 2 5 0 p . [ i n R u s s i a n ] (Федин М. А. М е т о ­ д и к а г о с у д а р с т в е н н о г о с о р т о и с п ы т а н и я с е л ь с к о х о з я й с т в е н н ы х к у л ь т у р . О б щ а я ч а с т ь . М . References/Литература Я р о в о й я ч м е н ь в З а п а д н о й С и ­ б и р и ( С е л е к ц и я , с е м е н о в о д с т в о , с о р т а ) : М о ­ н о г р а ф и я . О м с к : В а р и а н т - О м с к , 2 0 1 0 . 3 3 8 с . ) . I n s t i t u t e o f a g r i c u l t u r e a n d c h e m i c a l i z a t i o n . A l t a i r e s e a r c h I n s t i t u t e o f a g r i c u l t u r e a n d c r o p b r e e d ­ i n g . N o v o s i b i r s k , 2 0 0 0 , 5 2 p . [ i n R u s s i a n ] (Пи­ воваренный ячмень в З а п а д н о й С и б и р и : м е ­ т о д . р е к о м е н д а ц и и / М С Х Р Ф А П К . С и б - Н И И з Х и м . А Н И И З и С . Н о в о с и б и р с к , 2 0 0 0 . 5 2 с . ) . Manual for technological control i n b e e r b r e w i n g . M . : P u b l i s h i n g h o u s e " F o o d i n d u s t r y " , 1 9 6 7 , 2 3 4 p . References/Литература И. Г е н е т и к а п р о д у к т и в н о с т и и к а ч е с т в а з е р н а п и в о в а р е н н о г о я ч м е н я в у с л о в и я х С р е д н е г о П р и и р т ы ш ь я . Н о в о с и б и р с к , 2 0 0 5 . 1 3 2 с . ) Surin N. A., Lyakhov N. E. S e l e c t i o n o f b a r l e y i n S i ­ b e r i a . N o v o s i b i r s k , 1 9 9 3 , 2 9 2 p . [ i n R u s s i a n ] (СуринН. А.,ЛяховаН. Е. С е л е к ц и я я ч м е н я в С и б и р и . Н о в о с и б и р с к , 1 9 9 3 . 2 9 2 с . ) . Loskutov I. G., Kovaleva O. N., Blinova E. V. M e t h ­ o d o l o g i c a l g u i d a n c e d i r e c t o r y f o r s t u d i n g a n d m a i n t a i n i n g V I R ' s c o l l e c t i o n s o f b a r l e y a n d o a t . S t . P e t e r b u r g : V I R , 2 0 1 2 , 6 3 p p . [ i n R u s s i a n ] (Лоскутов И. Г., Ковалева О. Н., Блинова Е. В. References/Литература М е т о д и ч е с к и е у к а з а н и я п о и з у ч е н и ю и с о ­ х р а н е н и ю м и р о в о й к о л л е к ц и и я ч м е н я и о в с а . С П б . : В И Р , 2 0 1 2 . 6 3 с . ) . The state register o f s e l e c t i o n a c h i e v e m e n t s , a d m i t ­ t e d t o u s e : v a r i e t i e s o f p l a n t s M i n i s t r y o f a g r i c u l ­ t u r e o f t h e R u s s i a n F e d e r a t i o n . F G U " S t a t e C o m m i s s i o n o f t h e R u s s i a n F e d e r a t i o n " . M o s ­ c o w , 2 0 1 6 , 1 6 0 p . [ i n R u s s i a n ] (Государственный реестр с е л е к ц и о н н ы х д о с т и ж е н и й , д о п у щ е н н ы х к и с п о л ь з о в а н и ю : с о р т а р а с т е н и й / М С Х Р Ф . Ф Г У « Г о с у д а р ­ с т в е н н а я к о м и с с и я Р Ф » . М . , 2 0 1 6 . 1 6 0 p . ) . Выводы 1. Пивоваренный сорт ярового ячменя ‘Омский 100’ относится к высокоурожай­ ным сортам. Максимальная урожайность получена в 2015 г. - 6,6 т/га, прибавка к стандартному сорту ‘Омский 95’ соста­ вила 0,8 т/га. 1. Пивоваренный сорт ярового ячменя ‘Омский 100’ относится к высокоурожай­ ным сортам. Максимальная урожайность получена в 2015 г. - 6,6 т/га, прибавка к стандартному сорту ‘Омский 95’ соста­ вила 0,8 т/га. 2. Содержание белка в зерне сорта ‘Омский 100’, в среднем, 12,8%. По дру­ 98 Труды по прикладной ботанике, генетике и селекции, том 178, выпуск 4 References/Литература Malting barley i n W e s t e r n S i b e r i a : m e t h o d . r e c o m ­ m e n d a t i o n s / M i n i s t r y o f a g r i c u l t u r e o f t h e R u s ­ s i a n F e d e r a t i o n o f a g r i c u l t u r e . S i b e r i a n r e s e a r c h 99
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Optimization of aluminumand its alloys doping by ionic-beam-plasma coating
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MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/0121 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 E-mail: L-7755me@mail.ru Abstract. The surface morphology, chemical composition, microstructure, nanohardness, and tribological properties of systems were investigated. The paper considers the methodology offilmpplicationusingionic-beam irradiationby means of the installation‘Solo’ with different exposure modes. Irradiation modes which allow an increase in the microhardness of the material and a decrease in its wear rate are defined. Physical substantiation of this phenomenon is given. 1. Introduction Alloying is the best way to improve characteristics of metals. However, the alloying process demands a large amount of dopant, but the application of plasma methodsallows reducing this amount.In this method heating and cooling rates are sufficiently high for a rapid growth ofa crystal. In addition to it, there is a method of ion implantation allowing one to introducealloying atoms directly into the surface layers of solids [1].Silicon and titanium are the main alloying elements. Silicon is one of the main additives in creation of cast alloys (silumin). Silumins usually contain from 5 to 14% of Si, i.e. a few percent higher or lower than the eutectic concentration Sr and Sb [2], V and Mo [3], W [4] are normally used as additives. The addition of these elements influencesbeneficially on the structure and mechanical properties of alloys. y We have revealed the presenceof intermetallic compounds of aluminum and titanium (TiAl, Ti3Al, Al3Ti) in the alloys. The main disadvantage of these compounds is low plasticity and impact strength. This drawbackcan be reduced owing to a decrease of the grain sizes and a creationof a columnar structure. The aim of our study is to choose an optimal mode of sample influence, to analyze the structures and strength properties of commercially pure aluminum of the A7 grade and siluminof the АК12 grade alloyed with silumin (25 wt. %)and titanium. M Rygina1, O Krisina2, Yu Ivanov1,2, E Petrikova2, A Teresov2 1Tomsk Polytechnic University, 30, Lenina ave., Tomsk, 634050, Russia 2Institute of High Current Electronics, Siberian Branch of the Russian Academy of Sciences, 2/4, Akademicheskiyave., Tomsk, 634055, Russia 1Tomsk Polytechnic University, 30, Lenina ave., Tomsk, 634050, Russia 2Institute of High Current Electronics, Siberian Branch of the Russian Academy of Sciences, 2/4, Akademicheskiyave., Tomsk, 634055, Russia Optimization of aluminumand its alloys doping by ionic-beam- plasma coating M Rygina1, O Krisina2, Yu Ivanov1,2, E Petrikova2, A Teresov2 1Tomsk Polytechnic University, 30, Lenina ave., Tomsk, 634050, Russia 2Institute of High Current Electronics, Siberian Branch of the Russian Academy of Sciences, 2/4, Akademicheskiyave., Tomsk, 634055, Russia MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/0121 CSEM tribometer, Switzerland, at room temperature and air humidity. The wear volume of th materialwas estimatedafter conducting a profilometry of the formed track using a laser range optica profilometer ‘MicroMeasure 3D Station’ (Stil, France). The elemental and phase compositions of th modified surface were analyzed by means of an optical microscope (NEOFOT-32) and a scannin electron microscope (Philips SEM-515). 2. Materials Samples of commercially pure aluminum of the A7 grade, silumin of the AK12 grade, and silumin (25 wt. % Si) were used as amaterialfor study. Samples were subjected to electronic-ionic-plasma treatment on the‘SOLO’installation. At the first stage samples were evaporated witha thin silumin film (h = 0.5 µm and h = 1.0 µm) by the method of sputtering of the silumin АК25 sample. After it, samples were irradiated by a high-intensity pulsed electron beam at different irradiation modes. Another system is represented by theTi film /Al substrate system,the thickness of the Ti film being 0.5 µm. Thin (0.5 µm)Ti films were synthesized on a specialvacuum installation ‘TRIO’ in the low-pressure arc plasma [4].The film/substrate system was modified by the high-intense pulsed electron beam on the SOLO installation [5].The electron beam energy density was 10 J/cm2 and 15 J/cm2;the pulse repetition frequency was 0.3 Hz anda number of radiation pulses were varied in the range of 3…30. The samples were characterized by microhardness (a PMT-3 device, the indenter load is 0.1N). The study of the wear rate of the film/substrate system was conducted in the disc-pin geometry by means of a MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 3. Research Results and Discussion Let us analyze the results for the film (Al-25wt.% Si) / substrate (Al-12wt.% Si)system presented in Figure 1.The silicon inclusions in the thin near-surface layer have a globular shape, if the film thickness is 0.5 µm (Figure 1a, b), and an island shape, if the film thickness is 1.0 µm (Figure 1c, d). Table 1. Dependence of the hardnesson the mode exposure. Es =15J/cm , h=0.5µm 84 kg/mm Es =20J/cm2, h=0.5µm 104 kg/mm2 Es =15J/cm2, h=1µm 88.7 kg/mm2 Es =20J/cm2, h=1µm 165 kg/mm2 а b c d Figure 1.The surface structure of samples of the film (Al-25wt.% Si) / substrate (Al-12wt.% Si) systemirradiated by an electron beam pulse; a) ES = 15 J/cm2, h = 0.5 µm; b) ES = 20 J/cm2, h = 0.5 µm; c) ES = 15 J/cm2, h = 1 µm; d) ES = 20 J/cm2, h = 1 µm b d b а b d c c d c d Figure 1.The surface structure of samples of the film (Al-25wt.% Si) / substrate (Al-12wt.% Si) systemirradiated by an electron beam pulse; a) ES = 15 J/cm2, h = 0.5 µm; b) ES = 20 J/cm2, h = 0.5 µm; c) ES = 15 J/cm2, h = 1 µm; d) ES = 20 J/cm2, h = 1 µm Table 2. The wear resistance and friction coefficient of the surface layer of silumin (12 wt. %) modified by sputtered silumin and subsequent processing by the electron beam. able 2. The wear resistance and friction coefficient of the surface layer of silumin (12 wt. %) modified by sputtered silumin and subsequent processing by the electron beam. Table 2. The wear resistance and friction coefficient of the surface layer of silumin (12 wt. %) modified by sputtered silumin and subsequent processing by the electron beam. Mode V, 10-6 V0/V <µ> <µ0>/<µ> Silumin (12wt.%) 24322 mm3/(H·m) 0.612 15 J/cm2, 0.5 µm, Al-Si 18630 mm3/(H·m) 1.31 0.648 0.94 15 J/cm2, 1 µm, Al-Si 22228 mm3/(H·m) 1.10 0.632 0.97 20 J/cm2, 0.5 µm, Al-Si 19416 mm3/(H·m) 1.25 0.633 0.97 2 MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 f. 3. Research Results and Discussion Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 20 J/cm2, 1 µm, Al-Si 21124 mm3/(H·m) 1.15 0.628 0.97 Figure 2.Dependence of hardness H and Young's modulus E on the indenter load (a nanoindentation method) of the surface layer of the AK12 grade silumin modified by silumin sputtering (the layer thickness is 0.5 µm) and subsequent treatment with an electron beam (in the mode of 15 J/cm2, 150 ms, 5 imp., 0.3 Hz). Dash-dotted lines indicate the characteristics of the original AK12 grade silumin (12wt.%). Infigure b: 1 –siluminYoung's modulus 2 –silicon Young's modulus m) 1.15 0.628 0.97 Figure 2.Dependence of hardness H and Young's modulus E on the indenter load (a nanoindentation method) of the surface layer of the AK12 grade silumin modified by silumin sputtering (the layer thickness is 0.5 µm) and subsequent treatment with an electron beam (in the mode of 15 J/cm2, 150 ms, 5 imp., 0.3 Hz). Dash-dotted lines indicate the characteristics of the original AK12 grade silumin (12wt.%). Infigure b: 1 –siluminYoung's modulus 2 –silicon Young's modulus The data on microhardness for another film(25 wt.% of silumin)/substrate (Al) system are represented in Table 3. The data on microhardness for another film(25 wt.% of silumin)/substrate (Al) system are represented in Table 3. n Table 3. Table 3. Dependence of hardnesson the mode exposure. Processing mode Hardness Mode of processing 250 MPa Pure Al 425 MPa ES =15 J/cm2, h=0.5µm 370 MPa ES =20 J/cm2, h=0.5µm 548 MPa ES =15 J/cm2, h=1µm 470 MPa Figure 3.Dependence of hardness H and Young's modulus E on the indenterload (a nanoindentation method) of the surface layer of aluminum modified by the method of silumin sputtering (layer thickness is 1 µm) and subsequent treatment with an electron beam in the mode of 20 J/cm2, 150 ms, 5 imp., 0.3 Hz. Dashed-dotted lines indicate the characteristics of the original A7 grade aluminum. Table 3. Dependence of hardnesson the mode exposure. Table 3. Dependence of hardnesson the mode exposure. 3. Research Results and Discussion Processing mode Hardness Mode of processing 250 MPa Pure Al 425 MPa ES =15 J/cm2, h=0.5µm 370 MPa ES =20 J/cm2, h=0.5µm 548 MPa ES =15 J/cm2, h=1µm 470 MPa Figure 3.Dependence of hardness H and Young's modulus E on the indenterload (a nanoindentation method) of the surface layer of aluminum modified by the method of silumin sputtering (layer thickness is 1 µm) and subsequent treatment with an electron beam in the mode of 20 J/cm2, 150 ms, 5 imp., 0.3 Hz. Dashed-dotted lines indicate the characteristics of the original A7 grade aluminum. The wear resistance and friction coefficients of the surface of samples are given in Table 4. It has been established that high wear resistance belongs to samplestreated in the mode of20 J/cm2 and the film thickness of 0.5 µm. Thelowestfriction coefficient is acquired by the materialin the mode of 20 J/cm2 and the film thickness of1µm. 3 MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 MEACS2015 MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/0121 Table 4. The wear resistance and friction coefficients of the surface layer of commercially pure A7 grade aluminum modified by siluminsputtering and subsequent processing by the electron beam (150 ms, 5 Table 4. The wear resistance and friction coefficients of the surface layer of commercially pure A7 grade aluminum modified by siluminsputtering and subsequent processing by the electron beam (150 ms, 5 Table 4. 3. Research Results and Discussion The wear resistance and friction coefficients of the surface layer of commercially pure A7 grade aluminum modified by siluminsputtering and subsequent processing by the electron beam (150 ms, 5 imp., 0.3 Hz.) Mode V, 10-6, mm3/(Н·m) V0/V <µ> Pure Al 7592 - 0.483 15 J/cm2, 0.5 µm 6080 1.25 0.385 15 J/cm2, 1 µm 6566 1.16 0.4 20 J/cm2, 0.5 µm 5466 1.39 0.353 20 J/cm2, 1 µm 4790 1,59 0.311 The research results of the hardness of the thirdTi film/Al substrate system have shown that hardnessincreases more than three timesduringthe electron beam irradiation underthe mode of 15 J/cm2, aluminum modified by siluminsputtering and subsequent processing by the electron beam (150 ms, 5 imp., 0.3 Hz.) Mode V, 10-6, mm3/(Н·m) V0/V <µ> Pure Al 7592 - 0.483 15 J/cm2, 0.5 µm 6080 1.25 0.385 15 J/cm2, 1 µm 6566 1.16 0.4 20 J/cm2, 0.5 µm 5466 1.39 0.353 20 J/cm2, 1 µm 4790 1,59 0.311 The research results of the hardness of the thirdTi film/Al substrate system have shown that hardnessincreases more than three timesduringthe electron beam irradiation underthe mode of 15 J/cm2, 30 pulses, 50 µs, and 0.3 Hz (Figure4, curve 2). The research results of the hardness of the thirdTi film/Al substrate system have shown that hardnessincreases more than three timesduringthe electron beam irradiation underthe mode of 15 J/cm2, 30 pulses, 50 µs, and 0.3 Hz (Figure4, curve 2). Figure 4. The wear rate (curve 1) and the surface microhardness (curve 2) of the film (Ti)/substrate (Al) system after different modes of treatment: 1 – substrate (commercially pure A7 aluminum); 2…4 the film/substrate systems irradiated with the electron beam with parameters of 10 J/cm 2, 50 µs, 10 pulses (lower index 2);with parameters of 15 J/cm 2, 50 µs, 3 pulses (lower index 3); and with parameters of 15 J/cm 2, 50 µs, 30 pulses (lower index 4) Figure 4. The wear rate (curve 1) and the surface microhardness (curve 2) of the film (Ti)/substrate (Al) system after different modes of treatment: 1 – substrate (commercially pure A7 aluminum); 2…4 the film/substrate systems irradiated with the electron beam with parameters of 10 J/cm 2, 50 µs, 10 pulses (lower index 2);with parameters of 15 J/cm 2, 50 µs, 3 pulses (lower index 3); and with parameters of 15 J/cm 2, 50 µs, 30 pulses (lower index 4) Figure 4. References [1] Mashkov Yu, PoleschenkoKN,Povoroznyuk C H and Orlov PV 2000Friction and modification of materials tribosystems (M: Nauka) p 280 [2] Szymczak T,Gumienny G and Pacyniak T 2015Archives of Foundry Engineering15(1) 105–108 [3] Szymczak T,Gumienny G and Pacyniak T 2015ArchivesofFoundry Engineering15(4) 81–86 [4] Szymczak T, Gumienny G, Pacyniak T and Walas K 2015 Archives of Foundry Engineering15(3) 61–66 [5] GrinbergBA and IvanovMA2002 Intermetallic Ni3Al and TiAl compounds: microstructure and deformation behavior(UrB RAS: Ekaterinburg) 3. Research Results and Discussion In addition to the diffraction maximum of aluminum and titanium, the X-ray patterns show the diffraction maximums of the Al3Ti phase.Thevolume 4 IOP Publishing erials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 MEACS2015 IOP Publishing IOP Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/012137 Conf. Series: Materials Science and Engineering 124 (2016) 012137 doi:10.1088/1757-899X/124/1/0121 fraction of the intermetallic is ~20 %, titanium – 28 %. Irradiation underES = 15 J/cm2 (3 imp.) leads to a increase in the lattice parameter of aluminum up to a = (0.4050±0.0001) nm, which indicates th formation of alloy Al-Ti. fraction of the intermetallic is ~20 %, titanium – 28 %. Irradiation underES = 15 J/cm2 (3 imp.) leads to an increase in the lattice parameter of aluminum up to a = (0.4050±0.0001) nm, which indicates the formation of alloy Al-Ti. Figure 5. The surface structure of the film/substrate system irradiated with an electron beam under: a– 10 J/cm2, 10 imp., b– 15 J/cm2, 3 imp. Th l f ti f th Al3Tii t t lli d d t 3 % th l f ti f tit i Figure 5. The surface structure of the film/substrate system irradiated with an electron beam under: a– 10 J/cm2, 10 imp., b– 15 J/cm2, 3 imp. The volume fraction of the Al3Tiintermetallic compound reduces to ~3 %,the volume fraction of titanium emainsvirtually constant (~30 %). The volume fraction of the Al3Tiintermetallic compound reduces to ~3 %,the volume fraction of titanium remainsvirtually constant (~30 %). The volume fraction of the Al3Tiintermetallic compound reduces to ~3 %,the volume fraction of titanium remainsvirtually constant (~30 %). Acknowledgments g The work has been performedwith the support of the RNF grant (project № 14-29-00091). 4. Conclusion The maximum microhardness is achieved by means of influence on the surface of the silumin (25 wt. %) film/substrate (Al) system and the energy density of the electron beam ofES = 15 J/cm2. An increase of the energy density up to 20 J/cm2 (regardless of the deposited film thickness) leads to a decrease of the microhardness of the irradiated surface.A percentage of silicon in the surface changes in a similar way. The hardness of the modified layer depends on the percentage of silicon in it.A better wear resistance was shown by a sample irradiated with an electron beam underES = 20 J/cm2, h = 0.5 µm. The minimum friction coefficient was detected in the sample under ES = 20 J / cm2, h = 1µm. p S , µ The maximum microhardness is identified in the silumin (25 wt. %) film / silumin (12 wt. %) substrate system irradiated by the electron beam with the energy density of ES = 20 J / cm2. When using a substrate, wear resistance reduces irrespective of the irradiation modes and the layer thickness for 12 % silumin. The Ti film/Al substrate system subjected to high-energy impact under 15 J/cm2, 3 pulses, 50 µs, 0.3 Hz demonstrates a 7.5 time increase in the wear rate. The irradiation under 15 J/cm2, 30 pulses, 50 µs, 0.3 Hz provides a more than 3 time increase of the system microhardness of the material compared to the substrate. 3. Research Results and Discussion The wear rate (curve 1) and the surface microhardness (curve 2) of the film (Ti)/substrate (Al) system after different modes of treatment: 1 – substrate (commercially pure A7 aluminum); 2…4 the film/substrate systems irradiated with the electron beam with parameters of 10 J/cm 2, 50 µs, 10 pulses (lower index 2);with parameters of 15 J/cm 2, 50 µs, 3 pulses (lower index 3); and with parameters of 15 J/cm 2, 50 µs, 30 pulses (lower index 4) The best resulting properties are attainedduring the electron beam irradiation of the film/substrate system with parameters of 15 J/cm2, 3 pulses, 50 µs, and 0.3 Hz (Figure 4, curve 1); the wear rate of the modified layer is more than 7.5 times lower compared to the substrate. The best resulting properties are attainedduring the electron beam irradiation of the film/substrate system with parameters of 15 J/cm2, 3 pulses, 50 µs, and 0.3 Hz (Figure 4, curve 1); the wear rate of the modified layer is more than 7.5 times lower compared to the substrate. The surface alloy, while displaying high hardness, reveals a decline in wear resistance, which is apparently due to structural features of the formed film/substrate system. This is due to the appearance of intermetallic compounds in the structure. p Intermetallic compounds of aluminum and titanium (TiAl, Ti3Al, Al3Ti) feature a unique combination of properties: high values of specific strength, heat resistance, thermal stability, elastic modulus, creep resistance, and anomalous temperature dependence on the mechanical properties [1]. p p p p Typical images of the surface structure of the film / substratesystem irradiated with the intense electron beam are shown in Figure5. When irradiating with the electron beam with the energy density of ES = 10 J/cm2 (10 imp.) and 15 J/cm2 (3 imp.), the Ti film deposited on the aluminum surface is preserved. However, at the same time, the Ti film is fragmented withmicro-cracks (Figure5a, b).The sizes of fragments underES = 15 J/cm2are 3…4 times less than those in the sample irradiated underES = 10 J/cm2. A microcracks volume with cross-sectional dimensionsof ~10 microns is filledwith the melt of aluminum. The crystal lattice parameter of aluminum, when ES = 10 J/cm2,is a= (0.4041±0.0001) nm, which corresponds to the lattice parameter of pure aluminum. References References [1] Mashkov Yu, PoleschenkoKN,Povoroznyuk C H and Orlov PV 2000Friction and modification of materials tribosystems (M: Nauka) p 280 [2] Szymczak T,Gumienny G and Pacyniak T 2015Archives of Foundry Engineering15(1) 105–108 [3] Szymczak T,Gumienny G and Pacyniak T 2015ArchivesofFoundry Engineering15(4) 81–86 [4] Szymczak T, Gumienny G, Pacyniak T and Walas K 2015 Archives of Foundry Engineering15(3) 61–66 [5] GrinbergBA and IvanovMA2002 Intermetallic Ni3Al and TiAl compounds: microstructure and deformation behavior(UrB RAS: Ekaterinburg) 5
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A case study of enterprise identity management system adoption in an insurance organization
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A Case Study of Enterprise Identity Management System Adoption in an Insurance Organization Pooya Jaferian, David Botta, Kirstie Hawkey, Konstantin Beznosov University of British Columbia, Vancouver, Canada {pooya,botta,hawkey,beznosov}@ece.ubc.ca ABSTRACT This case study describes the adoption of an enterprise identity management(IdM) system in an insurance organization. We describe the state of the organization before deploying the IdM system, and point out the challenges in its IdM practices. We describe the organization’s requirements for an IdM system, why a particular solution was chosen, issues in the deployment and configuration of the solution, the expected benefits, and the new challenges that arose from using the solution. Throughout, we identify practical problems that can be the focus of future research and development efforts. Our results confirm and elaborate upon the findings of previous research, contributing to an as-yet immature body of cases about IdM. Furthermore, our findings serve as a validation of our previously identified guidelines for IT security tools in general. Categories and Subject Descriptors K.6.5 [Management of Computing and Information Systems]: Security and Protection; H.5.2 [Information Interfaces and Presentation]: UIs—Interaction Styles; H.5.3 [Information Interfaces and Presentation]: Group and Org. Interfaces—Collaborative Computing Keywords Identity management, Qualitative Research, Security Tools, Organizational Factors, Case Study 1. INTRODUCTION Identity management (IdM) comprises the processes and infrastructure for the creation and maintenance of user’s digital identities and the designation of who has access to resources, who grants that access, and how accountability and compliance are maintained [5, 7]. While IdM can be studied in different contexts (e.g., the Internet), the scope of IdM in this paper is enterprise identity management. Enterprise IdM includes tasks such as managing identities of the organization’s users, managing roles through their life-cycle, Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. To copy otherwise, to republish, to post on servers or to redistribute to lists, requires prior specific permission and/or a fee. CHIMIT ’09, November 6-7, 2009, Baltimore, Maryland, USA. Copyright 2009 ACM 1-60558-572-7/09/11 ...$10.00. assigning identities to roles, determining the resources each role has access to, and the auditing and reporting of information related to IdM in an organization. IdM has become an important aspect of IT security infrastructure in organizations, and some consider it to be the most important solution for enabling compliance with legislative requirements [19]. Further drivers of IdM adoption include cost reduction, better security, better access to information, and better agility during mergers and acquisitions [13]. However, the practice of IdM is challenging, both organizationally and technologically [13, 19]. Identifying these challenges and studying how they can be addressed are important steps toward improving IdM systems and practices in organizations. Despite the widespread and increasing adoption of IdM solutions, there are few available case studies that examine the practice of IdM in organizations. Two major studies of IdM are the Identity Project [19] (an academic survey of IdM practices in UK higher education institutions) and reports by the Burton Group [1] (a firm that provides IT research and advisory services to private clients). Both of these studies blend the results of their case studies into their reports and give recommendations for improving IdM practices and systems. Most recently, Bauer et al. [4] describe real life challenges in access control management as gleaned through interviews with policy professionals. Although not explicitly about IdM, Heckle et al. [9] discuss organizational challenges in implementing a single sign-on system without previously assisting end-users to develop an accurate mental model. Also, Post et al. [14] identify security controls as factor that interferes with end-users’ work and propose recommendations for alleviating this problem. In order to improve the usability of IdM systems, or propose new development, more case studies are needed to illuminate nuances of the issues that are already indicated by prior research, and to reveal topics for further research. In this paper, we present a case study of an insurance organization that has recently made two phases of a multiphase integrated IdM system operational. IdM is an important part of this organization’s security infrastructure. Our case study contributes to an as-yet immature body of cases about IdM. It provides a realistic picture of an organization during the various stages of deploying an IdM system. This picture helps identify practical problems that can be the focus of future research, and that suggest opportunities for new development. In addition, we describe the organization’s challenges and discuss their IdM solution in IdM Selection Table 1: Participant roles Participant Role SA-lead Security Administration group leader IdM-lead IdM Project leader SecA Security Analyst SSecC Senior Security Consultant comparison to related work such as the access control management challenges identified by Bauer et al. [4] and findings and recommendations from the Identity Project [19] and the Burton Group [1]. Furthermore, we discuss and validate a framework of guidelines and recommendations for IT security tools [10] ; these were selected from the literature based on their relevance to the practice of IT security management in general and we illustrate through the case study findings how they can be applied to IdM tools in particular. This information may be used by practitioners who must identify requirements for and evaluate IdM systems, as well as by developers to improve their IdM products. The remainder of this paper is organized as follows. We first explain our methods for performing the case study and analyzing the data in Section 2, before laying out the findings of our case study in Section 3. In Section 4, we discuss the findings from our case study and compare them with those from prior research; while in Section 5, we examine how our case study findings validate our previously developed guidelines for IT security tools in general. Finally, we conclude in Section 6 with the limitations of our research and Section 7 with a summary of our contributions and a discussion of our future work in this area. 2. METHODOLOGY We performed four semi-structured interviews with people from the Security Administration (SA) group who were involved in the selection and/or deployment of an IdM system in an insurance organization. The SA group leader (SAlead), IdM project leader (IdM-lead), and Security Analyst (SecA) have been involved during the entire process of IdM adoption in the organization, while the Senior Security Consultant (SSecC) was only involved in the selection process (see Table 1 for the role key). Each interview lasted from one to two hours and was conducted by two interviewers in the workplace of the participant. The interviews took place at different stages in the IdM deployment process (Fig. 1). Two interviews (SecA, SSecC) were conducted in 2006 and 2007 as a part of our previous project (HOT-Admin, see [8] for an overview of the themes of analysis), and two interviews (SA-lead, IdM-lead) were conducted in late 2008 to study IdM in particular. While the focus of the two HOT-Admin interviews was on IT security management in general, the importance of IdM in the organization led our participants to answer many questions in the context of IdM. The longitudinal aspect of data collection enables us to describe the state of the organization at different stages of IdM adoption. Interviews were audio-recorded, transcribed, and analyzed by two researchers. In addition to the interviews, the researchers analyzed the Request for Information and Qualification (RFIQ) that the organization issued when beginning the IdM selection process. This documentation of the orga- Deployment Phase I Deployment Phase II SSecC SecA (Sept 2006) (Nov 2007) SA-lead IdM-lead (Dec 2008) Figure 1: Interview timeline that shows when the interviews were conducted during the course of the IdM adoption process nization’s requirements for the IdM system allowed for triangulation at the level of data source, mitigating some bias inherent in the self-report data of interviews. Using two researchers provided triangulation at the level of analysis and increased confidence in the shared findings. For analysis, we performed a combination of direct interpretation and categorical aggregation as recommended by Stake [15]. During direct interpretation, we focused on a single instance of data, pulling it apart and re-shaping it in a more meaningful way. Conversely, during categorical aggregation, we put together snippets of information about a phenomena from different parts of a single interview or different interviews and elaborated the meaning of the phenomena based on the different evidence. We also looked for certain patterns by finding correspondence between different categories. To perform the above mentioned analysis, we employed both high-level and open coding techniques. We first coded the data in high level categories including “Stakeholders”, “Tools”, “Challenges”, “Tasks”, and “Interactions”. We then performed open-coding inside each category and coded the data with more fine-grained codes that emerged from the data itself. After coding the data, we revised our codes by collapsing similar codes into a single code. As an illustration, to study the “Role Discovery” challenge, we examined the problem by aggregating snippets of information about role discovery. To analyze the role of managers in IdM adoption challenges, we looked for coinciding codes of “Managers” and “Challenges”. This coding practice was performed using Qualrus 2.1, a qualitative analysis software that provides the ability to perform categorical aggregation and find correspondence. 3. IDM CASE STUDY 3.1 The organization The participating insurance organization had about 2,500 employees at the time of the study. Approximately 2,000 of them worked in the head office, and the rest in branch offices. The processing environment included a single IBM mainframe (z/OS), more than 200 Intel-based Microsoft Windows 2003 servers, and several UNIX (AIX) servers. The personal computers that were in use at both the head office and branch offices numbered around 3,000. The responsibilities of the organization’s central IT security group included developing policies, standards, and practices related to IT security, as well as managing digital identities and computer-related access control. At the time of this study, the organization had already contracted an IdM software vendor, and the IT security group had implemented the first two phases of their IdM plan: (1) self-service password recovery, and (2) basic provisioning, in which all employees had access to commonly needed services like e-mail and the Internet. Further phases were in development. Management of digital identities was centralized, while access control was distributed. The SA group handled the creation of digital identities, primarily using Active Directory. They also controlled access to certain kinds of information on the mainframe using Resource Access Control Facility (RACF), a software product from IBM that provides access control functionality in mainframe servers. The RACF rules indicate transactions at the level of software applications, and what user groups are permitted to access those transactions. However, access to local area network (LAN) resources, the internet, databases, etc., was controlled by departmental administrative groups. Prior to IdM deployment, the SA group developed the “Data Guardianship Policy” that governs who can access a resource in the organization. The SA group developed a list of resources in the organization. Each resource is associated with an owner, called a “data guardian”. Normally, the data guardian for a resource is the manager of the business unit to which the resource belongs. The data guardian is responsible for keeping that resource safe by deciding who can access it. As the manager may not have sufficient awareness about the necessary security requirements for a resource, or the time to respond to access requests, he or she can delegate authority to another person in the business unit. In this case, the person to whom the authority is delegated is called the “data steward.” The data-guardianship policy in our case study organization is an official policy which is documented and strictly enforced. The SA group performs periodic training for data guardians and data stewards to raise their awareness of policies and procedures. The organization also maintained a separate IdM system that allowed its customers to track their insurance claims through a website. The separation was to ensure there would be no accidental leakage of information. 3.2 Before deployment of an IdM System We now describe the state of the organization before deployment of an integrated IdM system, including the IdM processes in place and the challenges these processes posed. 3.2.1 The basic IdM process The basic steps in the organization’s prior identity management workflow (before full deployment of an integrated IdM system) were as follows: 1. Human resources created an ID for a new employee. 2. Both the SA group and the employee’s manager were notified. This notification was automatic, however, the SA group manually processed the ID. 3. A security administrator provided basic permissions to access the systems that are common to all employees, such as e-mail. 4. The employee’s manager requested access to the systems that are appropriate for that employee. This means that the manager needed to know which systems to request. The request was made by means of an electronic form, and was made to the SA group. Similarly, the manager might request additional, possibly temporary, access for an existing employee. 5. If the request was related to RACF and Active Directory (Application Layer Access), a security administrator deployed the request to the data guardian of the requested data. The data might be distributed, and thereby owned by several data guardians. In this case, the security administrator made multiple requests. If the request was not related to RACF and Active Directory, the security administrator forwarded the request to the pertinent DB/LAN administrator, who could implement the access. 6. The data guardian might delegate the request to a data steward. 7. The security administrator performed a follow-up cycle, to handle non-response or lag from data guardians. 8. If the data guardian or data steward granted permission, the security administrator would implement the access in RACF and Active Directory. 9. When an employee was terminated or his status changed, the employee’s manager was responsible for notifying the SA group. 3.2.2 Challenges in IdM workflow Challenges in the process motivated the organization to adopt an IdM system. To begin with, there could be a significant lag between when a new employee began work and when Human Resources completed processing the new employee and creating an ID (step 1), thereby reducing employee productivity. Once the ID was created in Active Directory, the SA group had to then repetitively enter it in other systems (step 2) – there was no central repository for IDs. For example, the same information had to be entered in RACF, UNIX, SQL databases, and so on (SA-lead, SecA). In step 4, the SA group provided a detailed multi-page form for managers to identify resources and request access. The managers were overwhelmed by the amount of information that they were expected to fill in (SecA). As already mentioned, a manager who requests access for an employee must have knowledge of the systems, and not all managers knew what to ask for. Frequently, managers would ask the SA group to make a new employee’s access the same as some another current employee’s access: “A manager who hired a new employee who knew that you had the access that you needed to do the job for him or her would say, ‘Oh, make this new employee’s access just like yours.’ And so then an employee would then inherit very high levels of privileges and access based on the success of a previous employee in terms of doing that job. [...] Historically here if you were an individual who started at [the organization] in 1930 (I’m exaggerating) by the time you retired and had 40 years you would have access to every single system that you had ever used in your entire lifetime with” (SA-lead). However, the SA group disallowed this kind of generalized request as it might provide more access than required for the employee; they required the managers to indicate the individual desired systems: The managers who didn’t know what to ask for then tried to work around the security requirement by asking for the list of accesses possessed by a current employee, and requesting access for every item on that list. Obtaining knowledge about access profiles (groups of access privileges) and presenting it to the requesting managers was in itself a significant challenge. The organization developed an application to automatically collect information about access privileges from heterogenous systems and put them in a database. The database grew very quickly and faced performance and availability problems (SecA). The organization had 400,000 RACF rules that were created by generations of security administrators over nearly 20 years. Each generation created rules in its own way. Succeeding generations, when failing to make sense of how the previous security administrators made rules, would invent their own way of making rules (SA-lead). Needless to say, the managers did not necessarily understand the esoteric access rules and system names – these had to be translated into language that the managers could easily understand: “[...] and we get this huge profile - here’s all the access the user has. We then have to translate that into more of an English format for the individual, saying this means this, this means this, and this means this. And then, they have to put that into a form of what they want” (SecA). Furthermore, there was no common terminology throughout the organization for requesting access, resulting in cases where it was difficult for the SA group to identify which systems in which divisions were requested: “[...] but it’s terminology we don’t know. We say what area is this in, is this in the assessment part, is this prevention, is this the claims? sometimes the user has no idea, they just say I don’t know I just have to get access to it. So we end up going to the security coordinators for all those divisions saying are you familiar with this app? Or whatever they are talking about. Oh yeah, this is actually this. Aha. Then we know, and then we can tell if the request has come to us in a form or an email and it’s approved, we can set up the access or send it to the area responsible for setting up the access” (SecA). In step 5, when the security administrator processed the request and deployed it to various data guardians, it was often difficult to identify the correct data guardians. Also, as mentioned, some data guardians would not respond in a timely fashion, or even not respond at all (making step 7 necessary). The security administrator could end up implementing the requested access in a piecemeal fashion, thereby decreasing employee productivity as the employee would then have to wait for access: “[...] and so the security administrators would then send notes, e-mail, to the data guardians saying, ‘Are you OK if we grant Bob or Jill access to the system?’ And then there would be that sort of follow-up cycle where the data guardian would ignore them or not respond or say I’m not the data guardian or that kind of crap. So there would often be a delay in terms of getting approval... so systems access to the employee would then build over time as individual data guardians would respond” (SA-lead). When an employee changed his or her role or department, the SA group would notify that employee’s new manager, and ask the manager to revise the employee’s access privileges. Due to a lack of accountability and/or lack of understanding the access profile of the employee, often the employee would accumulate unnecessary privileges (SA-lead, IdM-lead, SecA, SSecC). This issue was compounded by a trade union mentality: “So some people view that as an infringement upon their union rights. You can’t take things away from me. I have seniority. You can add to me, but you cannot take away from me. They don’t understand like the security concept of you’re doing this job now, you’re not doing this job, you don’t need that access anymore” (SSecC). Concerning temporary access, the SA group manually set re- minders in their e-mail client software about when revocation of access was due to happen. Sometimes the SA group forgot to either set a reminder or to act on a notification of a reminder (SecA). It was the managers’ responsibility to notify the SA group about termination or changes in the status of their employees (step 9). However, as managers did not suffer negative consequences for failing to notify the SA group, terminations often went unreported. The SA group compensated by obtaining automated notifications from Human Resources. However, the automated notifications were not entirely effective, because there are various stages of termination: “We were getting status change notifications regarding employee departures... not universally 100% effective because what can happen is employees can not quite depart. Meaning that you can go on severance, which means that you have a year, two years worth of severance and you haven’t departed” (SAlead). To solve these issues, the SA group would periodically query the system for accounts that had not been used for a while, and manually follow up on the results (SA-lead, IdM-lead). Apart from the steps of providing or revoking access, the SA group faced challenges around both audits and troubleshooting. It was difficult to perform audits on groups, because groups in RACF were highly granular. Each access request corresponds to multiple transactions and each transaction can be accessed by multiple groups. Therefore, the SA group was required to respond to an access request by making the user a member of different RACF groups; the combination of groups provides access to all required transactions. This made it difficult for SA group to later ascertain which group was associated with some requested access, altogether resulting in the personnel sometimes creating a new group, compounding the complexity. The issue was further magnified when the SA group would send reports to the data guardians about what groups the data guardians “owned”, the users in those groups, and the transactions to which the users had access. The reports were almost impossible for the data guardians to audit as they contained a great deal of RACF information. Periodically, the data guardians would ask for a list of who had permission to access to their data, and the SA group would “give them reams of paper, and that would confuse them” (SA-lead). Besides problems with determining who can have access to data, determining who accessed the data was almost impossible. For example, if a data guardian wanted to know who looked at a particular insurance claim, the SA group would not be able to easily identify the person. Not all the systems generated audit logs, because of the negative impact on performance (SA-lead). A similar issue occurred when someone would act as the backup for a manager and be expected to not abuse the manager’s access privileges. To double check whether or not abuse had occurred, the SA group would need to know whether the backup person actually accessed inappropriate data. Again, the lack of audit logs hampered this kind of investigation. In our case study organization, access requests were made by e-mail, and the historical record was of poor quality. Gaps in the historical record could result in miscommunication: “Well the real serious ones are where you go to a data guardian and say okay this is what the situation is and I wonder if this client can have access to this kind of data for this length of time and they say sure, then you go and give them the access, but you didn’t get it in writing [...] and this happened to me, once, I didn’t get an email [from the data guardian], I didn’t say hey, by the way can you send me an email that says you approve this, I forgot that part of it. I got the access granted, and everything happened [...] then halfway through, oh why is this access granted, um I came and explained to you, no you came and explained to me but I don’t remember saying yes, I was going to think about it, take it away, take it away. So we had to you know, and I got in a little bit of trouble and that was that.” (SSecC). When a user would initiate a trouble ticket about a lack of expected access, the SA group would have to manually check RACF, Active Directory, and so on, resulting in a heavy workload. This also is related to audit logs and traces not being recorded due to performance issues. Tracing every action would result in terabytes of mostly useless information (SecA). Concerning end-user issues, sometimes a manager would inappropriately delegate authority to an employee (i.e., by giving out his or her password) in order to have the employee perform a task, despite there being a process for delegation: “I think some of the executive are some of the worst offenders of doing some of that stuff [bypassing the process for requesting access]. We’ve had some executive that have given their own accounts to their admin assistants to process transactions when they’re away on vacation. Which is a clear breach of our policy which says that you should not, and will not do that” (IdM-lead). Also, users forgetting their passwords comprised the primary call volume of the support center. To summarize, managing accesses and identities before deployment of the IdM system was challenging. The challenges impacted the creation of IDs, access requests, provisioning of users, ongoing management of accesses, access terminations, auditing and troubleshooting, and daily tasks of users like authentication or delegation of authorities. Multiple stakeholders in the organization were impacted by the challenges, including security practitioners, managers, data guardians, and employees. Addressing these challenges was the main motive for the organization to deploy an IdM system. 3.3 The IdM deployment process We now depict the selection, deployment, and configuration of an integrated IdM system in the studied organization. We describe the prerequisites for deploying the IdM system, the process of selection a system (including system requirements and evaluation of different IdM system options), and the challenges encountered during deployment and configuration. 3.3.1 Pre-requisites for deploying an IdM solution Having a well-defined access control policy and a business process for executing that policy is a pre-requisite for successfully deploying an IdM system (SA-lead, SecA, IdMlead). As the SA-lead explained: “it’s a classic thing where you buy a tool and you think it’s going to solve your problems but if you don’t have the staff, and you don’t have the business routine already internally to grant and manage access then an identity management system isn’t going to help you.” The IdM-lead further identified deployment prerequisites: “there’s a wide variety of things that definitely need to be in place before you even look at going down the pure technology implementation. Understanding your own processes when it comes to identities, access administration, the various identity repositories you have is another very important step as well your HR processes, because that’s usually your integration point into the identity lifecycle of where they start and where they end and then understanding the tolerance or interaction from the business into your current processes as well.” In the studied organization, the SA group had developed a “data guardianship policy” for managing access to resources (see section 3.1). In addition, HR had a comprehensive list of jobs, which could be used as a basis for defining roles (RFIQ). The organization also had a well-defined business process for creating IDs, provisioning, and management of accesses: “The critical piece for us was understanding our processes and understanding the user IDs and the identities in your own organization. Fortunately for us, once again, through standardization early on all our identities and all our repositories are the same; so when it came to going to explore those repositories for matches and stuff like that, it was easy to do, there’s a lot of organizations that don’t enforce that standardization, or through acquisition of a another company the standards are different and so they merge their information systems and they get into a bit of a problem where, you know, you’ve got J Smith in one system and John Smith in another and they’ve just got miss-matches, so there’s a bit of a challenge there” (IdM-lead). As a result, the organization was ready to introduce a new technology that would employ available business processes to support IdM more effectively. There was also an understanding of the need to involve other stakeholders in the organization before deployment of the IdM system. In particular, it was felt that the stakeholders of the system should be made aware of the benefits of the new technology and understand what the system will offer (IdM-lead, SecA). The SA group followed two strategies to create awareness. First, through e-mail, they contacted all stakeholders who were affected by deployment of the new technology. They provided the stakeholders with a brief overview of the upcoming changes. Additionally, the organization’s internal news website was used to inform employees about the new technology. The second strategy was to deploy the system incrementally. The SA group deployed and made small pieces of the system operational in each iteration. This made the transition to the new technology smoother. The incremental approach showed the benefits of the IdM system to the stakeholders early in the deployment process: “One of the big ones right now is password self service. People today have to call the help desk to get passwords reset. Now the user can do that themselves, or will be able to within another two weeks” (SecA). Availability of staff that could handle the deployment of the IdM system was also a pre-requisite for the project. The SA-lead noted that they hadn’t started the project until one of their staff attained the knowledge to act as a security business analyst. He defined a security business analyst to be a person with knowledge in the areas of both computer security and business. The SA-lead creatively fostered the organization’s in-house development of security business an- alysts, because he was unable to find and hire one, despite his significant resources: “[...] rare as hen’s teeth are people who are security business analysts which are people who function as business analysts, perhaps they come from applications development or some other job, but who function as business analysts in terms of information security ... you can’t hire those people for love or money. I had ads in the paper, I had ads on monster, I had ads on any kind of electronic bulletin, I put an ad in the globe and mail for [expletive], which is like going back 20 years. You cannot hire security business analysts and so you have to grow you’re own” (SA-lead). The SA group was interested in having a reliable estimation of the project’s cost before starting the project. On the other hand, the vendors were reluctant to give a fixed price for their IdM system and to commit to this price until the end of the project: “We had a fairly big paranoia that a lot of the vendors said, ‘Well we can’t really give you a fixed price or any real definitive number on an implementation for what you’re asking for because we don’t know your organization, we don’t know the complexity, we don’t know various factors that may drive that cost’ ” (IdM-lead). To address this, the SA group hired a consulting firm to analyze the current state of the organization and to provide the IdM system vendor with a report of expected complexity of the project. Having this report, the vendor committed to provide its IdM system with a fixed price. Finally, the SA group manager stated that they did not start this project earlier because IdM systems were not mature, and the cost of the solutions could not justify their benefits: “[...] the tools weren’t ready; even now, in some cases, I think we’re about 3 weeks behind the developer for the vendor, [Vendor] “oh yeah, you can do this now...” oh, cool that’s what you sold us on 2 years ago, but it’s nice to know we can do it now.” (SA-lead). 3.3.2 Selecting an IdM system The stakeholders who were involved in the selection process were the SA-lead, the SSecC, the IdM-lead, the SecA and people from the IT department (for reviewing server infrastructure) (IdM-lead). Implementation of an IdM system depends on a good understanding of the business processes [1]. Nevertheless, no one from the business side participated in the selection process. However, it is likely the presence of a security business analyst (who was groomed in-house for the job), helped to compensate for this lack. The selection process consisted of multiple steps (SA-lead, IdM-lead). First, the selection team developed a set of requirements, on which they based their RFIQ. The requirements for the IdM system can be classified into functional and nonfunctional requirements. The functional requirements include centralized role-based access administration, role mining, integration with available infrastructure, workflow support (integrated workflow engine), and auditing and reporting. From the end-user point of view, requirements include self-serve password management, plus access request and delegation. Nonfunctional requirements of the system include customizability, scalability to about 5000 users, disaster recovery (including the ability to backup), performance, availability, and reliability (including 99.99% uptime), working over slow or unreliable links, and fail- ing gracefully while working with different repositories (e.g. RACF and Active Directory). After publishing the RFIQ, they received and processed paper-based proposals from vendors. The score by which they evaluated the proposals consisted of the vendor’s company profile, qualifications and experience (9%); implementation approach and timeline (10%); ability to meet functional requirements (40%); ability to meet non-functional requirements (25%); and estimated cost (16%). The SAlead highlighted the importance of easily integrating the IdM system with current infrastructure in their decision making process when discussing an unsuccessful vendor proposal: “[...] it had to do with compatibility with our existing infrastructure. That sounds simple but for example, [IdM vendor], has an identity management system but we don’t do [vendors proprietary database technology X], we’re not an [X] infrastructure, we don’t do [X], we do [alternative technology Y] and [alternative technology Z] and so the [IdM vendor] folk we found difficult to be compatible with our infrastructure because their proposal basically said, ‘you have to convert to [X].’ and the answer is, ‘no, sorry, go away.’ ” The scoring narrowed down their focus to three vendors, who were then invited to present their system. From those three, two were selected to provide their software for lab testing to evaluate whether they could do what their vendors claimed (IdM-lead). This testing took about two and a half months. The SA group did not test every single feature of the systems, but they did conduct a full installation and tested functions that were important to the organization. The remaining two candidate vendors were scored on the presentation (20%) and the technical validation (80%). Distinguishing features that determined the winner were the ability to analyze and mine roles (building roles based on existing user-permission relationships), usability, and integration with current infrastructure. None of the candidates had integrated role mining feature in their products (SAlead, IdM-lead), and this was considered a critical feature: “We actually came close to collapsing the entire bid at one point because neither vender had anything on the role management side or role analysis side, they had recommended we manage roles within their own products themselves but the way they managed roles was confusing, it wasn’t what we were trying to achieve.” (IdM-lead). 3.3.3 IdM deployment and setup process Deployment of the IdM system was an incremental process. Two operational milestones identified were the password self-service sub-system and role based management of accesses. The SA group decided to deliver password selfservice in the first stage to show the benefits of the IdM system to end-users and to obtain management support for the rest of the project. The hardware infrastructure was provided by the organization, and they used platform virtualization to deploy the IdM. At the time of writing, the organization ran three different servers on two boxes using VMWare. The deployment was performed collaboratively by the SA group and an integrator from the vendor (IdM-lead). Before starting the deployment phase, the vendor claimed that they could deploy and configure the IdM system in three months, but the SA group planned to finish the project in one year. In actuality, the project took around fifteen months complete. The SA group manager (SA-lead) believed that the project went over schedule due to their lack of awareness about the full scale of the project. He also noted that the vendor’s flexibility in providing support during deployment and configuration was key to keeping the overshoot down to a reasonable level. The vendor did not require the organization to pay for technical support during deployment, which helped the SA group to have technical support on demand. As a result, they could deploy the IdM system without an excessive budget for on-site support. The IdM-lead described IdM deployment as an extremely complicated process, attributing some of the complexity to the vendor’s growth: “oh it’s extremely complicated. And that’s one thing actually the vender for their next release of the product just tried to resolve; they recognize it is an issue for their clients and their customers. And this I largely believe this is through the acquisition path they’ve gone through to get to where they are today. But the product itself resides on multiple servers and has multiple components, it’s own internal directories and databases for auditing that are completely separate [...] And that’s partly just because they haven’t had enough time to integrate their acquisitions further into their core product” To enable role based access control, a complete and correct set of roles needs to be created. The creation of the roles was the biggest aspect of setting up the IdM system for the insurance organization. The SA group decided to build roles by following both a top-down and bottom-up role engineering approach (see [16] for a discussion of role mining). They started the role engineering process by developing a set of roles from existing user-permission assignments. This was a bottom-up approach that required mining existing userpermission assignments in different access control repositories. The SA-lead highlighted the importance of discovery in role-mining: “if you don’t know how to do discovery, if your tool can’t do discovery you’re committing the staff to 2-3 years work of heavy lifting to do discovery. So a tool that did discovery and managed roles potentially can save you years of effort.” The role mining engine in their IdM system could analyze different repositories in each system and find users with similar accesses (SecA). Consequently, the SA group collaborated with individuals from each business area to check the differences between those similar accesses and create a single role that corresponds to a single job description (a top-down approach). Additionally, the SA group collaborated with data guardians to determine which roles should be authorized to access each resource in the organization. 3.4 Benefits and challenges Our participants expected their IdM system to have several benefits, mainly reduced workload and role-based auditing. But the IdM system also brought its own challenges. At the time of this writing, only two components of the IdM system had been made operational throughout the organization, so the final verdict is still to come. The automation of provisioning was expected to speed up the process and thereby reduce unproductiveness of employees who were waiting (up to a week) for access to resources. The automation was also expected to reduce the workload of the SA group. Upon a new employee’s enrollment in the organization, HR creates a new entry in their system, and the HR system triggers an enrollment event, in which the employee is automatically (1) assigned a role (corresponding to the HR job code) in the IdM system, and (2) provided with access to basic things like e-mail. In the next phase, the automatic provisioning will include access privileges that are associated with the role. Similarly, when changing a role (e.g., an employee moving to a different department), access privileges will automatically change based on information in the HR system. Further provisioning is requested by the employee’s manager through an online form, bundling many individual requests into one step, thereby reducing the workload on the the SA group. In the next phase, the form will be workflow driven, and automatically inform data guardians of requests, thereby further reducing the security administrators’ workload of managing the requests. Additionally, a self-serve password feature, that enables end-users to reset their passwords by answering a set of challenge-response questions, is expected to dramatically reduce the number of calls to the help desk. Better reporting and compliance was another expected benefit from the system. The old reports about who can access a resource were difficult for data guardians to read and understand. For example, they included cryptic RACF rules. With the new IdM system and role based access control, data guardians will be provided with a list of roles that have access to their data. Furthermore, the IdM system would generate reports by mapping the cryptic or technical terms in the rules to business terms. It was expected that this would make it easier for the organization to observe compliance. Automated role-based access was expected to enable both end-users and security administrators to be more critical about roles. End-users could see a catalogue of potential access privileges that they could request, and security administrators could identify and correct inappropriate privileges. That is, security administrators would be “freed up” to become security analysts, engaging in questions of how roles should be built and structured, which was expected to be beneficial to the organization in terms of both effective identity management and employee retention. The SA-lead highlighted this benefit: “[It] allows the function of a security administrator to become smarter in the sense that they are now using the IdM system to grant access and they’re now able to do more consideration in terms of, ‘is this the right access, can I do an investigation? do I need to build a role?’ and so hopefully, I don’t plan on reducing the number of the security administrators but I plan on requiring them to behave differently in the sense they’re becoming more sophisticated in how they deal with things. So identity management helps us from an enterprise level in terms of managing identities better but also helps me from a business point of view that it frees up staff resources.” We now summarize the main challenges encountered to date by our case study organization during its deployment of the IdM system. Firstly, role engineering has remained a challenge. Despite the role mining component, creating welldefined and structured roles in the organization required collaboration with the business side of the enterprise. The collaboration comprised the major step in role engineering, far out-weighing the technical aspects: “The two biggest areas are, depending on what you’re trying to achieve with your identity management project but for us it’s been the role analysis and the working with the business for those role definitions takes a significant period of time. Especially if you want to get it right. And coming up with a plan, organizationally of how you want to structure roles, coming up with what those common attributes are, those types of things” (IdM-lead). Secondly, deploying the loosely integrated components of the IdM system was a challenge. The fragmented components required multiple boxes for deployment. Configuring and running all these components was not easy. Furthermore, updates included multiple executables, each of which could require up to a day to install. This state of affairs was likely due to many acquisitions in the IdM vendor market [1]. One of the IdM vendor’s partners was acquired by a competitor, resulting in one of the components being at risk of losing support in the future. Thirdly, the practice of rehearsal and planning has been challenging; the rehearsal environment cannot be completely identical to the production environment. Particularly, concerning importing policies into the rehearsal environment, some information (e.g., IDs in a policy database) is not preserved – the team has to make do with the same kind of objects, rather than replications of them: “Yeah, you know, when you’re trying to import group policies and stuff like that for example the object names are slightly different than what you’ve got in production because the SIDs don’t match so it’s going to cause errors and stuff like that” (IdM-lead). 4. COMPARISON WITH RELATED WORK Since IdM is part of IT security, our results can be compared to prior findings in this area. Werlinger et al. [17] identified and classified challenges in IT security. Two of them – “access control” and “security culture” – were reasons for our case study insurance organization to adopt an IdM system. Our findings show that IdM related activities are distributed across the organization and require communication and collaboration between different stakeholders. This confirms prior research that shows that IT security is distributed across the organization [12, 6] and requires collaboration among different stakeholders [11, 18]. More specific to IdM, our findings provide an example of how previous research on identity and access control management [4, 19, 1] plays out in a particular case, which not only strengthens the previous findings, but also may highlight some nuances that were not previously emphasized. Bauer et al. [4] studied challenges in access-control management in academic and non-academic organizations. The main focus of their study was on the ongoing management of accesses to file systems and physical environments. Their focus was limited to the process of access control management, while our research covers the whole process of identity management both without and with an integrated IdM system. Nevertheless, our findings about challenges in IdM process before deployment of the IdM solution corroborate their key findings. In particular, they identified a set of challenges that we also observed in our case study: management of exceptions, getting notification about policy change (em- ployees leave the organization or change their department), getting updated information about who is responsible for a resource, verifying requests, keeping records, and choosing a usable access control management interface. As one of their implications for design, Bauer et al. propose allowing policy makers to directly edit the implemented policy in order to address the challenge that those who set the policies find it difficult to view and understand the implemented policy. Our findings reveal that this strategy may not be feasible, at least for organizations such as our case study organization that have complex system architectures and policy histories. If the policy implementers themselves (those who deploy the policies like SPs), with their domain knowledge of the systems are having difficulty, it is unlikely that the (primarily) non-technical policy makers will be up to the task of implementing it themselves. The Identity Project [19] is a study of IdM practices in UK higher education institutions. The results of the Identity Project are based on a broad survey, which was validated and refined by 161 semi-structured interviews in the participating institutions. The Identity Project’s results are tuned to the improvement of business processes in the UK higher education sector. Being broad, the Identity Project findings can be complemented by case studies that aim at finergrained information. While we confirm many of the results of the Identity Project, we also give a more detailed picture of some of the challenges, their cause, and the way the insurance organization coped with them. Moreover, comparison of our results with Identity Project results can show how the type of an organization can impact the challenges it faces in IdM and provide support for the generalizability of Identity Project’s challenges to non-academic organizations. For example, The Identity Project identified nine major challenges to IdM: limited consensus on defining “identity management,” heterogeneity in IT infrastructure, limited de-provisioning, lack of formal procedures, lack of both common standards and central IdM administration, lack of IdM data quality, use of non-unique user credentials, lack of policy for reuse of identifiers, and lack of adherence to a code of practice for information security. Our case study organization experienced only two of the nine challenges (limited de-provisioning and heterogeneity in IT infrastructure). We surmise that some of the other challenges may be more particular to academic organizations; Werlinger et al. [17] show that academic freedom is a barrier to enforcing policies. The Burton Group commissioned several studies about various aspects of IdM, resulting in a body of reports for management that are summarized in a root document [1]. The Burton Group sources include their client organizations (e.g., six participating organizations in a survey and interviews about the implementation of roles), non-client organizations who use IdM (e.g., “discussions with approximately 20 mediumsize to very large enterprises” concerning federation), presentations by vendors of IdM products, and discussion with consultants from other advisory organizations. The results of these studies are targeted more toward businesses who plan to decide about adopting an IdM system. Our results confirms Burton group findings about pre-requisites for IdM success and the challenges an organization might face adopting an IdM system. Our results also confirm business drivers for IdM adoption, including the need for security, the need to observe regulation, the need to reduce cost, establishing new business models, and enhancing the user experience. In our case study, the primary driver for IdM was audits (SA-lead); that is, accountability. Concerning cost reduction, improvements are twofold. From the viewpoint of the security team, less effort is required for handling of data security by reducing labor-intensive, redundant activities, and increasing the quality of reporting. Furthermore, employee productiveness is increased as they wait less for access to resources. We identified a new business driver which is changing the role of security administrators to security analysts; this is expected to increase employee retention (SA-lead). 5. A VALIDATION OF GUIDELINES FOR IMPROVING IDM SYSTEMS In our prior research [10], we developed a framework of design guidelines for IT security tools that classified guidelines generated by prior research according to the challenges the guidelines address. For example, the lowest layer in the framework comprises general usability guidelines for IT security tools. The next two layers contain guidelines that are necessary due to the work environment of security practitioners, which is characterized by technological and organizational complexity (including guidelines to address general communication challenges, guidelines applicable to tools used in a process that involves other stakeholders, and guidelines applicable to tools used by distributed SPs). The upper layer of the framework contains guidelines that are grouped based on task properties of the tool, such as those that require intensive configuration and deployment and those used in a process that requires intensive analysis. The framework is intended to aid researchers and developers in selecting guidelines for the security tool under consideration. Since IdM systems are complex, involve multiple stakeholders, and involve extensive deployment and configuration, we expect guidelines that address technological complexity, diverse stakeholders, communication, distribution of IT security, and configuration and deployment to be meaningful with respect to IdM. Our case study confirms this by showing that a number of the guidelines from the framework came into play during the IdM deployment; in particular, the organization required the IdM system to be integrated with the current infrastructure, be customizable, support workflow, enable flexible reporting, enable data to be presented in multiple formats, provide different methods of interaction, support archiving, and support rehearsal and planning. We next briefly discuss how our observations shed light on how some of these more general guidelines can be applicable to the domain of IdM and where opportunities for future improvements in IdM technologies remain. Provide integration with the current infrastructure: this guideline was a decisive requirement for our case study organization. They rejected one bidding vendor on the grounds that the vendor required them to replace their infrastructure. One of the reasons that the successful vendor was selected was because it was willing to adapt some features to the organization’s way of doing things; that is, their IdM system was, in part, customizable, including customizing their documentation and providing customizable workflow and UI. Furthermore, the insurance organization developed much of its own software, and was able to integrate some of its own modules (such as role mining) with the system. Provide workflow support: the insurance organization expected workflow support to reduce the workload of maintaining the data guardian framework by automating repeated manual tasks, facilitating division of responsibilities between stakeholders, and allowing effective communication and collaboration required for IdM tasks. Afford flexible reporting and presentation of information: although the organizations’ executive management was aware of security and privacy issues, nevertheless, the rationale for the implementation of the IdM system was couched in the language of business. The SA group wanted to generate reports that would show the effectiveness of the group to the executive. Likewise, they wanted to replace technical terms (like esoteric IDs) with more natural terms in reports to data guardians about who could have (or did have) access to their data. In the same vein, managers had to know what systems their employees should access and often did not know, largely because of the technical terminology. Provide appropriate interaction methods for varying stakeholders: the insurance organization exhibited different interaction methods for different but related tasks. For example, managers requested access on behalf of their employees through an online, multi-page form, graphical interface (GUI), while the security administrators would deploy the requests to data guardians or network admins by e-mail. Additionally, in the RFIQ, the availability of command line interface (CLI) to allow communication with scripting tools was stated. Similarly, roles and access policies were perused differently depending on whether they were being edited by security analysts or explored by managers. Our case study shows that managers have trouble in understanding access policies. Using natural language or visualization techniques would be a viable solution to this problem. Incorporative archiving capabilities: the insurance organization’s determination to maintain accountability entailed archiving on a large scale, including through email and online forms for access requests. Our participants were not satisfied with the state of record keeping before deployment of the IdM system. In the RFIQ document, the organization stated the need for keeping time-stamped and tamper proof logs of user administration and workflow events. From a different perspective, the case study organization couldn’t determine who accessed the data as they need to store logs from different systems. Log summarization could be a solution to this problem. Enable rehearsal and planning: a main challenge for our case study organization for the IdM implementation was that the rehearsal environment could not be completely identical to the production environment. Also the IdM system was particularly difficult to update. Because of the critical nature of IT and IT security systems, planning and rehearsal are very important. The need to synchronize the rehearsal environment with the production environment may be seen as an opportunity for tool improvement. 6. LIMITATIONS As a limitation of our work, the findings are based on the self reports of the participants. While these interviews are rich sources of information about the IdM adoption process, the data reflects the participants’ interpretation of the process, which may not be accurate. We tried to alleviate this limitation by analyzing documents related to organizational structure, the SA group structure and responsibilities, as well as the RFIQ document. Another possible approach to address this limitation would be to perform naturalistic observation of the technology adoption process; however, as of yet, the insurance organization has not been willing to allow researchers to perform observation. Interview ingother involved stakeholders like managers or end-users would also increase the accuracy of the findings. Since our study was focused on one organization, our findings may not be generalizable. To address this problem, we compared our findings with related work to identify the portions of the work that strengthen or extend existing knowledge. Moreover, we tried to provide technical and organizational details about the context to facilitate the reader’s naturalistic generalization (i.e., generalizations that are intuitive, empirical, and based on personal direct and vicarious experience [15]). Finally, our data does not cover the state of the organization after the complete deployment of the IdM system. Therefore, we can’t argue whether the expected benefits are realized or not. A follow-up study is required to measure the benefits of the IdM system like service time, employee satisfaction, etc. 7. CONCLUSION AND FUTURE WORK In this paper we described our case study of an insurance organization that deployed an integrated IdM system. We studied the organization at different stages of this project: before deployment, in the process of deployment, and after deployment of the first phase of their project. Our description of the insurance organization’s stages of IdM deployment provides details about the organization’s expectations and challenges. The state of the organization before IdM adoption may provide awareness in other organizations about possible costs of using legacy IdM techniques. Additionally, the experience of the case study organization and the expected benefits of IdM adoption may motivate other organizations to migrate to an integrated IdM system, while helping them know the challenges they may face. Finally, comparison of our findings with related work builds confidence that, not only are some of the findings generalizable, but they extend existing knowledge about IdM and validate that some of the recommendations for IT security tools extend to IdM. For future work, we plan to continue interviews with various stakeholders during ongoing usage of the IdM system in the insurance organization. In particular, we are interested to learn whether the organization achieves the expected benefits. We are also performing more interviews in other organizations that have deployed an IdM system, which will help us to develop generalizable models of challenges, interactions, and recommendations for improvement of IdM solutions. Because role mining was a challenge for the insurance organization, we are interested in performing a detailed study on role mining in organizations, and identify the organizational and social barriers to role mining. Acknowledgments This work would not be possible without access to the HOT Admin and IdM project study participants. Members of LERSSE provided comments on preliminary versions of this paper. The HOT Admin project is supported by the NSERC Strategic Partnership Program, grant STPGP 322192-05. 8. REFERENCES [1] Root Document Enterprise Identity Management: Moving from Theory to Practice. Technical report, Burton Group (June 2005). [2] Bauer, L., Cranor, L. F., Reeder, R. W., Reiter, M. K., and Vaniea, K. Real life challenges in access-control management. In CHI . ACM, New York, NY, USA, 2009, 899–908. [3] Blum, D. Identity management - concepts and definitions. Burton Group (September 2005). 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An integrated view of human, organizational, and technological challenges of IT security management. Journal of Information Management & Computer Security, 17(1) (2009), 4–19. [16] Werlinger, R., Hawkey, K., Botta, D., and Beznosov, K. Security practitioners in context: Their activities and interactions with other stakeholders within organizations. International Journal of Human-Computer Studies, 67 , 7 (2009), 584–606. [17] Wright, J. Final progress reports. http://www.jisc.ac.uk/media/documents/programmes /einfrastructure/tidpfinalreport.pdf (November 2007).
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Atmos. Chem. Phys., 21, 9761–9777, 2021 https://doi.org/10.5194/acp-21-9761-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Large seasonal and interannual variations of biogenic sulfur compounds in the Arctic atmosphere (Svalbard; 78.9◦ N, 11.9◦ E) Sehyun Jang1, , Ki-Tae Park2,3, , Kitack Lee1,4 , Young Jun Yoon2 , Kitae Kim2,3 , Hyun Young Chung2,3 , Eunho Jang2,3 , Silvia Becagli5 , Bang Yong Lee2 , Rita Traversi5 , Konstantinos Eleftheriadis6 , Radovan Krejci7,8 , and Ove Hermansen9 1 Division of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang, 37673, Korea Polar Research Institute (KOPRI), 26 Songdomirae-ro, Yeonsu-gu, Incheon, 21990, Korea 3 Department of Polar Sciences, University of Science and Technology (UST), Incheon, 21990, Korea 4 Institute for Convergence Research and Education in Advanced Technology, Yonsei University, Seoul, 03722, Korea 5 Institute of Polar Science, ISP-CNR, via Torino, 155, Venezia Mestre, Venice, 30172, Italy 6 NCSR Demokritos, Environmental Radioactivity Laboratory, Ag. Paraskevi, 15341, Attica, Greece 7 Department of Environmental Science, Stockholm University, 10691 Stockholm, Sweden 8 Bolin Centre for Climate Research, Stockholm University, 10691 Stockholm, Sweden 9 Norwegian Institute for Air Research, Kjeller, Norway  These authors contributed equally to this work. 2 Korea Correspondence: Kitack Lee (ktl@postech.ac.kr) and Young Jun Yoon (yjyoon@kopri.re.kr) Received: 15 December 2020 – Discussion started: 2 February 2021 Revised: 18 May 2021 – Accepted: 25 May 2021 – Published: 29 June 2021 Abstract. Seasonal to interannual variations in the concentrations of sulfur aerosols (< 2.5 µm in diameter; non sea2− salt sulfate: NSS-SO2− 4 ; anthropogenic sulfate: Anth-SO4 ; 2− biogenic sulfate: Bio-SO4 ; methanesulfonic acid: MSA) in the Arctic atmosphere were investigated using measurements of the chemical composition of aerosols collected at NyÅlesund, Svalbard (78.9◦ N, 11.9◦ E) from 2015 to 2019. In all measurement years the concentration of NSS-SO2− 4 was highest during the pre-bloom period and rapidly decreased towards summer. During the pre-bloom period we found a 2− strong correlation between NSS-SO2− 4 (sum of Anth-SO4 2− 2− and Bio-SO4 ) and Anth-SO4 . This was because more than 50 % of the NSS-SO2− 4 measured during this period was Anth-SO2− , which originated in northern Europe and was 4 subsequently transported to the Arctic in Arctic haze. Unexpected increases in the concentration of Bio-SO2− 4 aerosols (an oxidation product of dimethylsulfide: DMS) were occasionally found during the pre-bloom period. These probably originated in regions to the south (the North Atlantic Ocean and the Norwegian Sea) rather than in ocean areas in the proximity of Ny-Ålesund. Another oxidation product of DMS is MSA, and the ratio of MSA to Bio-SO2− 4 is extensively used to estimate the total amount of DMS-derived aerosol particles in remote marine environments. The concentration of MSA during the pre-bloom period remained low, primarily because of the greater loss of MSA relative to Bio-SO2− 4 and the suppression of condensation of gaseous MSA onto particles already present in air masses being transported northwards from distant ocean source regions (existing particles). In addition, the low light intensity during the pre-bloom period resulted in a low concentration of photochemically activated oxidant species including OH radicals and BrO; these conditions favored the oxidation pathway of DMS to Bio-SO2− 4 rather than to MSA, which acted to lower the MSA concentration at Ny-Ålesund. The concentration of MSA peaked in May or June and was positively correlated with phytoplankton biomass in the Greenland and Barents seas around Svalbard. As a result, the mean ratio of MSA to the DMS-derived aerosols was low (0.09 ± 0.07) in the pre-bloom period but high (0.32 ± 0.15) in the bloom and post-bloom periods. There was large interannual variability in the ratio of MSA to Bio-SO2− 4 (i.e., 0.24 ± 0.11 in 2017, 0.40 ± 0.14 in 2018, and 0.36 ± 0.14 in 2019) during the Published by Copernicus Publications on behalf of the European Geosciences Union. 9762 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds bloom and post-bloom periods. This was probably associated with changes in the chemical properties of existing particles, biological activities surrounding the observation site, and air mass transport patterns. Our results indicate that MSA is not a conservative tracer for predicting DMS-derived particles, and the contribution of MSA to the growth of newly formed particles may be much larger during the bloom and post-bloom periods than during the pre-bloom period. 1 Introduction Aerosols alter the radiative properties of the Earth’s surface by means of direct (e.g., scattering and absorption of solar radiation) and indirect (e.g., cloud lifetime) effects and thereby contribute to climate change (Albrecht, 1989; Haywood and Boucher, 2000; Sekiguchi et al., 2003). Moreover, acidification of the Arctic Ocean has been enhanced because of the increasing addition of anthropogenic CO2 , facilitated by ocean freshening and greater air–sea CO2 exchange (Lee et al., 2011); and ocean acidification potentially impacts on the net production and fluxes of marine trace gases and so affects climate (Hoppkins et al., 2020). The recent acceleration of Arctic warming has highlighted the role of natural aerosols in influencing the radiative properties of the Arctic atmosphere (Dall’Osto et al., 2017; Willis et al., 2018). Nonetheless, current knowledge of the effect of aerosols on climate regulation and the mechanisms of formation of natural aerosols is far from comprehensive, and more alarmingly it is ambiguous (Mahowald et al., 2011; IPCC, 2013). Sulfurous compounds including SO2 , methanesulfonic acid, and hydroperoxymethyl thioformate in the atmosphere are the oxidation products of dimethyl sulfide (DMS). These effectively form new particles through homogeneous nucleation and clustering reactions that are closely linked to water vapor and ammonia (negative ion-induced ternary nucleation) and contribute to particle growth (Kulmala, 2003; Kulmala et al., 2004; Veres et al., 2020). Sulfuric acid is widely recognized as a driver of new particle formation (NPF) (Kulmala, 2003), whereas methanesulfonic acid (MSA) particles tend to condense onto particles that are already present (existing particles) and so contribute to particle growth (Wyslouzil, et al., 1991; Leaitch et al., 2013; Hayashida et al., 2017). However, recent studies have provided evidence for MSA involvement in new particle formation – for example, the reaction of MSA with amines or ammonia in the presence of water results in particle formation and growth (Dawson et al., 2012; H. Chen et al., 2015, 2016). MSA also indirectly contributes to NPF by enhancing the formation of H2 SO4 -amines clusters (Bork et al., 2014). Some studies have reported that MSA only increased the mass of particles and not their number (Hoffmann et al., 2016; Yan et al., 2020b), suggesting a minor role for MSA in NPF. The growth of particles following NPF is particularly crucial in generating cloud condensation Atmos. Chem. Phys., 21, 9761–9777, 2021 nuclei (CCN), which eventually lead to cloud formation. As a result, naturally produced gas molecules can promote NPF and subsequent growth of particles in the presence of sulfate and MSA (DMS oxidation products) (Chang et al., 2011a; Burkart et al., 2017). Hence, data on the quantities of non sea-salt sulfate (NSS-SO2− 4 ) and MSA and their variations are crucial in elucidating NPF and particle growth and ultimately the role of ocean phytoplankton in modulation of the radiative properties of the Arctic atmosphere. The origins of sulfate aerosols include sea-salt sulfate (SSSO2− 4 ), anthropogenic SO2 , volcanic SO2 , boreal production of natural precursor, and DMS (Bates et al., 1992a). Among those, DMS is produced through multiple biological processes occurring in pelagic and sympagic ecosystems (e.g., Kettle and Andreae, 2000; Stefels et al., 2007; Kim et al., 2010; Lee et al., 2012; Levasseur, 2013; Park et al., 2014a, 2019). Some of the DMS is ultimately released into the atmosphere through air–sea gas exchange processes. Airborne DMS is rapidly oxidized to SO2 via hydrogen abstraction by OH radicals, nitrate, and chlorine; to hydroperoxymethyl thioformate via hydrogen shift by OH radicals; and to MSA via OH addition by OH radicals and in part by halogen oxides (von Glasow and Crutzen, 2004; Barnes et al., 2006; Veres et al., 2020). Seasonal variations in the product ratio of DMS oxidized to MSA and biogenic sulfate (Bio-SO2− 4 ) over the Arctic region reflect the complexity of aerosol chemistry. The product ratio of DMS oxidation is highly variable and is affected by air temperature, relative humidity, precipitation, and solar radiation (Hynes et al., 1986; Yin et al., 1990; Bates et al., 1992b). Among those factors involved, air temperature is known to largely determine the oxidation pathways of DMS. At ambient temperatures the proportions of MSA and Bio-SO2− 4 are typically 0.25 and 0.75, respectively (Hynes et al., 1986). DMS is well known to be oxidized more to MSA at lower temperatures. The observed latitudinal variations in the product ratio of DMS oxidation are largely consistent with those predicted from the temperature dependence of the oxidation pathway of DMS (Hynes et al., 1986; Berresheim et al., 1990; Bates et al., 1992b), although equally available are reports on an absence of temperature dependence (Ayers et al., 1991; Prospero et al., 1991; Chen et al., 2012). The product ratio of DMS oxidation is a result of the net effect of multiple processes, including concentration of atmospheric oxidants and meteorological factors influencing DMS oxidation. Therefore, the ratio could vary considerably among seasons and years. To investigate DMS oxidation pathways in the Arctic atmosphere we measured sulfate aerosol concentrations at 3 d intervals from 2015 to 2019; this provided comprehensive datasets encompassing seasonal and interannual variations in sulfate and MSA concentrations in aerosol particles in the Arctic atmosphere. In particular, S isotope ratios were measured for all aerosol samples and were used to partition 2− the total NSS-SO2− 4 into anthropogenic sulfate (Anth-SO4 ) https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds 9763 aerodynamic equivalent diameter). The aerosol sampler was mounted on the roof of the Gruvebadet observatory. Particulate matter in the atmosphere was collected on a quartz filter over approximately 72 h at a flow rate of 1000 L min−1 , corresponding to a total air volume of 4320 m3 . The method of aerosol sampling has been described elsewhere (Park et al., 2017). 2.2 Figure 1. (a) Location of the aerosol sampling site (black star; Gruvebadet observatory; 78.9◦ N, 11.9◦ E) and the ocean domains (70–80◦ N, 25◦ W–50◦ E for Region 1; 50–70◦ N, 25◦ W–50◦ E for Region 2) defined for this study. Mean Chl-a concentration for (b) March and April, (c) May and June, and (d) July and August over the period of 2015–2019, overlaid with air mass trajectory clusters that represent the dominant pathways of air masses reaching the observation site. and Bio-SO2− 4 (the oxidative product of biogenic DMS). We also calculated the product ratio of MSA to biogenic sulfur aerosols (MSA + Bio-SO2− 4 : Bio-S-aerosol). Analysis of 2− Anth-SO2− , Bio-SO , and MSA concentration data, in con4 4 junction with data on air mass back trajectories, enabled the identification of the sources of S aerosols and elucidation of factors governing variations in their concentrations. 2 2.1 Materials and methods Sampling site and aerosol sampling Aerosol samples were collected at 50 m above sea level at the Gruvebadet observatory (78.9◦ N, 11.9◦ E; Fig. 1a) at Ny-Ålesund, Svalbard. Sampling covered the phytoplankton pre-bloom (defined as March to the second week of April), bloom (third week of April to the second week of June), and post-bloom periods (third week of June onwards). Division of these periods was subjectively made based on the mean chlorophyll-a (Chl-a) concentration in the Greenland and Barents seas near Svalbard. The period during which the concentration of Chl-a was > 0.5 mg m−3 was defined as the phytoplankton bloom period, whereas the periods when the concentration of Chl-a was < 0.5 mg m−3 prior to and following the bloom were defined as the pre-bloom and postbloom periods, respectively. Aerosol samples were collected at 3 d intervals using a high-volume sampler (HV-1000R; SIBATA, Japan) outfitted with a PM2.5 impactor (collecting particles < 2.5 µm in https://doi.org/10.5194/acp-21-9761-2021 Atmospheric DMS mixing ratio and major ions in aerosol samples The analytical system enabling measurement of atmospheric DMS mixing ratio at parts per trillion levels is equipped with a DMS trapping component, a gas chromatograph, and a pulsed flame photometric detector. The detection limit of the DMS system was close to 1.5 pptv with a sampling air volume of 6 L, and the description of the system can be found elsewhere (Jang et al., 2016). For determination of concentrations of major ions, a disk filter (47 mm diameter) was taken from a whole quartz filter (20.3 cm × 25.4 cm), soaked in 50 mL of Milli-Q water and sonicated in a bath for 60 min; aliquots of this solution were used for analysis. Milli-Q water used for the ion extraction was produced using a water purification system (Milli-Q Direct 16, Merck Millipore, USA). The concentrations of water-extractable inorganic anions and cations including MSA were measured using ion chromatography (Dionex ICS-1100, Thermo Fisher Scientific Inc., USA) fitted with an IonPac AS 19 column (Thermo Fisher Scientific Inc., USA). The instrumental detection limits were 0.02 µg L−1 for MSA and 0.02 µg L−1 for SO2− 4 . From replicate injections, the analytical precision was determined to be < 5 % (relative standard deviation). 2.3 Stable S isotope ratio in sulfate aerosols For measurement of stable S isotope ratio (δ 34 S) in an aerosol sample, half of the quartz filter was soaked in 50 mL MilliQ water and sonicated for 60 min. Then, 50–100 µL of 1 M HCl was added to the solution (resulting in a pH of 3–4), after which 100 µL of 1 M BaCl2 solution was injected into the solution, leading to gradual precipitation of BaSO4 . Following the completion of precipitation over 24 h, the BaSO4 precipitates were filtered onto a membrane filter and dried for another 24 h prior to S isotope ratio measurement. Each membrane filter was packed into a tin capsule and analyzed using an isotope ratio mass spectrometer (IsoPrime100; IsoPrime Ltd, UK) and an elemental analyzer (Vario MICRO cube; Elementar Co., Germany). Each filter treatment was carried out in a laminar flow hood to minimize contamination. International standard reference materials were used to measure the abundance of S isotope in the aerosols. We used NBS-127 (20.3 ± 0.4 ‰), IAEA-S1 (silver sulfide; −0.3 ± 0.3 ‰), and IAEA-S2 (silver sulfide; 22.7 ± 0.2 ‰) (Coplen and Krouse, 1998; Halas and Szaran, 2001; Santamaria-Fernandez et al., Atmos. Chem. Phys., 21, 9761–9777, 2021 9764 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds 2008) to prepare the calibration curve. NBS-127 was used as the primary standard reference material and was measured with every five samples. The resulting S isotope ratio of an aerosol sample (δ 34 S) was expressed (Eq. 1) as parts per thousand (‰) relative to the 34 S / 32 S ratio of a standard (Vienna-Canyon Diablo Troilite) (Krouse and Grinenko, 1991).      −1 δ 34 S(‰) = 34 S/32 S / 34 S/32 S standard sample × 1000. (1) 2− Among known sources, both Anth-SO2− 4 and Bio-SO4 are the main sources of sulfate aerosols in the Arctic environment (Udisti et al., 2016; Park et al., 2017). Data on the S isotope ratio of aerosol particles and the concentrations of major ions enabled estimation of the contributions of biogenic DMS (fBio ), anthropogenic SOx (fAnth ), and SS-SO2− 4 (fSS ) to the total SO2− 4 concentration. The concentration of 2− SS-SO2− 4 was estimated using the seawater ratio of SO4 to 2− + Na (0.252; Keene et al., 1986). The NSS-SO4 fraction of the total SO2− 4 was then calculated by subtracting the fraction 2− of SS-SO2− 4 from the total SO4 . The fraction of biogenic 2− SO4 was estimated by solving the following equations: fAnth + fBio + fSS = 1, 34 34 (2) 34 34 δ Ssample = fAnth δ SAnth + fBio δ SBio + fSS δ SSS , (3) h i h i 2− + + fSS = SO2− /Na · Na /SO . (4) 4 4 SS sample To solve Eqs. (2)–(4) we used the reported S isotope val2− ues of SS-SO2− 4 (21.0 ± 0.1 ‰), Anth-SO4 (5 ± 1 ‰), and 2− Bio-SO4 (18 ± 2 ‰) (Norman et al., 1999; Böttcher et al., 2007; Lin et al., 2012). Based on measurements of the S isotope ratio on aerosol samples, then we calculated the fraction of MSA (RBio = MSA/[MSA + Bio-SO2− 4 ]) in the total biogenic sulfur aerosols to evaluate the oxidative pathway of DMS to MSA or to Bio-SO2− 4 . In calculating RBio , some −3 data (∼ 23 data) having low Bio-SO2− 4 values (< 25 ng m ) 2− were not included because unusually low Bio-SO4 values resulted in biases in the RBio values (Table S1 in the Supplement). black carbon had common sources (i.e., fossil fuel combustion and forest burning) (Text S1; Figs. S1 and S2) (Massling et al., 2015; L. Chen et al., 2016). 2.5 Air mass origin, chlorophyll-a concentration, and meteorological parameters Both 8 d and monthly mean Chl-a concentration level-3 MODIS Aqua data were downloaded from the NASA OceanColor website (http://oceancolor.gsfc.nasa.gov/, last access: 5 August 2020) at a 4 km resolution. The three-dimensional 5 d (120 h) back trajectories were calculated using the Hybrid Single-Particle Lagrangian Integrated Trajectory model from the NOAA Air Resources Laboratory (Draxler and Hess, 1998). Meteorological parameters including solar radiation, relative humidity, and air temperature at each time point were also calculated along the air mass trajectories. The calculations were made based on meteorological data from the Global Data Assimilation System (at 1◦ latitude × 1◦ longitude resolution) produced by the National Centers for Environmental Prediction. Air masses were modeled to arrive at an altitude of 50 m above sea level at the Gruvebadet station at each hour of the study period. To identify the major air mass pathways prior to reaching the Gruvebadet station, the calculated air mass trajectories were grouped into several clusters using the k-means clustering algorithm. Monthly mean air temperature data at 900 hPa were obtained from the European Centre for Medium-Range Weather Forecasts Reanalysis 5 at a 30 km resolution. Sea level pressure data were obtained from the National Oceanic and Atmospheric Administration Physical Sciences Laboratory (http: //psl.noaa.gov/, last access: 30 July 2020). The retention time for air masses in each domain type (including the ocean, marginal ice zone, multi-year ice, and land) was calculated based on the sea ice index at 25 km resolution provided by the National Snow and Ice Data Center (Choi et al., 2019). Note that the marginal ice zone and multi-year ice represent the areas in which the sea ice cover is 15 %–80 % and > 80 %, respectively (Stroeve et al., 2016). The air mass exposure to chlorophyll (EChl ) was calculated to estimate the biological exposure history of air masses arriving at the observation site (Arnold et al., 2010; Park et al., 2018), according to Eq. (5): P120 2.4 Black carbon An aethalometer (model AE31; Magee Scientific Co., USA) installed at the Zeppelin station was used to analyze the concentration of equivalent black carbon by measuring lightabsorbing particles at a wavelength of 880 nm, as described by Eleftheriadis et al. (2009). The good congruence between the concentrations of Anth-SO2− 4 and black carbon measured during the pre-bloom period (March to April) indicates that variations in black carbon were reasonably consistent with 2− variations in Anth-SO2− 4 , reflecting that both Anth-SO4 and Atmos. Chem. Phys., 21, 9761–9777, 2021 EChl = t=1 Chl n , (5) where Chl is the 8 d mean Chl-a concentration within a radius of 25 km at a given time point (t) along the 5 d air mass back trajectory, and n is the total number of time points for which valid Chl-a values were available. https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds 9765 Figure 3. Monthly data during the measurement years (2015–2019) for (a) black carbon (BC), (b) atmospheric DMS mixing ratio, (c) sulfur isotope measurements (δ 34 S), (d) NSS-SO2− 4 , (e) Anth- Figure 2. (a) Atmospheric DMS mixing ratios measured at the Zeppelin station, Svalbard, in 2015, 2016, 2017, 2018, and 2019. (b) Stable isotope composition of sulfate aerosols. Three end-member values; δ 34 SSS = 21 ± 0.1 ‰ for sea-salt sulfates; δ 34 SAnth = 5 ± 1 ‰ for anthropogenic sulfate; and δ 34 SBio = 18 ± 2 ‰ for biogenic sulfates. 3 3.1 Results Atmospheric DMS mixing ratio The mixing ratio of atmospheric DMS, the precursor of BioSO2− and MSA, showed considerable (several orders of 4 magnitude) variability at daily to weekly intervals during the bloom and post-bloom periods (Figs. 2a and 3b). As confirmed in other studies (e.g., Arnold et al., 2010; Park et al., 2013; Mungall et al., 2016), the atmospheric DMS mixing ratio generally corresponded to the phytoplankton biomass in the oceans surrounding Svalbard (Figs. 2a and S3). During the bloom period the maximum monthly mean mixing ratio of DMS occurred in May 2015 (68.4 ± 86.8 pptv); an increase in the DMS mixing ratio continued until August of that year, reflecting the persistent phytoplankton biomass producing DMS in the vicinity of Svalbard. Based on our atmospheric DMS concentration data, we conclude that DMS was ubiquitous in the Arctic atmosphere from the phytoplankton bloom to post-bloom periods (Park et al., 2013). https://doi.org/10.5194/acp-21-9761-2021 2− SO2− 4 , (f) Bio-SO4 , (g) MSA, (h) Bio-S-aerosol, and (i) MSAto-Bio-S-aerosol ratio (RBio ) during the measurement years (2015– 2019). Solid lines and red crosses represent the median and mean values of the data, respectively. 3.2 S isotopic composition (δ 34 S) and sources of sulfate aerosols The δ 34 S values for sulfate aerosols ranged from 2.2 ‰ to 17.6 ‰ between March and August (Fig. 2b). In all years of measurement, the δ 34 S values were low in April or earlier months, rapidly increased towards May to June, and remained high towards August (Fig. 3c). As warming progressed, the trend of increasing δ 34 S in the sulfate aerosols was broadly consistent with the increasing mixing ratio of atmospheric DMS. The δ 34 S values for the pre-bloom, bloom, and post-bloom periods averaged over 5 years were 7.5 ± 2.6 ‰, 9.5 ± 2.8 ‰, and 11.3 ± 2.8 ‰, respectively, reflecting an increasing enrichment in the heavier 34 S towards summer. The maximum monthly mean δ 34 S (13.5 ± 2.6 ‰) occurred in July 2018, whereas the lowest mean (3.7 ± 1.8 ‰) occurred in April 2019. The mean pre-bloom δ 34 S value in 2017 (9.2 ± 1.8 ‰) was higher than in 2018 (5.9 ± 1.2 ‰), whereas the mean bloom and post-bloom δ 34 S values were marginally lower in 2017 (11.0 ± 2.0 ‰) than in 2018 (12.5 ± 2.8 ‰). On monthly scales the greatest contribution of Bio-SO2− 4 occurred in August 2018 (59.4 ± 17.2 %) (Fig. S4). The proportion of Bio-SO2− among all SO2− particles was 4 4 18.1 ± 16.6 % during the pre-bloom period and then sharply increased to 37.2 ± 21.0 % during the bloom and postbloom periods, whereas the contribution of anthropogenic Atmos. Chem. Phys., 21, 9761–9777, 2021 9766 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds SO2 was 79.2 ± 16.9 % during the pre-bloom period and 57.9 ± 21.4 % during the bloom and post-bloom periods. Anth-SO2− 4 was found to be the largest contributor to total SO2− during all three periods (Fig. S4), which was consis4 tent with the previous findings (Li and Barrie, 1993; Norman et al., 1999; Udisti et al., 2016). 3.3 2− NSS-SO2− 4 , Anth-SO4 , and biogenic sulfur aerosols There were considerable seasonal and interannual variations in the concentrations of S aerosols including NSS-SO2− 4 , 2− Anth-SO4 , and Bio-S-aerosol (Figs. 3d–h, 4 and 5). In all years of the study the seasonal mean NSS-SO2− 4 concentration reached a maximum during the pre-bloom period (857 ± 520 ng m−3 ), decreased rapidly towards summer, and eventually dropped to a quarter of the maximum value during the post-bloom period (212 ± 120 ng m−3 ). We also found that the NSS-SO2− 4 concentration in the months prior to May varied by as much as a factor of 3 (1015 ± 586 in 2015 versus 291 ± 93 ng m−3 in 2019). The highest monthly mean NSS-SO2− concentration (1309 ± 131 ng m−3 ) was 4 recorded in March 2017, and the lowest was in July 2018 (165 ± 128 ng m−3 ). The concentration of Anth-SO2− 4 showed a temporal trend similar to that of NSS-SO2− 4 , with the highest monthly mean concentration (678 ± 450 ng m−3 ) occurring during the pre-bloom period, followed by a trend of decrease for the bloom (369 ± 236 ng m−3 ) and postbloom (114 ± 78 ng m−3 ) periods. During the pre-bloom period, when the chlorophyll-a concentration remained lower than 0.5 mg m−3 in waters around Svalbard, the concentration of Bio-SO2− 4 was unexpectedly high (180 ± 213 ng m−3 ), reaching 743 ng m−3 in 2016 (Fig. 4c). During the phytoplankton bloom period, the seasonal mean concentration of Bio-SO2− was 4 highest (184 ± 190 ng m−3 ). As summer approached, the Bio-SO2− 4 concentration decreased slightly during the postbloom periods (98 ± 68 ng m−3 ; Fig. 4c). In contrast to the trend for Bio-SO2− 4 , the MSA concentration remained low (< 30 ng m−3 ) during the pre-bloom period and rapidly increased during the transition from the pre-bloom to bloom periods (Figs. 3g and 5a). An elevated MSA concentration was maintained during much of the bloom and post-bloom periods, and then it decreased slightly to near the detection limit by the end of August. The highest monthly mean MSA concentrations were found in May (81.4 ± 58.1 ng m−3 ) and June (81.9 ± 56.5 ng m−3 ), which broadly agree with previous MSA measurements at Svalbard (Becagli et al., 2019). The annual mean concentrations of MSA (March to August) varied slightly among years (46.2 ± 35.9 ng m−3 in 2017, 63.5 ± 52.9 ng m−3 in 2018, and 55.4 ± 45.5 ng m−3 in 2019). The Bio-S-aerosol concentration increased with the onset of the spring bloom and stayed at moderate levels until June (Figs. 3h and 5b). The concentration of Atmos. Chem. Phys., 21, 9761–9777, 2021 2− Figure 4. Aerosol concentrations for (a) NSS-SO2− 4 (total SO4 2− 2− minus SS-SO2− 4 ), (b) Anth-SO4 , and (c) Bio-SO4 . The colored solid lines indicate 15 d moving average values. Bio-S-aerosol during the bloom period (252 ± 197 ng m−3 ) was slightly higher than that during post-bloom period (149 ± 91 ng m−3 ), and the highest monthly concentration of Bio-S-aerosol was found in April or May in all measurement years. The total concentrations of Bio-S-aerosol during the bloom and post-bloom periods were comparable in all 3 years (214 ± 124 ng m−3 in 2017, 204 ± 174 ng m−3 in 2018, and 160 ± 153 ng m−3 in 2019). 3.4 Ratio of MSA to Bio-S-aerosol (RBio ) In all years of this study the RBio values derived from δ 34 S data were lowest during the pre-bloom period and increased in the transition to the spring bloom, as biogenic DMS production peaked (Figs. 3i and 5c). The RBio value varied by a factor of 3 over seasons, showing maximum values during the bloom period (0.32 ± 0.17) and lowest values during the pre-bloom period (0.09 ± 0.07). The highest mean RBio (0.49 ± 0.05) was found in June 2018, whereas the lowest RBio (0.08 ± 0.01) was found in March 2017. There were large interannual variations in the seasonal mean RBio https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds 9767 tio) make data on the MSA-to-NSS-SO2− 4 ratio more widely available. 4 4.1 Figure 5. Aerosol concentrations of (a) MSA and (b) Bio-S-aerosol (MSA + Bio-SO2− 4 ). (c) Variations in the ratio of MSA to Bio-Saerosol (RBio ). The colored solid lines indicate 15 d moving mean values. (0.24 ± 0.11 in 2017, 0.40 ± 0.14 in 2018, and 0.36 ± 0.14 in 2019) during the bloom and post-bloom periods. Similar RBio values were also reported at Ny-Ålesund. For example, Udisti et al. (2016) reported a MSA-to-Bio-SO2− 4 ratio of 0.33 (RBio = 0.25) during the spring–summer period in 2014. This ratio was derived from a multi-seasonal asymptotic value in a plot between the MSA-to-NSS-SO2− 4 ratio and the MSA concentration. Implicit in this calculation is the assumption that the fraction of Bio-SO2− 4 in the to2− tal NSS-SO4 aerosols is overwhelming when the MSA-toNSS-SO2− 4 ratio approaches the asymptotic value (Udisti et al., 2016; Park et al., 2017). Other investigators also reported comparable RBio values in other Arctic environments: 0.18– 0.20 at the central Arctic Ocean (Chang et al., 2011b; Leck and Persson 1996), 0.28 at the eastern Antarctic Plateau (Udisti et al., 2012), and 0.28 at Alert (Norman et al., 1999). These RBio values were all derived from a multi-seasonal asymptotic value in a plot between the MSA-to-NSS-SO2− 4 ratio and MSA concentration. The analytical accessibilities associated with measurements of MSA and NSS-SO2− 4 concentration (i.e., less laborious and requires fewer aerosols than is needed for the technique measuring the S isotope rahttps://doi.org/10.5194/acp-21-9761-2021 Discussion Factors affecting variations in the S aerosol concentration in the Arctic atmosphere Seasonal variations in NSS-SO2− 4 aerosols were strongly associated with variations in Anth-SO2− 4 . In particular, the tight association of these parameters indicates that Anth-SO2− 4 aerosols were the largest contributor to NSS-SO2− during the 4 pre-bloom period, when the intrusion of Arctic haze is considerable (Figs. S4 and S5). During the transition from the pre-bloom to bloom periods, the input of Anth-SO2− 4 particles to our study area rapidly decreased because of weakening of the northward transport of air masses (containing Anth-SO2− 4 ) from Europe and increasing removal of AnthSO2− aerosols by increasing precipitation as the seasons 4 progress (Li and Barrie, 1993) (Fig. 4b). The decreasing input of Anth-SO2− 4 particles to the observation site during the bloom and post-bloom periods was also independently confirmed by the trend of decrease in the measured black carbon concentration at our observation site (Figs. 3a and S2). The large interannual variability in NSS-SO2− from 4 March to April was strongly associated with changes in the trajectory of the air masses reaching Svalbard and the sea level pressure along those air mass trajectories (Fig. 6). More explicitly, the higher concentrations of NSS-SO42− particles in 2015 (1015 ± 586 ng m−3 ) resulted from the greater input of pollutants (Anth-SO2− 4 ) from northern Europe via the intensified southwesterly wind, whereas the opposite occurred in 2018 and 2019 (634 ± 266 for 2018 and 291 ± 93 ng m−3 for 2019). Unusual elevation of the Bio-SO2− 4 concentration was occasionally found in the oceans surrounding Svalbard during the pre-bloom period in 2016 and 2017, despite low biological activity (as indicated by DMS mixing ratios of < 10 pptv) (Figs. 4c and S6). The spikes in the Bio-SO2− 4 concentration 2− likely originate from Bio-SO4 aerosols that were produced in distant ocean regions (e.g., the North Atlantic Ocean, the Norwegian Sea, and further south of 50–70◦ N and 25◦ W– 50◦ E) and then carried into the Arctic via a northward transport of air masses. Analysis of air mass back-trajectory data showed that the elevated values of Bio-SO2− 4 during the prebloom period in 2016 and 2017 resulted from air masses from lower-latitude regions reaching Svalbard rather than originating locally from the oceans around Svalbard, while the much lower Bio-SO2− 4 concentrations in 2018 probably resulted from an absence of air masses originating from distant DMS source regions during the pre-bloom period (Figs. 4c and S7). Atmos. Chem. Phys., 21, 9761–9777, 2021 9768 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds Figure 6. Sea level pressure (SLP) overlaid with wind vectors during March to April in (a) 2015, (b) 2016, (c) 2017, (d) 2018, and (e) 2019. Red stars indicate the location of the sampling site (Gruvebadet observatory; 78.9◦ N, 11.9◦ E). The MSA concentration remained low during the prebloom period (i.e., no apparent high peaks), largely because of the greater removal of MSA relative to Bio-SO2− 4 aerosols during long-range transport to Svalbard from the distant source regions to the south. For example, MSA tends to more easily condense onto existing particles (Hoppel, 1987; Pszenny et al., 1989) because of its higher vapor pressure and is thus more rapidly removed from the atmosphere with larger particles through wet deposition; this results in greater loss of MSA relative to SO2− 4 . Greater enrichment of MSA occurs in super-micron-sized particles than in submicron particles (Legrand and Pasteur, 1998). The higher ratios of MSA to NSS-SO2− 4 in rainwater and fresh snow than in aerosol particles is also indicative of the greater removal of MSA (Berresheim et al., 1991; Jaffrezo et al., 1994). The production mechanism of MSA (via DMS oxidation by OH radicals) (Gondwe et al., 2004) could also lower the MSA concentration during the pre-bloom period, when the low levels of OH radicals (as a result of low light conditions) resulted in less MSA production. The elevations of MSA occurred in May or June, when the production of OH radicals was high and associated with increasing solar radiation and biological production (Fig. S8 and S9a). The concentrations of Bio-S-aerosol during the bloom and post-bloom periods were comparable in all 3 years (214 ± 124 ng m−3 in 2017, 204 ± 174 ng m−3 in 2018, and Atmos. Chem. Phys., 21, 9761–9777, 2021 160 ± 153 ng m−3 in 2019), despite differing phytoplankton biomass (derived from Chl-a) among those years (Fig. S9b). This mismatch has been reported previously and suggests that estimations of marine organic aerosols based on Chl-a data only are unreliable (Rinaldi et al., 2013). In particular, the summer DMS-driven aerosols produced from the Barents Sea were not proportional to the Chl-a concentrations (Becagli et al., 2016). Different compositions of phytoplankton species in different ocean domains (Greenland Sea versus Barents Sea) could also result in changes in DMS production because phytoplankton have differing cellular levels of dimethylsulfoniopropionate (DMSP; a precursor of DMS) and the DMSP cleavage enzyme (enabling the transformation of DMSP to DMS) (Park et al., 2014b). The DMS production capacity in the Greenland Sea (where prymnesiophytes dominate) was found to be 3-fold higher than that in the Barents Sea (where diatoms dominate) (Park et al., 2018). Other studies have also reported that the concentrations of MSA or Bio-SO2− 4 do not always follow the atmospheric DMS mixing ratio, highlighting the involvement of other factors in the oxidation of DMS to MSA or Bio-SO2− 4 (Read et al., 2008; Yan et al., 2020a). Therefore, the amounts of DMS produced and its oxidation products may not be solely explained by variations in the ocean biomass. In the Arctic summer atmosphere the low abundance of large particles (i.e., Aitken and accumulation mode) could probably enhance the formation of new particles via the gasto-particle conversion process and the ultimate initiation of CCN formation (Boy et al., 2005; Dall’Osto et al., 2018). The concurrent increase in biogenic sulfate aerosols and smallsized particles (3–10 and 10–100 nm, respectively) reported for the Arctic atmosphere in May (Park et al., 2017) is a prime example that biogenic DMS is a major contributor to NPF. A model study reported that DMS enhanced the mass of sulfate particles in the size range 50–100 nm in regions north of 70◦ N (Ghahremaninezhad et al., 2019). During the bloom and post-bloom periods a decline in anthropogenic sources and an increase in oceanic DMS source strength resulted in the transition of major sulfate sources from Anth-SO2− 4 to Bio-SO2− , which highlights the increasing importance of 4 biogenic sulfur aerosols in the summer Arctic atmosphere. Biogenic organic aerosols in the high Arctic were reported to contribute considerably to the concentrations of ultrafine and CCN particles from summer to early autumn when anthropogenic source is lowest (Dall’Osto et al., 2017; Lange et al., 2019). Nonetheless, Anth-SO2− 4 contributed considerably to the total SO2− budget during the post-bloom period, 4 indicating that even in summer the Anth-SO2− 4 transported from Europe or local emissions can exert a significant influence on the sulfate budget in the Arctic atmosphere (Fig. S4) (L. Chen et al., 2016; Gogoi et al., 2016; Dekhtyareva et al., 2018). https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds 4.2 4.2.1 9769 Factors influencing the DMS oxidation pathways to either MSA or Bio-SO2− 4 (RBio ) Seasonal variations in RBio Our data spanning 5 years show two distinctive trends in RBio among seasons or years. The first is that the values of RBio during the bloom and post-bloom periods (0.32 ± 0.15) were a factor of 3 higher than the pre-bloom values (0.09 ± 0.07) (Fig. 5c). The large seasonal difference in RBio could be explained by known factors including the concentration of OH radicals (directly influenced by light intensity), air temperature (determining the oxidation pathway of DMS to either MSA or Bio-SO2− 4 ), the chemical properties of existing particles (e.g., the black carbon concentration) (e.g., Saltzman et al., 1986; Gondwe et al., 2004; Yan et al., 2020b), and biological activities near observation site. Among those, a major factor is the concentration of OH radicals. BrO radicals also help facilitate the addition pathway in the oxidation of DMS, even at concentrations > 1 pptv level (von Glasow and Crutzen, 2004). It has been hypothesized that the reactive bromines produced photochemically and heterogeneously at sea ice and snowpack surfaces lead to the BrO enrichment over ice-covered regions (Abbatt et al., 2012; Fernandez et al., 2019). Therefore, a high light intensity would favor the oxidation pathway of DMS to MSA, because this pathway is effectively mediated by photochemically activated species including OH and BrO. The solar radiation (51.3 ± 36.1 W m−2 ) over the distant DMS source regions during the pre-bloom period was much lower than over the Greenland Sea and the Barents Sea during the bloom (243.0 ± 63.4 W m−2 ) and post-bloom (222.5 ± 70.5 W m−2 ) periods (Fig. 7). The low OH radical and reactive bromine concentrations during the pre-bloom period probably lowered the production of MSA from DMS oxidation (i.e., weakening the addition pathway) and thereby resulted in the lower RBio value (0.09 ± 0.07) than was found during the bloom (0.32 ± 0.17) and post-bloom (0.32 ± 0.13) periods (Table S2). Consequently, solar radiation was likely to be a major driver of the seasonal RBio change in the Arctic atmosphere. The chemical properties of existing particles could influence the seasonal variations in RBio . Explicitly, the uptake of gaseous MSA onto particles was found to be sensitive to the chemical properties of those particles (Yan et al., 2020b). In particular, hydrophobic and acidic particles in the atmosphere tended to hinder the adhesion of gaseous MSA to particles, while alkaline sea-salt particles tended to accelerate the adhesion process (Pszenny, 1992; Jefferson et al., 1998; Yan et al., 2020b). Elemental carbon particles emitted from fossil fuel combustion are highly hydrophobic, and sulfates in the aerosol particles are acidic. However, only a small proportion of the anthropogenic particles formed in the polluted coastal and urban sites was found to be associated with MSA (Gaston et al., 2010; Yan et al., 2020b) formed https://doi.org/10.5194/acp-21-9761-2021 Figure 7. Five-year (2015, 2016, 2017, 2018, and 2019) mean radiation (red) and RBio (blue) during the pre-bloom, bloom, and postbloom periods. The solid line and dotted line represent median and mean value of each data in a box plot, respectively. from the oxidation of aqueous DMS catalyzed by iron and vanadium (Gaston et al., 2010; Moffett et al., 2020). Therefore, the air masses (rich in black carbon and sulfate) that originate from northern Europe probably have PM2.5 particles containing low MSA concentrations, despite the fact that those air masses swept through productive ocean areas during the pre-bloom period (Fig. 5a). In contrast, during the bloom period we found an elevation of the MSA concentration, primarily as a result of two reinforcing processes: the greater DMS oxidation to MSA, and the enhanced condensation of gaseous MSA to the existing particles under less hydrophobic and acidic conditions. For each group of RBio values, the lower concentrations of black carbon and sulfate resulted in the greater uptake of gaseous MSA and thereby resulted in higher RBio values (Fig. 8). We also found significant inverse correlations between black carbon and RBio (r = −0.79; Fig. 9a) and between total SO42− and RBio (r = −0.73; Fig. 9b); these tight correlations substantiate the importance of the chemical properties of atmospheric particles in determining the rate of uptake of gaseous MSA by the particles present in air. The number of samples measured during the bloom and post-bloom periods was higher in the groups having large RBio values (Fig. 8c). A strong positive correlation between monthly mean RBio and the air mass exposure to chlorophyll (EChl ) was observed during the study period (r = 0.82). The retention time of air masses over the ocean and marginal ice zone (i.e., DMS source regions) was also positively correlated with RBio values (r = 0.54). The RBio values decreased with decreasing air mass retention time over the land and multi-year ice regions (i.e., the non-DMS-source regions). The concentration of MSA was positively correlated with the mean Chl-a concentration in areas surrounding the observation site, but no similar clear correlation was found between Bio-SO2− 4 and Chl-a (Fig. S9). The absence of a correlation between Atmos. Chem. Phys., 21, 9761–9777, 2021 9770 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds Figure 8. Plots of the seasonal (a) black carbon (BC) concentration versus RBio , (b) the total SO2− 4 concentration versus RBio , and (c) the number of samples included in each RBio group. The solid and dotted lines represent the median and mean values of the data in the box plots, respectively. Bio-SO2− 4 and Chl-a indicates that the concentration of BioSO2− measured at the observation site included sulfur com4 pounds produced locally and in distant regions, because the greater atmospheric residence time of Bio-SO2− 4 (relative to MSA) indicates greater intrusion of Bio-SO2− 4 into the observation site. Hence, air masses that have been extensively exposed to local biological activities are likely to have higher RBio values. Therefore, the seasonal variations in RBio measured at Ny-Ålesund were probably controlled by the concentration of OH radicals (largely determined by light intensity), the chemical properties of the particles containing black carbon and sulfates, and biological activities surrounding the observation site. Another established factor that could affect the seasonal variations in RBio is air temperature. However, we found no association between RBio and air temperature (see Text S2). The RBio values in the present study, and those determined in other high-latitude regions including Barrow in Alaska (USA) and Neumayer station in the Antarctic coastal region, consistently pointed to the highest RBio values occurring in Atmos. Chem. Phys., 21, 9761–9777, 2021 Figure 9. Scatter plots of monthly mean RBio values as a function of (a) the monthly mean black carbon (BC) concentration, (b) the monthly mean total SO2− 4 concentration, (c) the air mass retention time over the ocean and the marginal ice zone (MIZ), (d) the air mass retention time over multi-year ice and land areas, and (e) the monthly mean air mass exposure to chlorophyll (EChl ). Error bars and the black solid line represent 1σ and the best fit, respectively. summer (Li et al., 1993; Legrand and Pasteur, 1998; Norman et al., 1999). We found that seasonal variability in RBio measured in the Arctic region can be better explained by light conditions, the chemical properties of particles, and biological activities near the observation site than by air temperature. Specifically, the RBio values measured during the pre-bloom period poorly represent the oxidative conditions of DMS in the Arctic atmosphere, because of the considerable intrusion of anthropogenic pollutants from the distant northern Europe. Thus, the RBio values measured during the bloom and post-bloom periods probably more accurately represent the ratio of the oxidation products of DMS produced in the ocean regions surrounding Svalbard under the less polluted conditions of the Arctic atmosphere. 4.2.2 Interannual variations in RBio The second distinctive trend is the interannual difference in RBio . The RBio values measured in 2017 were much lower than the values in other years (2015, 2016, 2018, and 2019; Fig. 10). One explanation for large interannual variations in RBio is the difference in the condensation of gaseous MSA onto particles in the Arctic atmosphere. As noted above, the chemical properties of particles largely determine the rate of https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds Figure 10. RBio (a–c) and the black carbon (BC) and total SO2− 4 concentrations (d–f) during pre-bloom, bloom, and post-bloom periods. Error bars represent 1σ . MSA condensation onto them (Jefferson et al., 1998; Yan et al., 2020b). During the pre-bloom and bloom periods in 2017, higher concentrations of black carbon and sulfate were found relative to other years, and consequently lower RBio values were found in 2017 (Fig. 10). However, we found no discernible interannual difference in the concentrations of black carbon (8.3 ± 4.9 ng m−3 in 2017 and 9.5 ± 6.1 ng m−3 −3 in 2017 in other years) and total SO2− 4 (235 ± 101 ng m and 232 ± 134 ng m−3 in the other years) during the postbloom period (Fig. 10f). To our surprise, during the postbloom period the RBio value (0.22 ± 0.07) in 2017 was only half the rate measured in the other years (0.39 ± 0.11). The lack of an association of black carbon and sulfate concentrations with RBio values indicates that factors other than chemical properties of existing particles affected the interannual variation in RBio values measured during the post-bloom period. Air temperature difference may explain the interannual variations in RBio during the post-bloom period (Fig. S11). However, the lack of correlation (e.g., Bates et al., 1992b) we found between RBio values and the mean temperatures of air masses along the entire pathway to Svalbard (Fig. S11b) is consistent with the results of other studies (Savoie et al., 1992; Legrand and Pasteur, 1998; Zhan et al., 2017; Moffett et al., 2020), implying that variations in air temperature were not a driver of determining the DMS branching ratio. We also found no discernible differences in solar radiation and relative humidity between year of 2017 and other years; thus, neither solar radiation nor relative humidity showed any association with RBio (Fig. S11c–d). Thus, no meteorological factors adequately explain the interannual variations in RBio during the post-bloom period. The concurrent measurements of DMS and MSA during summer in the Southern Ocean reinforce our finding that temperature and relative humidity https://doi.org/10.5194/acp-21-9761-2021 9771 Figure 11. Air mass exposure to chlorophyll (EChl ) (a–c) and the air mass residence times over the ocean and marginal ice zone (MIZ) and the multi-year ice and land areas (d–f) during pre-bloom, bloom, post-bloom periods. Error bars represent 1σ . have negligible effects on the conversion of DMS to MSA (Yan et al., 2020a). Analysis of air mass back-trajectory data indicated that the air mass exposure to chlorophyll (EChl ) in 2017 (0.44 ± 0.21) was 30 % lower than in other years (0.63 ± 0.35). The mean retention time of air masses over the sea ice and land areas (i.e., non-DMS source regions) in 2017 (40.9 ± 27.9 h) was 25 % longer than that estimated for other years (32.3 ± 19.2 h), whereas the mean retention time of air masses over the ocean and marginal ice regions (i.e., the DMS source regions) was lower in 2017 (79.1 ± 27.9 h) than in other years (87.7 ± 19.2 h) (Fig. 11). Hence, the 2017 RBio values were 40 % lower than those in 2018 and 2019, probably because more air masses swept over non-DMS source regions. As sulfate and MSA particles have different roles in terms of particle formation and growth, the importance of RBio is worth highlighting. Sulfate particles (including sulfuric acids) are known to produce 4–6 times more submicron-sized particles than MSA, leading to a 10-fold stronger cooling effect via scattering of solar radiation (i.e., a direct effect), whereas the impacts of sulfate and MSA particles on cloud microphysics (i.e., an indirect effect) are comparable (Hodshire et al., 2019). Our findings of considerable seasonal or interannual variations in RBio indicate that the conventional approach of using asymptotic values to determine the oxidation products of DMS and to evaluate the contribution of biogenic sources to the total sulfur budget at particular locations (e.g., Norman et al., 1999; Udisti et al., 2012, 2016) is problematic. Atmos. Chem. Phys., 21, 9761–9777, 2021 9772 5 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds Conclusion and implication This study shows that in the Arctic atmosphere extensive production of the oxidation products of DMS (i.e., Bio-SO2− 4 and MSA) occurred from the onset to the termination of phytoplankton blooms between 2015 and 2019. Anth-SO2− 4 was found to be the largest contributor to total sulfate aerosols during the pre-bloom periods, as a result of the influence of Arctic haze. Its contribution was comparable to that of Bio-SO2− 4 during the bloom and post-bloom periods. We also found large interannual variations in anthropogenic and biogenic sulfur aerosols. Moreover, the ratio of MSA to BioSO2− 4 (RBio ) tended to be higher (0.32 ± 0.15) in summer than in early spring (0.09 ± 0.07). Our results imply that NPF and subsequent growth of those particles to form CCN are governed by both Bio-SO2− 4 and MSA when RBio is high (bloom and post-bloom periods), but that when RBio is low (pre-bloom period) MSA makes only a small contribution to particle growth and other molecules with low-volatility vapors (e.g., highly oxygenated organic molecules) are more involved in particle growth near Svalbard. The large interannual variability of RBio further indicates that condensational growth following NPF can be affected by MSA or other molecules with low-volatility vapors, depending on the branching ratio of DMS oxidation. In modeling studies (Vallina et al., 2006, 2007) the annual contribution of biogenically induced CCN to total global CCN has been estimated to be greater than 30 %, and up to 80 % in the austral summer in the Southern Ocean. This is similar to findings for the Northern Hemisphere, where BioSO2− 4 particles accounted for greater than 60 % of CCN in late spring (May and June) in the North Atlantic (Sanchez et al., 2018). An acceleration of sea ice retreat and an increase in melt ponds in the Arctic Ocean will increase biogenic DMS production, resulting in a greater contribution of biogenic S aerosols to atmospheric aerosol formation and climate regulation (Arrigo et al., 2008; Gourdal et al., 2018; Park et al., 2019; Galí et al., 2019). Another important factor that may be involved in the formation of biogenic CCN is changes in the atmospheric concentrations of OH, NOx , and BrO; these are likely to be affected by future climate change and increasing anthropogenic perturbations (e.g., sea ice decline, increasing reduced carbon emissions) (Alexander and Mickley, 2015). Our measurements primarily focused on the particle phase of sulfur species (particles < 2.5 µm) but did not cover the initial phase of DMS oxidation and particle growth (i.e., nano size scales), including the concentration of the oxidants and gas-phase composition of sulfur species. Therefore, the integrated study of both the gas and particle phases of sulfur compounds (including gaseous MSA, SO2− 4 , and hydroperoxymethyl thioformate), ocean colors, and sea ice properties will help define the climate-relevant impacts of oxidation products of biogenic DMS in the Arctic environment. Atmos. Chem. Phys., 21, 9761–9777, 2021 Data availability. All data needed to draw the conclusions in the present study are presented in this report and/or the Supplement. For additional data related to this study, please contact the corresponding author (Kitack Lee; ktl@postech.ac.kr). Supplement. The supplement related to this article is available online at: https://doi.org/10.5194/acp-21-9761-2021-supplement. Author contributions. SJ, KTP, YJY, and KL designed the data analysis and wrote the manuscript. SJ, KTP, and EJ performed the data evaluation and analyses. KK and HYC performed the ion chromatograph measurements. KE provided the black carbon data. RT and SB were involved in aerosol sample collection. BYL, RK, and OH contributed to the interpretation of the results. Competing interests. The authors declare that they have no conflict of interest. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements. We thank the Sverdrup Research Station staff of the Norwegian Polar Institute for assisting us in maintaining the atmospheric DMS analysis system at the Zeppelin station. Financial support. 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S.: Nitrogen and sulfur species in aerosols at Mawson, Antarctica, and their relationship to natural radionuclides, J. Atmos. Chem., 14, 181–204, https://doi.org/10.1007/BF00115233, 1992. Sekiguchi, M., Nakajima, T., Suzuki, K., Kawamoto, K., Higurashi, A., Rosenfeld, D., Sano, I., and Mukai, S.: A study of the direct and indirect effects of aerosols using global satellite data sets of aerosol and cloud parameters, J. Geophys. Res., 108, 4699, https://doi.org/10.1029/2002JD003359, 2003. Stefels, J., Steinke, M., Turner, S., Malin, G., and Belviso, S.: Environmental constraints on the production and removal of the climatically active gas dimethylsulphide (DMS) and implica- Atmos. Chem. Phys., 21, 9761–9777, 2021 tions for ecosystem modelling, Biogeochemistry, 83, 245–275, https://doi.org/10.1007/s10533-007-9091-5, 2007. Stroeve, J. C., Jenouvrier, S., Campbell, G. 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Lincei, 27, S85–S94, https://doi.org/10.1007/s12210-016-05177, 2016. Vallina, S. M., Simó, R., and Gassó, S.: What controls CCN seasonality in the Southern Ocean? A statistical analysis based on satellite-derived chlorophyll and CCN and model-estimated OH radical and rainfall, Global. Biogeochem. Cy., 20, GB1014, https://doi.org/10.1029/2005GB002597, 2006. Vallina, S. M., Simó, R., Gassó, S., de Boyer-Montégut, C., Del Río, E., Jurado, E., and Dachs, J.: Analysis of a potential “solar radiation dose–dimethylsulfide–cloud condensation nuclei” link from globally mapped seasonal correlations, Global. Biogeochem. Cy., 21, GB2004, https://doi.org/10.1029/2006GB002787, 2007. Veres, P. R., Neuman, J. A., Bertram, T. H., Assaf, E., Wolfe, G. M., Williamson, C. J., Weinzierl, B., Tilmes, S., Thompson, C. R., Thames, A. B., Schroder, J. C., Saiz-Lopez, A., Rollins, A. W., Roberts, J. M., Price, D., Peischl, J., Nault, B. A., Møller, K. H., Miller, D. O., Meinardi, S., Li, Q., Lamarque, J., Kupc, A., Kjaergaard, H. G., Kinnison, D., Jimenez, J. L., Jernigan, C. M., Hornbrook, R. S., Hills, A., Dollner, M., Day, D. A., Cuevas, C. A., Campuzano-Jost, P., Burkholder, J., Bui, T. P., Brune, W. H., Brown, S. S., Brock, C. A., Bourgeois, I., Black, D. R., Apel, E. C., and Ryerson, T. B.,: Global airborne sampling reveals a previously unobserved dimethyl sulfide oxidation mechanism in the marine atmosphere, P. Natl. Acad. Sci. USA, 117, 4505–4510, https://doi.org/10.1073/pnas.1919344117, 2020. von Glasow, R. and Crutzen, P. J.: Model study of multiphase DMS oxidation with a focus on halogens, Atmos. Chem. Phys., 4, 589– 608, https://doi.org/10.5194/acp-4-589-2004, 2004. Willis, M. D., Leaitch, W. R., and Abbatt, J. P.: Processes controlling the composition and abundance of Arctic aerosol, Rev. Geophys., 56, 621–671, https://doi.org/10.1029/2018RG000602, 2018. Wyslouzil, B. E., Seinfeld, J. H., Flagan, R. C., and Okuyama, K.: Binary nucleation in acid–water systems. I. Methanesulfonic acid–water, J. Chem. Phys., 94, 6827–6841, https://doi.org/10.1063/1.460261, 1991. Yan, J., Zhang, M., Jung, J., Lin, Q., Zhao, S., Xu, S., and Chen, L.: Influence on the conversion of DMS to MSA and SO2− 4 in the Southern Ocean, Antarctica, Atmos. Environ., 233, 117611, https://doi.org/10.1016/j.atmosenv.2020.117611, 2020a. https://doi.org/10.5194/acp-21-9761-2021 S. Jang et al.: Large seasonal and interannual variations of biogenic sulfur compounds Yan, J., Jung, J., Zhang, M., Bianchi, F., Tham, Y. J., Xu, S., Lin, Q., Zhao, S., Li, L., and Chen, L.: Uptake selectivity of methanesulfonic acid (MSA) on fine particles over polynya regions of the Ross Sea, Antarctica, Atmos. Chem. Phys., 20, 3259–3271, https://doi.org/10.5194/acp-20-3259-2020, 2020b. Yin, F., Grosjean, D., and Seinfeld, J. H.: Photooxidation of dimethyl sulfide and dimethyl disulfide. I: Mechanism development, J. Atmos. Chem., 11, 309–364, https://doi.org/10.1007/BF00053780, 1990. https://doi.org/10.5194/acp-21-9761-2021 9777 Zhan, J., Li, W., Chen, L., Lin, Q., and Gao, Y.: Anthropogenic influences on aerosols at Ny-Ålesund in the summer Arctic, Atmos. Pollut. Res., 8, 383–393, https://doi.org/10.1016/j.apr.2016.10.010, 2017. Atmos. Chem. Phys., 21, 9761–9777, 2021
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Methodical peculiarities of state regulation of the wayside protective zones in Ukraine
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PAPER • OPEN ACCESS PAPER • OPEN ACCESS IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering This content was downloaded from IP address 80.64.83.98 on 21/04/2020 at 22:06 Methodical peculiarities of state regulation of the wayside protective zones in Ukraine I O Novakovska1, P F Zholkevskyi1, M P Stetsiuk1 and N F Ishchenko2,3 1Department of Land Management and Cadastre, National Aviation University, 1 Avenue Cosmonaut Komarov, Kyiv, Ukraine 2Institute of Agroecology and Nature Management of NAAS, 12 Metrolohichna Street, Kyiv, Ukraine Methodical peculiarities of state regulation of the wayside protective zones in Ukraine To cite this article: I O Novakovska et al 2019 IOP Conf. Ser.: Mater. Sci. Eng. 708 012016 View the article online for updates and enhancements. TRANSBUD-2019 IOP Conf. Series: Materials Science and Engineering 708 (2019) 012016 IOP Publishing doi:10.1088/1757-899X/708/1/012016 Conf. Series: Materials Science and Engineering 708 (2019) 012016 doi:10.1088/1757-899X/708/1/0120 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. nder licence by IOP Publishing Ltd 1 3Email: natalkai@ukr.net Abstract. The sizes of the areas of influence, protected zones, and reserved technological areas are contemplated here. There is also an analysis of modern use and following the wayside zones of motorways. The situation with pollution of roadside strips, surface waters, soils, groundwater and roadside vegetation has been analyzed. Solutions on lands protection and creation of prerequisites for the motorway protected zones formation are provided. It has been substantiated that the solution of these problems should be based on the application of mathematical methods and computer simulation with the use of remote sensing and requires detailed geographic research to the level of specific, quantitative justification of transport infrastructure projects in order to ensure optimal land use, recultivation. The comparative analysis of the regulatory requirements of the location highways elements of the and Ukraine, Belarus and the USA roadside territories has been conducted. The advantages of creating space data infrastructure for roadside establishment are elaborated. Establishing the sizes and configuration of the protection zones in the structure of the project documentation for the construction (reconstruction) of highways, as well as their indication in the town planning documentation, on cadastral plans will lead to the introduction of responsibility at the legislative level. 1. Introduction A At present stage of the development of the country, the transport branch is among the prospective ones. The quality of transport arterials predetermines economic, social, ecologic, cultural aspects of the functioning of a country, its future growth, and its position on an international stage. The conditions and development of a motorway network create a tight connection between investment, integration, social and economic, economic and ecological, anti-crises, civilization processes in society. In the meantime, the transport branch, despite all its positive aspects for the economic and social development of a country, has a significantly negative effect on the environment. This refers to harmful exhaustive fumes, emission of substances related to the use of tires and breaks, salts for melting snow, disposal of used materials and spare parts, as well as noise contamination to nature [1]. The matter of a holistic and comprehensive evaluation of the influence of transport infrastructure, able to synthesize adequately the whole scope of manifestation of this object of engineering infrastructures is the one less studied nowadays. Limited is the number of attempts to raise these questions, and the same of the solutions, although fragmented and quite schematic. Scientific researches, and practical ones even more, are focused on the exclusively economic assessment of 1 TRANSBUD-2019 onf. Series: Materials Science and Engineering 708 (2019) 012016 doi:10.1088/1757-899X/708/1/0120 isolated consumable features of territories. The lands are studied as those used for farming, forest activities, building motorways, and other projects. 2. Presentation of the main research material Considering how important is it to protect people and environment, adjacent lands, and different objects of nature against the harmful influence of transport, laws stipulate the creation of protective zones lengthwise the transport systems. The state regulates the protective zones by a range of rules and regulations, inter alia by the respective environmental demands to the motorways. y esigning. GBN V.2.3-218-007:2012 [2] divide them as follows: an influence wayside, ctive wayside, a reserve-engineering wayside. p y g g y The sizes of the influence, protective, and reserve-engineering waysides are provided in table 1 [2]. The sizes of the influence, protective, and reserve-engineering waysides are pro Table 1. Approximate sizes of influence, protective, and reserve-engineering waysides. Table 1. Approximate sizes of influence, protective, and reserve engineering waysides. Territory, adjacent to a motorway The distance from the edge of a traffic way to different classes of natural objects, m I II III Upon condition of free distribution of the influence Influence waysides 3000 2000 600 A protective wayside 300 150 60 A reserve-engineering wayside 30 12 7,5 In the case of hindrances (terrain, forests, built-up environment, landscaped areas) Influence waysides 1500 1000 600 A protective wayside 200 90 30 A reserve-engineering wayside 30 12 7,5 pp , p , g g y Territory, adjacent to a motorway The distance from the edge of a traffic way to different classes of natural objects, m I II III Upon condition of free distribution of the influence Influence waysides 3000 2000 600 A protective wayside 300 150 60 A reserve-engineering wayside 30 12 7,5 In the case of hindrances (terrain, forests, built-up environment, landscaped areas) Influence waysides 1500 1000 600 A protective wayside 200 90 30 In the case of hindrances (terrain, forests, built-up environment, landscaped areas) p p Influence waysides 1500 1000 600 A protective wayside 200 90 30 A reserve-engineering wayside 30 12 7,5 A reserve-engineering wayside The sizes of the influence waysides of motorways depend on the results of prognostic assessment of harmful emissions of contaminators, and the distribution of physical influence. [2]. Road engineering structures, like bridges, pipe culverts, drainage gutters, etc. cause water erosion of soils on roadside territories. The surface drainage caused by the precipitations, melting snow, and watering roads washes different soluble and insoluble substances and brings them to adjacent territories. A concentration of these contaminators depends on the frequency of cleaning streets, the intensity of traffic and precipitations, the length of a previous period without precipitations. Any wayside is specific not only by contamination to surface waters, but also by the same to soils, soil waters, and roadside-growing plants. Contamination of the ground surface with transport and road emissions aggregates gradually, depending on the number of vehicles passing a highway, a road, a motorway. It remains for a very long period, even after removal of the body of a road (in case of a road, highway, the motorway is closed or a road and its asphalt cover are removed completely). Different chemical elements, especially metals, tend to be accumulated by the soils. Then plants digest them and pass through a food chain to animals and people. Some chemicals dissolve and come with ground waters to rivers and other water mains. They can get to the human body in drinking water. The practice proves that small-dispersed solid particles and toxic components in the soil are as less concentrated, as bigger is the distance from a road surface [3]. Steadily developing is now the situation with illegally ploughed waysides. Leaseholders and proprietors illegally cultivate lands along the roads. They grow crops and sell their harvest to the food industry. As a rule, there are the most widespread cultures: sunflowers, corn, and wheat. The examples of such illegal cultivation are observed in every region of our country. This situation is illustrated by figure 1. 2 IOP Publishing TRANSBUD-2019 onf. Series: Materials Science and Engineering 708 (2019) 012016 doi:10.1088/1757-899X/708/1/0120 (a) (b) Figure 1. Use of the wayside: a - Odessa region, b - Kiev region. (a) (b) Figure 1. Use of the wayside: a - Odessa region, b - Kiev region. The picture demonstrates that the adjacent lands are seeded just next to the easement areas of motorways. There is an enormous quantity of such examples. A reserve-engineering wayside Neither the proprietors, not the leaseholders meet the respective rules. This causes contamination of the agricultural plants with heavy metals; a domino effect brings them to human bodies in a daily food, like bread, oil, butter, eggs, meat, and others. Low quality of food thus leads to many severe diseases. The occurrence of the diseases and distribution thereof are determined by environmental and social-economic factors. Their importance grows steadily due to the way of life, level of income, housing conditions, and structure of nutrition. In such conditions, topical become the matters of protection of the lands. To create the preconditions for the protected areas around the roadways, and to improve and eliminate the situation, it is important to take measures on the level of the law to make such use of the lands strictly forbidden: 1. to implement a system of strict fines for the landlords and users of the lands for the illegal ploughing; In such conditions, topical become the matters of protection of the lands. To create the preconditions for the protected areas around the roadways, and to improve and eliminate the situation, it is important to take measures on the level of the law to make such use of the lands strictly forbidden: it is important to take measures on the level of the law to make such use of the lands strictly forbidden: 1. to implement a system of strict fines for the landlords and users of the lands for the illegal ploughing; y 1. to implement a system of strict fines for the landlords and users of the lands for the illegal ploughing; p g g 2. to obligate the landlords and the users of the lands to cultivate the plants lengthwise the motorways, intended for production of alternative types of fuel if there is no greenery to hold back the harmful emissions; 2. to obligate the landlords and the users of the lands to cultivate the plants lengthwise the motorways, intended for production of alternative types of fuel if there is no greenery to hold back the harmful emissions; 3. to increase the size of the waysides, especially for growing crops; 4. to elaborate a method for assessment of damage, caused by unauthorized use of the land 4. to elaborate a method for assessment of damage, caused by unauth 5. A reserve-engineering wayside to elaborate rules and standards for the sphere of protection and use of the lands; 6. to create an automatic informational system for revealing the cases of land laws infringements, especially unauthorized use of the lands; 7. to ensure the implementation of reserving the lands for the needs of road transport and infrastructure. 7. to ensure the implementation of reserving the lands for the needs of road transport and infrastructure. First, the solution of these tasks shall be based on the wide use of mathematic methods and computer-aided simulation with the means of remote sounding. It demands detailed engineering- geographic investigations to reach the level of a certain quantitative-grounded project of transport infrastructure to ensure optimal land management and re-cultivation. The new IT-technologies are supported by the databases of digital cartographic information and by the modern digital methods of topography-geodesy and GPS measurements, remote sounding of lands, digital photogrammetry, promoting the development of geo-informational cartography that is the functioning environment thereof. All these results in a database of digital geographic information, able to become a new kind of geo-basic information product to be applied to study the influence of transport objects on the natural environment. First, the solution of these tasks shall be based on the wide use of mathematic methods and computer-aided simulation with the means of remote sounding. It demands detailed engineering- geographic investigations to reach the level of a certain quantitative-grounded project of transport infrastructure to ensure optimal land management and re-cultivation. The new IT-technologies are supported by the databases of digital cartographic information and by the modern digital methods of topography-geodesy and GPS measurements, remote sounding of lands, digital photogrammetry, promoting the development of geo-informational cartography that is the functioning environment thereof. All these results in a database of digital geographic information, able to become a new kind of geo-basic information product to be applied to study the influence of transport objects on the natural environment. Among the main advantages of the space data infrastructure for definition of the waysides, there are less expenses on collection, processing, and support of geo-basic data, improved quality and speed 3 IOP Publishing IOP Conf. Series: Materials Science and Engineering 708 (2019) 012016 g doi:10.1088/1757-899X/708/1/012016 onf. Series: Materials Science and Engineering 708 (2019) 012016 doi:10.1088/1757-899X/708/1/0120 of upgrade of the data, a surplus effect thanks to the new technology of accumulating information from the different sources and remote access thereto. A reserve-engineering wayside The main purpose thereof is to ensure equal and equivalent access to geo-informational resources on traffic and road infrastructure for state bodies, commercial institutions, and the public. of upgrade of the data, a surplus effect thanks to the new technology of accumulating information from the different sources and remote access thereto. The main purpose thereof is to ensure equal and equivalent access to geo-informational resources on traffic and road infrastructure for state bodies, commercial institutions, and the public. The Resolution of the Cabinet of Ministers of Ukraine of March 30, 1994 № 198 “On Approval of the Unified Rules for Reparation and Maintenance of Motorways, Streets, Railroad Crossings, Rules on Use and Protection” (as amended) provides the following definition of the waysides: 1. the waysides of the motorways (suburban) on the areas of the roads of state importance of I, II, and III category, built or being built in circumvention of cities, towns, and villages shall be not less than 100 meters; 1. the waysides of the motorways (suburban) on the areas of the roads of state importance of I, II, and III category, built or being built in circumvention of cities, towns, and villages shall be not less than 100 meters; 2. the waysides of the approaches to regional centers and big industrial centers shall be not less than 50 meters; 2. the waysides of the approaches to regional centers and big industrial centers shall be not less than 50 meters; 3. the waysides of the areas of roads of state importance between the settlements shall be not less than 32.5 meters [4]. 3. the waysides of the areas of roads of state importance between the settlements shall be not less than 32.5 meters [4]. There are some restrictions to economic activities within the limits of the areas as indicated, although they are not regulated by laws. The waysides (the waysides are controlled areas, road-side protection areas, etc.) of the motorways vary in different countries. Their width may be from 20 to 150 meters. Analysis of the statutory requirements to elements of placement of the motorways and waysides in Ukraine, Belarus, and the USA has shown that sizes of waysides and road-adjacent territories in Belarus, except for road construction elements, stipulates placement of service objects, while in Ukraine road service objects are located out of the waysides. A reserve-engineering wayside In Belarus, construction of the motorways stipulates designed waysides for any objects thereon to be controlled by the road’s owner. The waysides are intended to ensure traffic safety, proper conditions for reconstruction and overhaul of the roads, considering perspectives of development. The width of motorways in Belarus is up to 100 meters to both sides from the axis thereof; in settlements, waysides are the land plots towards the borders of an existing built-in environment [5]. In Belarus, the road owner's territory also includes a reserved area – a land plot, reserved for future building or reconstruction of the motorway [6]. In Ukraine, albeit the waysides are stipulated, their management lacks legislative regulation. As for parameters of placement elements of road facilities in the USA and Ukraine, it was discovered that junctions in the USA are not very frequent. The distance between them along autostrada equals 100 km (or one hour of driving), while in Ukraine they are each 35 km on the 1st category roads. Stops in the USA are placed on crossroads, while in Ukraine inversely they are located more than 50 meters far from the crossroads. Thus, to sum up, the comparison of the regulative requirements of Ukraine with the same of other countries, we could point out the basic ones. Some parameters of motorway elements in different rules and regulations of Ukraine argue each other; the distance between the service objects in the USA is regulated more, than in our country. In Ukraine, while designing a motorway, only a way for placement road constructive elements is to be ensured, while territory for road facility buildings is not stipulated, unlike Belarus. We have discovered the need for regulative support of the respective management and maintenance of the waysides and reserve zones [7]. While establishing the waysides, it is worth to emphasize that the land plots within the limits of the waysides indicated are not withdraws (purchased); to prescribe certain restrictions for building economic objects in indicated areas; to stipulate a possibility to preserve agricultural lands. A reserve-engineering wayside To ensure protection of roads against destruction in parts thereof endangered with soil displacements and erosion, some special protective areas have been already prescribed by the “Instruction on the procedure of allocation and management of waysides of the motorways in the Ukrainian SSR” by Council of the Ministers of Ukrainian Soviet Socialistic Republic in August 4, 1962, № 876 (as amended) without including thereof into the right of way [8]. g g y The article 112 of the Land Code of Ukraine prescribes the creation of protective zones longwise the transport lands to protect them against negative anthropogenic impact. Namely, in areas specific 4 IOP Publishing onf. Series: Materials Science and Engineering 708 (2019) 012016 doi:10.1088/1757-899X/708/1/0120 for soil displacements, landslides, washouts, mudslides, snow-wreaths, and other dangerous impacts, protective areas are stipulated longwise the lands of railway transport [9]. Although the procedure of allocation of the areas described above, their sized and regime of management still have not been defined by the Cabinet of Ministers of Ukraine [10]. Although the procedure of allocation of the areas described above, their sized and regime of management still have not been defined by the Cabinet of Ministers of Ukraine [10]. Protection of the motorways against the water and wind erosions, imbibition, landslides, avalanches, snow-banks are as important, as protection of the railways. The road facilities include already counter-avalanche and counter-mudslides structures and plants, drainage and water treatment plants for motorway protection. It is expedient to say that sizes and configuration of protective zones are stated by design documentary for building (reconstruction) of the motorways, while borders thereof shall be indicated in city-planning documentary and documentary on land development and cadaster plans. As for restriction of activities in the protective areas, they shall prohibit deforestation, construction of any building not related to the protection of the roads, any activities promoting processes of erosion and waterlogging of the lands. 3. Conclusion The use of the waysides causes a range of questions not regulated by the laws. To solve the tasks of land protection and the creation of preconditions for stable land management in the country, we offer a law-based implementation of the measures able to improve the situation. As a result of research, it has been proved that the land granting for reservation for autostrada‘s construction gives the opportunity to include them in the documentation of land management. Defining sizes and configurations of the protective zones within design documentary for construction (reconstruction) of motorways, as well as indicating thereof in city-planning documents, land-development documentary, and cadaster plans shall support the implementation of responsibility on the level of laws. References [1] Boichenko S, Zaporozhets O 2017 Transport ecology manual. (Ukraine: Center for Educational Literature) р 508 [2] Ecological Demands to Motorways. Designing. GBN V.2.3-218-007:2012. Dated Aug.06, 2012 307 [1] Boichenko S, Zaporozhets O 2017 Transport ecology manual. (Ukraine: Center for Educational Literature) р 508 2 l i l d i i G 2 3 218 00 2012 d A 06 2012 [ ] , p p gy ( Literature) р 508 [2] Ecological Demands to Motorways. Designing. GBN V.2.3-218-007:2012. Dated Aug.06, 2012 307 [2] Ecological Demands to Motorways. Designing. GBN V.2.3-218-007:2012. Dated Aug.06, 2012 307 [3] Perovych L, Vanchura L 2011 The Impact of the Motor Transport on Contamination of the Land Resources (Ukraine: Modern Achievement of Geodesy Science and Industry) 21 102- 109 [4] On Approval of the Unified Rules for Reparation and Maintenance of Motorways, Streets, Railroad Crossings, Rules on Use and Protection of 30 03 1994 № 198 g [5] Novakovskaya I O, Ischenko N F 2017 Problems of land allocation and use for the needs of the road economy. (Ukraine: Modern issues of economics and law) 1 (5.6) 135-144 [6] Novakovs'ka I, Ishenko N 2017 Transport strategy of Ukraine in the context of European integration Latvia: Collective Monograph 248 [7] Black W R 2010 Sustainable Transportation: Problems and Solutions. The Guilford Press 1 edition January 4 p 299 [8] On Motorways of Ukraine: the Law of Ukraine of September 08 2019 № 2862-IV [9] On Transport: the Law of Ukraine of November 10 1994 № 232/94-ВР [10] On Regulation of City-Planning Documentary: the Law of Ukraine of February 17 2011 5
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The Effectiveness of PAlliative Split COurse RAdiotherapy (PASCORA) Regimen in Non-metastatic Head and Neck Cancer Patients who are Treated with Palliative Intent- A Retrospective Single Centre Study
Asian Pacific journal of cancer biology
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Abstract However, there are no standardised RT regimens which are recommended in these patients. In our centre, we commonly practice PAlliative Split COurse Radiotherapy (PASCORA) which is 22.5 Grays (Gy) in 5 fractions on 5 days with one fraction per day. Corresponding Author: Dr. Umesh Velu Room No 8, Department of Radiotherapy, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal Udupi, 576104 India. Email: umesh.vellu@manipal.edu Asian Pac J Cancer Biol, 7 (4), 315-322 Submission Date: 09/29/2022 Acceptance Date: 11/12/2022 Asian Pac J Cancer Biol, 7 (4), 315-322 Asian Pac J Cancer Biol, 7 (4), 315-322 Submission Date: 09/29/2022 Acceptance Date: 11/12/2022 Umesh Velu1, Preethi S Shetty2, Anshul Singh1, Shirley Salins1, Krishna Sharan1 Umesh Velu1, Preethi S Shetty2, Anshul Singh1, Shirley Salins1, Krishna 1Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education (Mahe), Manipal, India. 2Department of Surgical Oncology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education (Mahe), Manipal, India. The Effectiveness of PAlliative Split COurse RAdiotherapy (PASCORA) Regimen in Non-metastatic Head and Neck Cancer Patients who are Treated with Palliative Intent- A Retrospective Single Centre Study The Effectiveness of PAlliative Split COurse RAdiotherapy (PASCORA) Regimen in Non-metastatic Head and Neck Cancer Patients who are Treated with Palliative Intent- A Retrospective Single Centre Study DOI:10.31557/APJCB.2022.7.4.315 DOI:10.31557/APJCB.2022.7.4.315 Abstract Introduction: We at our centre practice a PAlliative Split COurse RAdiotherapy (PASCORA) of 22.5Gy in 5 fractions followed by a gap of 4 weeks and then again repeat 22.5Gy in 5 fractions for locally advanced squamous cell carcinoma patients treated with a palliative intent. Aim was t0 assess the symptomatic relief at 3 months following PASCORA regimen. Materials & Methods: 49 Patients with LAHNSCC between January 2014 to January 2021, planned for PASCORA regimen were evaluated. Symptomatic relief was assessed on an objective scale. OS was determined using Kaplan Meir survival curves. Results: Median age was 61 years, multiple comorbidities (37%) were the most commonly documented reason for these patients being treated with a palliative intent. 25% of our patients had an excellent symptomatic relief, 26% of our patients had a good symptomatic relief and 31% had a partial relief. Median OS was 38 months in patients who had an excellent symptomatic relief and 3-8 months in patients with no or partial symptomatic relief ( p value=0.000) 6% of our patients had Grade 3 /4 RTOG toxicity. Conclusion: PASCORA regimen offers a good symptomatic relief with good local control rates and acceptable level of toxicity and comparable OS. Keywords: Palliative radiotherapy- head and neck cancers- split course radiotherapy 315 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 ntroduction According to the GLOBOCON 2020 data, head and neck (HN) cancers are the most common malignancies approximately 1.9 Lakhs cases per year) seen in India 1]. The locally advanced HNC patients are treated with a combination of surgery, radiotherapy (RT) and hemotherapy. Many randomized controlled trials have hown benefit with the use of chemoradiotherapy (CTRT) r anti Epidermal Growth Factor receptor (EGFR) therapy ombined with RT resulting in better local control rates nd an overall survival rates [2-4]. However, these reatment regimens are directed towards patients intended or curative intent. For patients who are not fit to undergo he proposed radical treatment there isn’t any available evidence based guidelines to offer standard of care. A patient can be deemed unfit for a radical intent treatment either due to poor performance status or due to locally advanced stage at presentation. Usually such patients are offered a multitude of palliative Radiotherapy (RT) regimens followed by supportive therapy. The supportive care including pain medications, airway protection and feeding tubes are most commonly provided for such patients. Assessments The PTV volumes were assessed during both the phases of RT. PTV1 and PTV2 were recorded and the difference between the two was calculated. The patients were assessed clinically before each phase of split course of RT. If there was increase in the volume (in cc) of PTV2 compared to PTV1, the disease was defined as progressive disease. If there was no increase in the volume when compared to PTV1, the disease was defined as stable disease. If there was decrease in volume of PTV2 when to compared to PTV1 by 50% or less, it was termed as partial response. If there was a decrease in the volume of PTV2 when compared to PTV1 by 50-70%, it was termed as good response. If there was a decrease in the volume of PTV2 when compared to PTV1 by more than 70% it was termed as excellent response. After the completion of phase 2 of PASCORA the patients were followed up on a monthly basis for the first 3 months and 3 monthly subsequently. The patients were assessed for symptomatic relief by objective scale (Table 1) and clinically for tumour response on each follow up visit. RT toxicity profile was assessed based on Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) acute and late morbidity scoring criteria [6]. Results We analysed a total 144 patients with non metastatic HN cancers who were treated with a palliative intent from January 2014 to January 2020. Of these, 84 patients were Materials and Methods Between January 2014 to January 2020, the patients with histologically proven squamous cell carcinoma of non-metastatic HN cancer, deemed unfit to receive radical intent treatment and planned to be treated by the PASCORA regimen were identified from our RT data base. The patient’s hospital records were accessed after ETHICS committee approval from Medical Records Department (MRD) of our institute. All patients were staged using American Joint Committee on Cancer (AJCC) 7th Edition [5]. The patients demographics, tumour staging, and performance status were documented. Radiotherapy Proceduref All patients were offered RT according to the department protocol. They were immobilized in supine position with a HN mould and neck placed in neutral position. The image acquisition was done by a non-contrast CT scan of HN region, 3 mm slice thickness i.e. from vertex to 2 cm below the clavicular head. The Gross Tumour Volume (GTV) was defined as all radiologically visible gross disease including the lymph nodes. Prophylactic nodal irradiation was avoided. A margin of 1 cm was given around the GTV to generate PTV 1 (Planning Target Volume 1) in 1st split course. The patients were prescribed a radiation dose of 22.5Gy in 5 fractions to 95% iso dose and delivered over 5 consecutive days with 1 fraction per day. This was followed by a 4-week gap and a second session of 22.5Gy in 5 fractions to 95% iso dose PTV 2 (Planning Target Volume 2) in 2nd split course and was delivered over a period of 5 consecutive days with 1 fraction per day. The patients were planned by 6MV photon beams using 3DCRT technique. The dose constraint used for spinal cord was Dmax (maximum dose to spinal cord) 30Gy with EQD2 of 36.56Gy for late effects with alpha/ beta ratio of 2 (Figure 1). Statistics The statistical analysis was carried using SPSS version 26. The overall survival (OS) was defined as the time duration from the date of diagnosis of the disease till the time the patient was alive from any cause. OS was determined using Kaplan Meir survival curves. The patients who had a disease progression or recurrence were censored. Log Rank test was used to determine the differences in Survival based on several factors. The statistical significance was declared at 10%. Introduction evidence based guidelines to offer standard of care. A patient can be deemed unfit for a radical intent treatment either due to poor performance status or due to locally advanced stage at presentation. Usually such patients are offered a multitude of palliative Radiotherapy (RT) regimens followed by supportive therapy. The supportive care including pain medications, airway protection and feeding tubes are most commonly provided for such patients. However, there are no standardised RT regimens which are recommended in these patients. According to the GLOBOCON 2020 data, head and neck (HN) cancers are the most common malignancies (approximately 1.9 Lakhs cases per year) seen in India [1]. The locally advanced HNC patients are treated with a combination of surgery, radiotherapy (RT) and chemotherapy. Many randomized controlled trials have shown benefit with the use of chemoradiotherapy (CTRT) or anti Epidermal Growth Factor receptor (EGFR) therapy combined with RT resulting in better local control rates and an overall survival rates [2-4]. However, these treatment regimens are directed towards patients intended for curative intent. For patients who are not fit to undergo the proposed radical treatment there isn’t any available In our centre, we commonly practice PAlliative Split COurse Radiotherapy (PASCORA) which is 22.5 Grays (Gy) in 5 fractions on 5 days with one fraction per day. 31 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Corresponding Author: Dr. Umesh Velu Room No 8, Department of Radiotherapy, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal Udupi, 576104 India. Email: umesh.vellu@manipal.edu apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients this regimen with an alpha/beta of 10 was calculated to 65.23Gy. The biologically 2 Gy equivalent dose (EQD2) was calculated to be 54.38 Gy. Then a treatment gap of 4 weeks followed by second dose of 22.5Gy/5 fractions on 5 days with one fraction per day. In this study we have retrospectively analysed the efficacy of this regimen in patients who were unfit for radical intent treatment. 316 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Radiation Toxicity There were no deaths noted amongst the study population. RT induced toxicities are summarised in Table 6. Grade 1 dermatitis were seen in all of our patients at the end of the regimen. 2 patients developed a Grade 3 mucositis following which they required nasogastric tube insertion. 1 patient developed Grade 3 oesophageal toxicity which was managed conservatively. 1 patient developed Grade 4 dermatitis which was managed by a short hospital admission. Radiobiology The calculated Biological Equivalent Dose (BED) for 316 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Figure 1. rt Schema Representation Figure 1. rt Schema Representation Figure 1. rt Schema Representation 316 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 316 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Table 2. Patient Characteristics Number Percentage (%) Gender Male 39 79 Female 10 21 Age Less than 70 years 41 84 More than 70 years 8 16 ECOG –Performance score 1 35 71 2 10 20 3 4 9 Addiction Tobacco alone 14 28 Alcohol alone 1 2 Tobacco + Alcohol 26 53 None 8 17 Comorbidities Single 24 49 Multiple 18 37 None 7 14 Site Oral Cavity 7 14 Oropharynx 14 28 Laryngopharynx 11 22 Hypopharynx 14 28 Metastasis of Unknown Origin 3 8 Table 1. Objective Classification of Symptomatic Relief Extent of symptom relief (scale 0-100%) 0 No relief Less than 50 Partial relief 50-75 Good relief More than 75 Excellent relief Table 1. Objective Classification of Symptomatic Relief Extent of symptom relief (scale 0 100%) Unknown primary of the neck was seen in 3 patients (6%).f The various reasons for palliative intent offered to these patients were firstly, the presence of multiple co-morbidities (18 patients, 37%), secondly due to poor performance status (14 patients, 28%), thirdly patient refusal of radical intent treatment (9 patients, 19%) and finally due to advanced age i.e. >70 years (8 patients, 16%) (Table 4). All 49 patients completed the planned PASCORA regimen without any treatment delays. The PTV1 and PTV 2 were analysed and compared. 10% of our patients had increase in size of the PTV 2 when compared to PTV 1. About 31% of patients had a less than 30% reduction in size of PTV 2. 47% of our patients had a 30%-50% reduction in size of PTV 2. About 10% of our patients had a reduction of 50 to 75% in PTV 2. While 2% of our patient showed a response of more than 75%. These findings are summarised in Table 5. Clinical Response Patients with primary at oropharynx and unknown primary neck had the best clinical relief (78.57% and 100% respectively) when compared to those with hypopharynx primary (14. 28%) (p value = 0.005). Patients with N2 disease (80.95%) had better clinical relief compared to those with N3 disease (27.27%) (p value = 0.001). Response Number Percentage (%) Increase in size 5 10 <30% size reduction 15 31 30-50% reduction in size 23 47 50%-75% reduction in size 5 10 More than 75% reduction in size 1 2 Table 5. Radiological Response with respect to PTV Volumes (PTV1-PTV 2) Grade Number Percentage Grade 1/ 2 49 100 Grade 3/ 4 3 6% Table 6 . RTOG Radiation Toxicity Table 5. Radiological Response with respect to PTV Volumes (PTV1-PTV 2) Figure 2. Kaplan Meir Survival Curves based on Symptomatic Relief with Life Table Response Number Percentage (%) Increase in size 5 10 <30% size reduction 15 31 30-50% reduction in size 23 47 50%-75% reduction in size 5 10 More than 75% reduction in size 1 2 Table 5. Radiological Response with respect to PTV Volumes (PTV1-PTV 2) Grade Number Percentage Grade 1/ 2 49 100 Grade 3/ 4 3 6% Table 6 . RTOG Radiation Toxicity (1/3) patients (p value = 0.004). Patients with primary at oropharynx and unknown primary neck had the best clinical relief (78.57% and 100% respectively) when compared to those with hypopharynx primary (14. 28%) (p value = 0.005). Patients with N2 disease (80.95%) had better clinical relief compared to those with N3 disease (27.27%) (p value = 0.001). (1/3) patients (p value = 0.004). Patients with primary at oropharynx and unknown primary neck had the best clinical relief (78.57% and 100% respectively) when compared to those with hypopharynx primary (14. 28%) (p value = 0.005). Patients with N2 disease (80.95%) had better clinical relief compared to those with N3 disease (27.27%) (p value = 0.001). (1/3) patients (p value = 0.004). Patients with primary at oropharynx and unknown primary neck had the best clinical relief (78.57% and 100% respectively) when compared to those with hypopharynx primary (14. 28%) (p value = 0.005). Patients with N2 disease (80.95%) had better clinical relief compared to those with N3 disease (27.27%) (p value = 0.001). Figure 2. Survival Data The estimated median overall survival is 25 months for a median follow up of 22 months (Figure 2). On univariate analysis the overall survival was found to be influenced by site of primary tumour, nodal stage and extent of symptom relief. On Cox regression analysis, extent of symptom relief was the only significant variable with a median survival of 38 months in patients having excellent symptom relief while those with no or partial symptom relief had survival of only 3 to 8 months respectively. This was found to be statistically significant (p value = 0.000) (Table 8 and Figure 3). A patient who is offered a palliative intent of treatment usually suffer with an array of symptoms due to local infiltration of the disease. These can be pain, bleeding from primary tumour site, dysphagia, stridor & mental distress. There are no fixed set guidelines to manage a patient intended for palliative treatment. The treatment modalities offered should be able to alleviate the patient’s symptoms without causing additional morbidities or mortalities. Surgery as a palliative option for a patient is not offered since a complete excision of the lesion would not be possible without causing significant morbidity in a locally advanced malignancy [8,9]. Several trials have tried to address the palliative symptoms with chemotherapy alone [10]. Most of these trials included patients with metastasis or recurrence HN cancers and the Clinical Response Kaplan Meir Survival Curves based on Symptomatic Relief with Life Table There are several reasons due to which such patients are treated with a palliative intent. The predominant ones being multiple co morbidities, poor performance status, advanced age at presentation and patients refusal of radical intent of treatment due to socio-economic constraints. Either one or a combination of these factors play a crucial role in deeming these patient as palliative intent. f Clinical Response The clinical responses were documented based on symptomatic relief and clinical examination along with indirect laryngoscopic assessment. The findings are summarised in Table 7. In our study we found that 25% of patients had an excellent symptomatic relief (>75% relief), 26% had a good symptomatic relief, 31% had a partial symptomatic relief (<50% relief) and 18% of had no relief in their symptoms. The extent of symptom relief on univariate analysis was found to be determined to be affected by ECOG status, site of primary and nodal (N) stage. ECOG 1 patients had better symptomatic relief (23/35) when compared to ECOG 2 (1/9) and ECOG 3 treated with other regimens of palliative radiotherapy such as 20Gy in 5 fractions or 30 Gy in 10 fractions and thus excluded from the study. Finally, 60 patients planned for PASCORA regimen were included in our study. Of the 60, 11 patients were excluded from the study (3 had disease progression after 1st course of RT, 2 patients defaulted after 2 fractions of RT and 4 patients follow up data was not available). 317 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 N1 N2 N3 Total T2 0 5 5 10 T3 0 6 15 21 T4 6 9 0 15 MUO 0 1 2 3 Total 6 21 22 49 Table 3. Tumor Characteristics Reason Number Percentage Multiple Co-Morbidities 18 37% Performance status 14 28% Patients refusing Radical intent 9 19% Advanced Age 8 16% Table 4. Documented Reason for Palliative Intent Table 3. Tumor Characteristics Table 3. Tumor Characteristics The patient demographics are summarised in Table 2. The median age of patients was 61 years (36-92 years). The ECOG performance status was 2 or more in 14 patients (29%). Majority of the patients had one or more co-morbidities (86%). The most common sites were oropharynx (28%) and hypopharynx (28%) followed by laryngopharynx (22%). Table 4. Documented Reason for Palliative Intent The tumour characteristics have been elaborated in Table 3. All patients had advanced HNC cancers AJCC stage IVA - 27 (55.2%) and Stage IVB - 22 (44.8%). apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiother (1/3) patients (p value = 0.004). Discussion The 5-year survival rates in locally advanced HNC are reported to be as high as 40-60% [7]. Unfortunately not all patients however will be eligible for a curative intent. 318 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Type of response Number Percentage (%) No relief 9 18 Less than 50% relief-Partial Relief 15 31 50-75% relief- Good relief 13 26 More than 75% relief- Excellent Relief 12 25 Table 7. Symptomatic Relief Type of response Median Overall Survival in months No relief 3 Less than 50% relief-Partial Response 8 50-75% relief- Good response 26 More than 75% relief- Excellent Response 38 Overall 25 Table 8. Survival Data Based on Symptom Relief 318 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Type of response Number Percentage (%) No relief 9 18 Less than 50% relief-Partial Relief 15 31 50-75% relief- Good relief 13 26 More than 75% relief- Excellent Relief 12 25 Table 7. Symptomatic Relief Type of response Median Overall Survival in months No relief 3 Less than 50% relief-Partial Response 8 50-75% relief- Good response 26 More than 75% relief- Excellent Response 38 Overall 25 Table 8. Survival Data Based on Symptom Relief apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients Table 9. 319 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Grade III mucositis-26% Grade III dysphagia- 11% Discussion Review of Literature of the Various Regimens of Palliative Radiotherapy (PRT) Study Number of participants PRT dose regimen Results Drawbacks 1) Split course Regimens Velu et al (this study) 49 22.5Gy in 5#-->4 wk gap 22.5Gy in 5# Median Survival 25 months -No standard QOL questionnaire used for symptom relief since this was retrospective study -Objective assessment of response Paris et al [14] 33 3.7Gy BID (14.4Gy /4 Fr) for 2 daysà (3-4 weeks gap × 3 cycles) Average survival-4.5 months -Only 40-50% patients were able to complete the entire course of RT -Increase in treatment time compromised the tumor control probability Lok et al [15] 75 Median Survival-5-7 months Corry et al [16] 30 Median OS-5.7 months, PFS- 3.1 months Porceddu et al [17] 30 6Gy in 1 fraction (Fr) à3 days gap ×5 cycles if good response + 6Gy boost Objective response -80% Grade III mucositis-26% Grade III dysphagia- 11% Kancherla et al [18] 33 20Gy in 5 Frà 2 weeks gap 20Gy in 5 Fr Median OS- 9 months Murthy et al [19] 126 4Gy/ Fr every alternate day upto 8 fractions (32Gy/8Fr) if good response 20Gy in 4Fr 3.2% CR ,41%PR for primary 3.2%CR for node -Only 26% patients were eligible for the boost phase - Only 43 of the 126 patients data were available during the first follow up 2) Split Course Regimens with Chemotherapy Minnatel et al [20] 58 25Gy/10Frà 2 week gapà 25Gy/10Fr with Concurrent Inj Bleomycin Median OS-7mo 80% symptom relief Grade III mucositis -47% of patients Carascosa et al [21] 20 3.7Gy BID (14.4Gy / 4 Fr) for 2 daysà(3-4 weeks gap × 3 cycles with Inj Paclitaxel 1 hour before every course Median OS- 4 months, CR in 14% Grade 3 mucositis in 14% Gamez et al [22] 21 3.7Gy BID (14.4Gy / 4 Fr) for 2 daysà(3-4 weeks gap × 3 cycles with Inj Cetuximab or Carboplatin 1 hour before each course of RT Median OS- 7 Mo, 24% CR -35% had Grade II mucositis -Included recurrent and metastatic patients 3) Single course of PRT ( 10 or less than 10 Fractions) Mohanti et al [13] 574 20Gy in 5 Fr 4 weeks gap if >50% responseà convert to Radical dosing Median OS-6.7 Mo 100% Grade 2 mucositis in all the treated patients Saikat et al [23] 33 40Gy 10 Fr with 2 Fr/week for a total of 4 weeks Median OS -7Mo 88% symptom relief 27% of patients needed treatment breaks to recover from the toxicity Paliwal et al [24] 50 20Gy in 5 Fr 92% symptom relief after 1 month Follow Up Only 1 month follow up period Fortin et al [25] 32 25Gy in 5 Fr Median OS-6.5 Mo 14% of Grade III toxicity 4) Single course of PRT ( More than 10 Fractions) Agarwal et al [26] 110 40Gy in 16 Fr Median OS 12 Mo, CR in 10% 63% Grade III mucositis, 3% with Grade IV mucositis Grade II xerostomia 54% Al-mamgani [27] 158 50Gy in 16 Fr OS at 3 years-17%, Response rate-71% 16 patients received Induction chemotherapy Grade III mucositis- 63% Grade III dermatitis-71% -No standard QOL questionnaire used for symptom relief since this was retrospective study -Objective assessment of response -Only 40-50% patients were able to complete the entire course of RT -Increase in treatment time compromised the tumor control probability Grade III mucositis-26% Grade III dysphagia- 11% Grade III mucositis-26% Grade III dysphagia- 11% -Only 26% patients were eligible for the boost phase - Only 43 of the 126 patients data were available during the first follow up Grade III mucositis -47% of patients Grade 3 mucositis in 14% -35% had Grade II mucositis -Included recurrent and metastatic patients 63% Grade III mucositis, 3% with Grade IV mucositis Grade II xerostomia 54% 16 patients received Induction chemotherapy Grade III mucositis- 63% Grade III dermatitis-71% 319 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients Figure 3. Split course Radiotherapy Historically the most commonly used palliative radiotherapy regimen (RT0G 8502 regimen- QUAD SHOT) has been 3 cycles of 14Gy-14.8Gy given over 2 days with twice daily fractions (3.7Gy/fraction BID) followed by a gap of 3-4 weeks between each cycle. Paris et al [14], evaluated 33 patients using this regimen and reported an average survival of 4.5 months. Lok et al [15], evaluated 75 patients on this regimen and reported a median OS of 5-7 months. However, in this study about 45% of patients were non squamous cell carcinoma patients. Corry et al [16] evaluated 30 patients with this regimen and reported a median OS of 5.7 months and PFS of 3.1 months. However, only 53% of patients were able to complete all 3 cycles of QUAD shot regimen in this study. Also in the treatment QOL which was used to assess the patient’s satisfaction after the completion of 3 courses of RT, only 10 out of the 30 felt the treatment course was worthwhile. In our study all 49 patients planned for PASCORA regimen completed the planned course of radiation. Figure 3. Clinical Response vs Site of Primary response rates have been as low as 20-30% [11]. Majority of the patients (approximately 60%) in India fall under the below poverty line (BPL) category [12]. The affordability of these chemotherapy drugs thus becomes a huge challenge for them. In this scenario it is very essential to have a palliative treatment option which is more affordable and less toxic, at the same time comparable to other modalities with respect to symptom relief. RT used in a palliative setting is affordable when compared to other modalities such as surgery or chemotherapy. The tumour control rates are superior and with acceptable toxicity profile when compared to chemotherapy. Porceddu et al [17] reported a split course RT in which 30 patients were evaluated. The patients received a dose of 30Gy in 5 fractions with each fraction being at least 3 days apart. The patients who responded well received an addition dose of 6Gy as boost. They reported an objective response of 80%. But there was also a higher incidence of grade 3 mucositis 26% and grade 3 dysphagia 11%. One of the most widely used palliative RT dose regimen is 20Gy in 5 daily fractions [13]. Although it offers a good alternative to palliative chemotherapy. Discussion Clinical Response vs Site of Primary by significant symptom relief ( 51% patients having good to excellent relief and 31% having partial relief) and the same translating to an increased overall survival (26 - 38 months in good and excellent relief patients respectively). Also the advantage of using a split course regimen is that it reduces the acute radiation toxicity and yet at the same time delivers a high dose of RT to the tumour. The EQD2 in our study was calculated to be 54.38 Gy. With a gap of 4 weeks and using the radiation dose lost per day was calculated to be only 4.5Gy. Additionally the patient didn’t have to incur added charge for delivering the second palliative course, thus being cost effective. With the intent of treatment being to alleviate the symptoms rather than cure, to achieve more than 50% symptomatic relief in more than half of our study patients was significant. It was achieved without any financial burden or repeated hospital visits. With this regimen it was found that the RT dose was adequate to achieve a good tumour control rates and symptomatic relief rates. 320 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Single course Radiotherapy (more than 10 fractions) Agarwal et al [26], evaluated 110 patients with palliative RT of 40Gy in 16 fractions. They reported a PFS at 12 months to be 55.1%. Complete response (CR) was seen in 10% of patients. 63% of patients developed grade III and 3% with grade IV mucositis. It was also seen that 14% patients had grade III dermatitis and grade 2 xerostomia in 54% of the patients. Carrascosa et al [21], evaluated 20 patients in QUAD shot regimen of RT along with administration of intravenous Paclitaxel 1 hour before every course of RT. They reported the grade 3 mucositis rate of 14%. Al-mamgani et al [27] evaluated 158 patients with palliative RT of 50Gy in 16 fractions. They reported an overall response rate of 71% and OS at 3 years with 17%. It should be noted that 16 patients received induction chemotherapy. Grade III mucositis was seen in 65% of patients and grade III dermatitis was seen in 71% of patients. Gamez et al [22], analysed 21 patients with QUAD shot regimen of palliative RT along with administration of concurrent Carboplatin or Cetuximab 1 hour prior to each course of RT. Of the 21 patients 6 were metastatic malignancies, 7 had recurrent diseases. 35% of these patients had grade 2 mucositis which again is a significantly higher number. As we can see there have a wide array of RT regimens which have been offered when it comes to palliative RT in HN cancers. Each regimen has its own advantage and disadvantage which have been summarised in Table 9. Ideally a non-metastatic HN cancer patient who is treated with a palliative intent should receive a regimen which offers a good local control rate, has the least toxicity profile and results in an acceptable overall survival. From the above trials it’s clear that combining a chemotherapy regimen to a split course RT regimen results in more toxicities overshadowing the cure rates. In a financially constrained country like India the affordability of drugs like Paclitaxel, Carboplatin or Cetuximab is an issue which cannot be over looked. Moreover, these patients are unable to complete the entire regimen due to the toxicities related to the concurrent chemotherapy. The shortcomings of our study are that the patients were not evaluated for symptom relief with a standard QOL questionnaire since this was retrospective study. Hence the response evaluation maybe biased. Single course Radiotherapy (more than 10 fractions) Also an objective assessment of response evaluation by imaging was not done due to financial burden on our patients. Single course Radiotherapy (10 or less than 10 fractions) Mohanti et al [13], analysed 574 non metastatic HN cancer patients retrospectively. All patients were given a palliative RT dose of 20Gy in 5 fractions over 1 week. They reported a median OS of 6.7 months and partial response in 37% of the patients. In conclusion, with this study we would like summarize that with the PASCORA regimen of RT of 22.5Gy in 5 fractions followed by a break of 4 weeks and a second course of 22.5Gy in 5 fractions, offers a very good local control rates with acceptable level of toxicity and better overall survival rates. However, to further prove our claim a prospective trial will have to be conducted. p p Saikat et al [23], evaluated 33 inoperable HN cancer patients with a palliative RT dose of 40Gy in 10 fractions with 2 fractions per week. They reported a median overall survival of 7 months and symptomatic relief in 88% of their patients. It was observed that 27% of the patients could not completed the entire course of RT. The total duration of the treatment was 5 weeks. 27% of patients also required radiation breaks in order to recover from the toxicity. There was no comment on the actual duration of treatment in the patients who needed treatment breaks. Although tumoricidal dose was delivered, there was also the risk of treatment delays and potential increase in toxicity which would invariably translate to higher hospital admissions and more financial burden. apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients available for the first follow up. This reflects that thes RT regimens might have resulted in significant morbidity Split Course Radiotherapy with Chemotherapy available for the first follow up. This reflects that these RT regimens might have resulted in significant morbidity Fortin et al [25], in a phase 2 study evaluated 32 patients who were planned for palliative RT with a dose of 25Gy in 5 fractions. They reported a median overall survival of 6.5 months. available for the first follow up. This reflects that these RT regimens might have resulted in significant morbidity Minnatel et al [20] evaluated 58 patients with 50Gy/10 fractions of RT given over a split course regimen of 25Gy in 10 fractions with a gap of 2 weeks and then another 25Gy in 10 fractions. Injection Bleomycin 30mg was given concurrently over twice a week for first 3 weeks of treatment. They reported grade 3 mucositis in 27 of 58 patients (47%), which is a significant number considering the fact that the intent was to palliative with a lower rates of toxicity. 321 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 Split course Radiotherapy In the AIIMS trial it was seen that the patients who had a 50% or more objective regression had received a total dose of 70Gy. However, in a scenario where the patient is treated with a palliative intent, it is unsure if the dose escalation to 70Gy is warranted. Kancherla et al [18] retrospectively evaluated 33 patients who received a palliative split course RT of 20Gy in 5 fractions over a week followed by a gap of 2 weeks and then a second course of 20Gy in 5 fractions over a week. They reported a median OS of 9 months. Murthy et al [19] evaluated 126 patients with a palliative radiotherapy dose of 32 Gy in 8 fractions given over 2 weeks and patients who responded well were given an additional dose of 20Gy in 4 fractions. It was noted in this study that only 26% of the total patients planned were eligible for the additional radiotherapy. Also of note is that out of the original 126 patients only 43 patients were The biggest hurdle for using a split course regimen in a general setup is increase in the total treatment time. The traditional principles of radiobiology state that as the treatment time increases it invariably leads to a decrease in local tumour control. However excellent local control was achieved in our study as demonstrated 320 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 m Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients [Palliative chemotherapy of head and neck cancer: present status and future development]. Laryngo- Rhino- Otologie. 2006 03;85(3):172-178. https://doi. org/10.1055/s-2005-921107 23. Das S, Thomas S, Pal SK, Isiah R, John S. Hypofractionated Palliative Radiotherapy in Locally Advanced Inoperable Head and Neck Cancer: CMC Vellore Experience. Indian Journal of Palliative Care. 2013 05;19(2):93-98. https://doi. org/10.4103/0973-1075.116709 12. https://www.business-standard.com/article/economy-policy/ coronavirus-impact-over-100-million-indians-could-fall- below-poverty-line-120041700906_1.html. 24. Paliwal R, Patidar A, Walke R, Hirapara P, Jain S, Raj-Bardia M. Palliative Hypo-fractionated Radiotherapy in Locally Advanced Head and Neck Cancer with Fixed Neck Nodes. Iranian journal of cancer prevention. 2012 09 03;5:178-82. 13. Mohanti BK, Umapathy H, Bahadur S, Thakar A, Pathy S. Short course palliative radiotherapy of 20 Gy in 5 fractions for advanced and incurable head and neck cancer: AIIMS study. Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology and Oncology. 2004 06;71(3):275-280. https://doi.org/10.1016/j. radonc.2004.03.009 25. Fortin B, Khaouam N, Filion E, Nguyen-Tan PF, Bujold A, Lambert L. Palliative Radiation Therapy for Advanced Head and Neck Carcinomas: A Phase 2 Study. International Journal of Radiation Oncology, Biology, Physics. 2016 06 01;95(2):647-653. https://doi. org/10.1016/j.ijrobp.2016.01.039 14. Paris KJ, Spanos WJ, Lindberg RD, Jose B, Albrink F. Phase I-II study of multiple daily fractions for palliation of advanced head and neck malignancies. International Journal of Radiation Oncology, Biology, Physics. 1993 03 15;25(4):657-660. https://doi.org/10.1016/0360- 3016(93)90012-k 26. Agarwal JP, Nemade B, Murthy V, Ghosh-Laskar S, Budrukkar A, Gupta T, D’Cruz A, Pai P, Chaturvedi P, Dinshaw K. Hypofractionated, palliative radiotherapy for advanced head and neck cancer. Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology and Oncology. 2008 Oct;89(1):51-56. https://doi. org/10.1016/j.radonc.2008.06.007 15. Lok BH, Jiang G, Gutiontov S, Lanning RM, Sridhara S, Sherman EJ, Tsai CJ, McBride SM, Riaz N, Lee NY. Palliative head and neck radiotherapy with the RTOG 8502 regimen for incurable primary or metastatic cancers. Oral Oncology. 2015 Oct;51(10):957-962. https://doi. org/10.1016/j.oraloncology.2015.07.011 27. Al-mamgani A, Tans L, Van rooij PHE, Noever I, Baatenburg de jong RJ, Levendag PC. Hypofractionated radiotherapy denoted as the “Christie scheme”: an effective means of palliating patients with head and neck cancers not suitable for curative treatment. Acta Oncologica (Stockholm, Sweden). 2009;48(4):562-570. https://doi. org/10.1080/02841860902740899 16. Corry J, Peters LJ, Costa ID, Milner AD, Fawns H, Rischin D, Porceddu S. The ‘QUAD SHOT’--a phase II study of palliative radiotherapy for incurable head and neck cancer. Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology and Oncology. 2005 Nov;77(2):137-142. https://doi.org/10.1016/j. radonc.2005.10.008 17. m Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients and Oncology. 2007 Dec;85(3):456-462. https://doi. org/10.1016/j.radonc.2007.10.020 5. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Annals of surgical oncology. 2010 06 01;17(6):1471-1474. https://doi.org/10.1245/s10434- 010-0985-4 and Oncology. 2007 Dec;85(3):456-462. https://doi. org/10.1016/j.radonc.2007.10.020 18. Kancherla KN, Oksuz DC, Prestwich RJD, Fosker C, Dyker KE, Coyle CC, Sen M. The role of split-course hypofractionated palliative radiotherapy in head and neck cancer. Clinical Oncology (Royal College of Radiologists (Great Britain)). 2011 03;23(2):141-148. https://doi. org/10.1016/j.clon.2010.09.006 6. Cox JD, Stetz J, Pajak TF. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC). International Journal of Radiation Oncology, Biology, Physics. 1995 03 30;31(5):1341-1346. https://doi. org/10.1016/0360-3016(95)00060-C 19. Murthy V, Kumar DP, Budrukkar A, Gupta T, Ghosh- Laskar S, Agarwal J. Twice-weekly palliative radiotherapy for locally very advanced head and neck cancers. Indian Journal of Cancer. 2016 03;53(1):138-141. https://doi. org/10.4103/0019-509X.180847 7. Pulte D, Brenner H. Changes in survival in head and neck cancers in the late 20th and early 21st century: a period analysis. The Oncologist. 2010;15(9):994-1001. https://doi. org/10.1634/theoncologist.2009-0289 20. Minatel E, Gigante M, Franchin G, Gobitti C, Mascarin M, Bujor L, Barzan L, Trovò MG. Combined radiotherapy and bleomycin in patients with inoperable head and neck cancer with unfavourable prognostic factors and severe symptoms. Oral Oncology. 1998 03;34(2):119-122. https:// doi.org/10.1016/s1368-8375(97)00073-0 8. Jang DW, Teng MS, Ojo B, Genden EM. Palliative surgery for head and neck cancer with extensive skin involvement. The Laryngoscope. 2013 05;123(5):1173-1177. https://doi. org/10.1002/lary.23657 21. Carrascosa LA, Yashar CM, Paris KJ, Larocca RV, Faught SR, Spanos WJ. Palliation of pelvic and head and neck cancer with paclitaxel and a novel radiotherapy regimen. Journal of Palliative Medicine. 2007 08;10(4):877-881. https://doi. org/10.1089/jpm.2006.0192 9. Roland NJ, Bradley PJ. The role of surgery in the palliation of head and neck cancer. Current Opinion in Otolaryngology & Head and Neck Surgery. 2014 04;22(2):101-108. https:// doi.org/10.1097/MOO.0000000000000031 10. Rajendra A, Noronha V, Joshi A, Patil VM, Menon N, Prabhash K. Palliative chemotherapy in head and neck cancer: balancing between beneficial and adverse effects. Expert Review of Anticancer Therapy. 2020 01;20(1):17-29. https://doi.org/10.1080/14737140.2020.1708197 22. Gamez ME, Agarwal M, Hu KS, Lukens JN, Harrison LB. Hypofractionated Palliative Radiotherapy with Concurrent Radiosensitizing Chemotherapy for Advanced Head and Neck Cancer Using the “QUAD-SHOT Regimen”. Anticancer Research. 2017 02;37(2):685-691. https://doi. org/10.21873/anticanres.11364 11. Hennemann B. m Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients Porceddu SV, Rosser B, Burmeister BH, Jones M, Hickey B, Baumann K, Gogna K, Pullar A, Poulsen M, Holt T. Hypofractionated radiotherapy for the palliation of advanced head and neck cancer in patients unsuitable for curative treatment--”Hypo Trial”. Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology 322 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 References 1. https://gco.iarc.fr/today/data/factsheets/populations/356- india-fact-sheets.pdf. 2. Pignon J, Maître A, Maillard E, Bourhis J. Meta-analysis of chemotherapy in head and neck cancer (MACH-NC): an update on 93 randomised trials and 17,346 patients. Radiotherapy and Oncology: Journal of the European Society for Therapeutic Radiology and Oncology. 2009 07;92(1):4-14. https://doi.org/10.1016/j.radonc.2009.04.014 3. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. The New England Journal of Medicine. 2006 02 09;354(6):567-578. https://doi.org/10.1056/NEJMoa053422 3. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, Jones CU, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. The New England Journal of Medicine. 2006 02 09;354(6):567-578. https://doi.org/10.1056/NEJMoa053422 Paliwal et al [24], evaluated 50 patients treated with palliative intent with RT of 20Gy in 5 daily fractions. The patients were assessed for symptomatic relief after 1 month of treatment. They reported 92% symptomatic relief in their patients. The short follow up may not be adequate to determine the local response or radiation induced toxicity in such patients. 4. Lacas B, Bourhis J, Overgaard J, Zhang Q, Grégoire V, Nankivell M, Zackrisson B, et al. Role of radiotherapy fractionation in head and neck cancers (MARCH): an updated meta-analysis. The Lancet Oncology. 2017;18(9):1221- 1237. https://doi.org/10.1016/S1470-2045(17)30458-8 321 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4 321 apjcb.waocp.com Umesh Velum, et al: Palliative Split Course Radiotherapy in Non-Metastatic Locally Advanced Head and Neck Cancer Patients This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. This work is licensed under a Creative Commons Attribution- Non Commercial 4.0 International License. 322 Asian Pacific Journal of Cancer Biology• Vol 7• Issue 4
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IMPLEMENTATION OF THE SMART SOLAT CAMP FOR SPECIAL NEEDS STUDENTS IN YAN DISTRICT
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ABSTRACT Learning in Islamic Education is the ultimate goal in enhancing the character and faith for muslim’s students. Among the things that are emphasized in the subjects of Islamic Education is the prayer of worship. Children with special needs are not exempt from performing prayers as long as fulfilling obligations and can perform solah just as much as they can. Parents and teachers play a role in guiding children with special needs so they can perform the prayers. This study aims to examine the approaches used by teachers of Islamic education in implementation of the worship service through the Special Education Smart Prayer Camp Model which is available in the J-QAF programme at three Special Education Class Learning (SECL) in Yan district involving special education teachers of Islamic education. The servay methodology used interviews and observations to involve qualitative data. Because of that, the implementation of the J-QAF programme in Special Education is one of the government's initiatives to special needs students. The J-QAF’s programme is part of the efforts of the Special Education Division to produce human capital and further the gap education that is the goal of the Ministry of Education Malaysia. Keywords: Islamic education, solah, Special Education Smart Prayer Camp, j-QAF IMPLEMENTATION OF THE SMART SOLAT CAMP FOR SPECIAL NEEDS STUDENTS IN YAN DISTRICT aMohd Noor Akmal Bin Hashim aSekolah Kebangsaan Jelutong Barat, Georgetown, Pulau Pinang, Malaysia bUniversiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia 553 DOI : https://zenodo.org/record/6909978 Published by https://publication.seameosen.edu.my/index.php/icse/issue/view/4 © 2021 SEAMEO SEN Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs 1.1 Problems Of The Study Research problems are very important in a study that involves the reason or reason for the research is conducted. Through this study, researchers found that there are still many students with special needs who have not mastered the basics of prayer even though they are 12 and 13 years old. This matter is considered a concern because students with special needs who study in KPKP schools should have the potential to improve their abilities at least at the basic level. The main problem that arises stems from parents who play a big role in guiding and educating children to get used to performing prayers in daily life. Religious values in a child are inculcated starting from the education of parents from birth. As the Malay saying goes, let the curved bamboo shoots. Parents who begin to shape their children to either be pious human beings or vice versa (Zainuddin & Norazmah 2011). Due to this problem, it was found that the implementation of the Bestari Solat Special Education Camp in schools experienced several constraints, among others through the factor of the arrival of students who did not attend the Bestari Solat Camp program. This may be due to the perception of the parents of students with special needs themselves who assume that children with special needs do not need prayer and are guaranteed heaven. Prayer is a pillar of religion. Thus, every Muslim individual should carry out the duties that have been commanded by Allah s.w.t. Prayer acts as a strengthening wall to the awakening of Islam. In addition, the success of a religion depends on the strength of its adherents' worship. Every Muslim is obliged to establish prayers even in any physical situation, place and atmosphere, so that in war situations it is also obligatory to establish prayers (Al-Ghazali 1994). Similarly, those involving the limitations of a person's physical and mental condition, such as students with special needs, are not spared in fulfilling the obligation of prayer so that they also get the same or more benefits than typical children. Therefore, considering that the Ministry of Education Malaysia places great emphasis on the Special Education Smart Prayer Camp (KBS) Model Program in improving and advancing the mastery of Islamic Education subjects in Special Education. 1.1 Problems Of The Study Therefore, a detailed study is needed to identify the improvement and mastery of students with special needs through the implementation of the Special Education Smart Prayer Camp (KBS). 1. Introduction Every child is born with a different intelligence that allows the child to learn and adapt to the environment. This intelligence of the mind is based on the perfect senses and will help children explore things around them better. In order to produce knowledgeable and independent individuals, children from an early age should need a beneficial educational process because learning from such education is something that begins with stimuli received from the senses which are then processed through psychomotor and finally stored as knowledge and experience. (Mohd Sharani 2004). No less so for children with special needs, the basics and values of religion are also important to be inculcated and practiced for the purpose of enslaving themselves to the divine nature. The main basis that is highly emphasized is the practice of prayer that is symbolic to Muslims regardless of individuals with special or typical needs (Hamdi 2011). There are many problems associated with students with special needs who still do not practice prayer even at the basic level. This refers to the achievement statistics of students with special needs with learning difficulties in the subject of Islamic Education in the field of prayer worship in the teaching and facilitation session (PdPc) 553 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs the lowest and lowest with a blank achievement percentage (Examination Board 2003). the lowest and lowest with a blank achievement percentage (Examination Board 2003). 1.3 Research Questions Based on the objectives of the study, the results of this study in response to the following questions: 1. What is the approach of Special Education Islamic Education teachers in Yan District in implementing the KBS Special Education program. . What is the approach of Special Education Islamic Education teachers in Yan pp p District in implementing the KBS Special Education program. p g p p g 2. What is the teacher's perception of students' interest in the involvement of the KBS Special Education program. p p g 3. What are the problems faced by teachers for students with special needs in the implementation of the KBS Special Education model program. g 3. What are the problems faced by teachers for students with special needs in the implementation of the KBS Special Education model program. 2. Research Methodology This study uses a qualitative research approach. Special Education Islamic education teachers were selected by the researcher as respondents of the study involving three schools of Special Education Learning Class (KPKP) in Yan district, Kedah. The selected schools are in rural areas where the distance of each school is not very far, about 5 to 10 kilometers. Because the study respondents did not involve many people, the researcher used observation and interview instruments to obtain the study data. This study uses a descriptive qualitative approach. Research that uses descriptive studies aims to describe the conditions and phenomena that occur in a study (Majid 2005). The design of this study is to obtain the relationship between the variables that have been stated by the researcher. The study design that is usually used to provide a systematic description of the facts or characteristics of a population or field of choice precisely is a descriptive study design (Sidek 2005). Studies that use observation and interview methods are qualitative in nature because they are usually more accurate and state the actual situation of the study being implemented. 1.2 Objectives Of The Research This study aims to find out the practices, perceptions and problems faced by Islamic Education teachers in Special Education Learning Classes (KPKP) in implementing the Special Education Prayer Smart Camp Model Program. S C f S S The Smart Prayer Camp Model Program for Students with Special Needs in particular, the objectives of this study are: 1. To identify the practices or approaches of Special Education Islamic Education teachers in Yan District in implementing the Special Education Bestari Solat (KBS) program. 1. To identify the practices or approaches of Special Education Islamic Education teachers in Yan District in implementing the Special Education Bestari Solat (KBS) program. 2. To know the teacher's perception of students' interest in the involvement of KBS Special Education program. 554 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs 3. Identify the problems faced by teachers for students with special needs in the implementation of the KBS Special Education model program. 3. Identify the problems faced by teachers for students with special needs in the implementation of the KBS Special Education model program. 3. Findings Of The Research Table 1 discusses the background of Special Education Islamic Education teachers viewed in terms of gender, as many as 3 people (50%) are male teachers and the rest are female teachers (50%). While in terms of the age range of Special Education Islamic Education teachers is between 30 to 50 years. Overall, the majority of Islamic education teachers in special education are in the age range of 30 to 40 years (66.7%). This was followed by only 2 people (33.2%) of them aged between 41 to 50 years. In terms of position status, Special Education Islamic Education teachers with a permanent status are all inclusive (100%). However, it differs in terms of the level of approval obtained. A total of 3 (50%) Special Education Islamic Education teachers have a Diploma in Education through Postgraduate Teaching Courses, while 1 (16.7%) Special Education Islamic education teachers have a Bachelor of Education (ISMP). The rest are 2 teachers (33.3%) with a degree in Education Course Mode During Holidays (KDC). 555 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs In terms of experience teaching Islamic education in special education, only one (16.7%) teacher has 4 years of teaching experience. Then followed by 2 people (33.3%) teachers with teaching experience for more than 5 years. While a total of 3 people (50%) who have been teaching for more than 9 years. The Islamic Education teachers who teach Special Education Classes (KPKP) in this study all have academic qualifications and are holders of bachelor's degrees from various institutions of higher learning. Only one (16.7%) teacher continued his studies abroad from Al-Azhar University, Egypt while the rest with a majority of 5 people (83.3%) were students from within the country or local universities. While studying at Institutions of Higher Learning, the specialization taken by Islamic education teachers who teach in Special Education classes is Islamic Studies which is 4 people (50%), followed by specialization in Arabic only one (16.7%) and specialization in Education Islam is also one (16.7%). 3. Findings Of The Research There are 66.3% of teachers who are able to discipline students to perform prayer activities through this program. Table 2 shows the detailed information of the findings on teachers' perceptions regarding the interests of students with special needs with learning difficulties when participating in the Bestari Solat Camp. A survey of the interests of students with special needs through their participation in the Bestari Solat Camp program through the perceptions of Special Education Islamic education teachers and they also contributed ideas on approaches taken to attract students in learning the theory and practice of prayer. The majority of special education Islamic education teachers (83.3%) stated that they would observe the behavior of each student in the practice of prayer. They (66.7%) also strongly agree or agree that the use of study aids can attract students to prayer activities. In addition, all (100%) Special Education Islamic education teachers allocate more time to strive to increase the feelings of love and interest of students with special needs with learning difficulties in performing worship and prayer activities either outside or in the classroom. Table 2: Persepsi Guru Pendidikan Islam Pendidikan Khas Terhadap Minat Murid dalam Model Kem Bestari Solat Pendidikan Khas Pernyataan Tidak setuju Kurang setuju Tidak pasti Setuju Amat setuju f % f % F % f % F % Murid suka mengikuti program Kem Bestari Solat 0 0 0 0 1 16.7 1 16.7 4 66.7 Murid mendisiplinkan diri ketika aktiviti solat 16. 7 1 1 16.7 0 0 2 33.3 1 33.3 Murid-murid bersungguh- sungguh menunaikan solat 0 0 0 0 1 16.7 0 88.3 0 0 Murid-murid menyoal cara-cara menunaikan solat dengan sempurna 0 0 2 33.3 1 16.7 3 50 0 0 Murid-murid tidak berminat menunaikan solat di sekolah 1 16.7 2 33.3 1 16.7 1 16.7 1 16.7 Table 2: Persepsi Guru Pendidikan Islam Pendidikan Khas Terhadap Minat Murid dalam Model Kem Bestari Solat Pendidikan Khas The aspect of imparting knowledge (66.7%) is the main aspect that is emphasized in performing activities related to prayer. Negative reinforcement or fines are also among the approaches used by the majority of teachers (83.2%) to encourage students with special needs with learning difficulties to pray in addition to motivating refer to table 3. 3. Findings Of The Research For teachers who have a degree in Islamic Studies and Arabic is required to take a Diploma in Education at any Institute of Teacher Education as the main condition to work in the field of teaching. Table 1: Latar Belakang Guru Pendidikan Islam Pendidikan Khas Perkara F % Jantina Lelaki Perempuan 3 3 50 50 Umur 30-40 tahun 41-50 tahun 4 2 66.7 33.3 Jawatan Tetap Latihan Sandaran 6 0 0 100 0 0 Pengalaman mengajar pendidikan Islam di Pendidikan Khas 4 tahun 5-8 tahun 9 tahun ke atas 1 2 3 16.7 33.3 50 Tahap Pendidikan Tertinggi (Akademik) Diploma Ijazah Sarjana Muda 0 6 0 100 Universiti Luar Negara Tempatan 1 5 16.7 83.3 Pengkhususan Pengajian Islam Pendidikan Islam Bahasa Arab 4 1 1 66.7 16.7 16.7 Table 1: Latar Belakang Guru Pendidikan Islam Pendidikan Khas Findings obtained from the views and monitoring of Special Education Islamic education teachers on the interest of students with special needs with learning difficulties towards the Bestari Solat Camp program as a whole showed that, most (66.7%) of Islamic education teachers who teach Special Education informed that students showed interest in participating in the Bestari Camp Special Education Prayers. Teachers (83.3%) were also able to identify that the students also showed seriousness in each activity carried out in the KBS. In addition, there are a handful of students who are able to dare to ask questions if there is confusion and lack of clarity about manners and the correct way to establish prayers (50%). Teachers responded to questions about the response of students with special needs regarding their interest in the suggestions and instructions to perform the midday prayer at school before returning home. As a result of the action, there are 2 people (33.3%) Islamic education teachers told the researcher that there are students who are less interested in performing prayers in school. Therefore, the initiative to 556 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs implement the Bestari Solat Camp for students with special needs with learning difficulties can be used as an initiative for students who are less interested in praying. 3. Findings Of The Research Table 5: Problems and constraints in the Implementation of the Special Education Prayer Smart Camp Table 5: Problems and constraints in the Implementation of the Special Education Prayer Smart Camp Table 5: Problems and constraints in the Implementation of the Special Education Prayer Smart Camp 3. Findings Of The Research However, teachers try to emphasize the aspect of student appreciation and disagree that they emphasize academic achievement only in prayer activities Table 3: Pendekatan Pengajaran Solat Pernyataan Tidak setuju Kurang setuju Tidak pasti Setuju Amat setuju F % f % f % f % F % Saya lebih mengutamakan penghayatan berbanding pencapaian akademik murid dalam aktiviti solat 2 33. 3 2 33. 3 0 0 1 16. 7 1 16.7 Table 3: Pendekatan Pengajaran Solat 557 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Aspek utama yang saya tekankan dalam aktiviti solat ialah aspek pengetahuan 0 0 1 16. 7 1 16. 7 3 50 1 16.7 Saya memerhatikan perlakuan murid dalam amalan solat mereka 0 0 0 0 1 16. 7 2 33. 3 3 50 Penggunaan BBB dapat menarik minat murid terhadap aktiviti solat 0 0 1 16. 7 0 0 2 33. 3 2 33.3 Konsep peneguhan negatif sebagai antara galakan melakukan amalan solat 0 0 0 0 1 16. 7 3 50 2 33.3 Saya menyediakan peruntukan masa yang lebih bagi menarik minat murid dalam aktiviti solat 0 0 0 0 0 0 4 66. 6 2 33.3 Based on table 5, it is found that, overall, the Bestari Solat Camp for students with special needs with learning difficulties went smoothly and well according to plan. Most (83.3%) of Special Education Islamic education teachers agreed that the schedule of the Special Education Smart Prayer Camp program arranged at the school level was appropriate for the place, in addition to the number of students present and the appropriate time allocation of 66.7% also agreed. Islamic Education teachers who act as implementers are also satisfied with the support and cooperation shown by the school (100%) and help supervise the Special Education Prayer Smart Camp program such as the provision of places and facilities needed (83.3%). Cooperation from parents (66.7%) had a good impact so that they themselves better understood and helped to diversify the approach depending on the inclinations of the special needs students involved. Other teachers (50%) also play a role in the success of each activity carried out by providing direct cooperation from before, during and after the implementation of the Bestari Solat Camp. 4. Discussion And Recommendations Based on the findings of the study presented, the Bestari Solat Camp program for students with special needs has a positive impact in improving their identity and developing themselves into pious people. With the existence of this program in series at the school level, can further increase the interest of students to get involved in each activity provided. Next was a success for the teachers and the school administrators 558 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs themselves for being able to solve the complexities faced as in this study. Through the various approaches highlighted by the teachers, can provide fun to students with special needs to continue to receive all the learning provided. Each student with special needs with different abilities and cognitive is seen and scrutinized on the activities that are suitable for them so that there are no limitations of involvement and leakage of knowledge to them. Such activities are strengthening and rehabilitative activities (Mok Soon Sang 1996). themselves for being able to solve the complexities faced as in this study. Through the various approaches highlighted by the teachers, can provide fun to students with special needs to continue to receive all the learning provided. Each student with special needs with different abilities and cognitive is seen and scrutinized on the activities that are suitable for them so that there are no limitations of involvement and leakage of knowledge to them. Such activities are strengthening and rehabilitative activities (Mok Soon Sang 1996). Play activities that are suitable for students with special needs make teaching and facilitation activities (PdPc) more practical and interesting. Students with special needs will receive learning more effectively when they learn something in line with their needs and interests. Furthermore, the increased use of various learning aids can make the teaching of prayer by teachers more effective and interesting (Ahmad 2004). Islam places great emphasis on its people to be excellent human beings in this world and the hereafter. Thus, Islamic Education is not merely theoretical and practical learning, but more to the value and appreciation of every moment of life. 4. Discussion And Recommendations Teachers of Islamic Education play a major role as a connector to the da'wah and teachings delivered by the Prophet Muhammad s.a.w by the command of Allah s.w.t. As explained in this study, Islamic Education teachers who teach students with special needs with learning difficulties allocate more teaching time outside the classroom to provide exposure in getting used to istiqamah practicing prayer in daily life. Al-Ghazali (2004) gives the view that the concept of prayer needs to be clearly understood in daily life so that the prayers performed are more meaningful and accepted in charity by Allah s.w.t. Therefore, students with special needs are also not exempted in receiving the revelation of appreciation about prayer so that the prayers performed are not only ritual and behavioral. Referring to the findings of the study found, support and encouragement from various parties can increase the enthusiasm of teachers to continue to serve as dedicated educators. However, it should be noted that the shortcomings and constraints that exist must be resolved so that the matter does not recur. Preliminary planning needs to be done so that there are no financial problems in the process of implementing the Bestari Solat Camp as well as providing training courses to certain individuals so that there is no shortage of facilitators in each activity carried out. 5. Conclusion The success of a program depends on the individual who implements the program. Thus, through this study found, the Bestari Solat Camp program has a positive impact on improving the ability of students with special needs with learning difficulties. With the cooperation of all responsible parties are able to ensure the effectiveness in the implementation of the program. As a result, this study is able to achieve the desired objectives of the study. In order to reduce the limitations and burden of teachers and students with special needs, various efforts and initiatives are made to further strengthen the implementation of the Bestari Solat Camp program get strong support, both materially and morally from various parties. Continuous efforts must be maintained so that the implementation of the Special Education Smart Prayer Camp program shows an increase in cognitive ability as well as behavioral changes for students with special needs. 559 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Zainuddin Sharif & Norazmah Mohamad Roslan 2011. Faktor-faktor yang mempengaruhi remaja terlibat dalam masalah sosial di Sekolah Tunas Bakti, Sungai lereh, Melaka. Journal of Education Psychology & Counseling. 1(115- 140). Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs References Abd. Rasyid Hj.Ahmad 1995. “Aspek-aspek Pendidikan Dalam Solat”, Jurnal Usuluddin Bil. 2. Ahmad Mohd. Salleh 2004. Pendidikan Islam: Falsafah, Sejarah Dan Kaedah Pengajaran Dan Pembelajaran. Shah Alam: Penerbit Fajar Bakti Al-Ghazali, Al-Imam Abi Hamid Muhammad bin Muhammad 1994. Ihya’ Ulumuddin. Beirut: Dar al-Kheir. Asmawati Suhid, Lukman Abd Mutalib 2009. Tinjauan Terhadap Pelaksanaan Kem Bestari Solat. Journal of Islamic and Arabic Education 1(1). Bangi: Universiti Kebangsaan Malaysia. Fakulti Pendidikan 1998. Universiti Malaya. ”Isu Khas Kaedah Kualitatif dalam Penyelidikan Pendidikan”. Jurnal Pendidikan. Jil. 19, 1998. Hamdi Ishak 2011. Amalan Pengajaran Guru Pendidikan Islam Di Sekolah Kebangsaan Pendidikan Khas (Masalah Pembelajaran): Satu Kajian Kes. Tesis Doktor Falsafah, Fakulti Pendidikan: Universiti Kebangsaan Malaysia. Jabatan Pendidikan Islam Dan Moral 2005. Buku Panduan Dasar, Pelaksanaan Dan Pengurusan Kurikulum j-QAF. Kuala Lumpur: Kementerian Pendidikan Malaysia. Jabatan Pendidikan Islam dan Moral 2007. Buku Panduan Kursus Pemantapan Pelaksanaan Kurikulum Pendidikan Islam KBSR. Kuala Lumpur: Kementerian Pendidikan Malaysia. Jabatan Pendidikan Islam dan Moral 2004. Buku Panduan Pelaksanaan Model-model Pengajaran dan Pembelajaran dan Kokurikulum. Kuala Lumpur: Kementerian Pendidikan Malaysia. Kamarul Azmi Jasmi et al 2011. “Kajian Kes Penggunaan Kaedah Pengajaran dan Pembelajaran Guru Cemerlang Pendidikan Islam (GCPI) Sekolah Bandar Dan Luar Bandar: Satu Kajian Perbandingan,” Jurnal Teknologi 56. Kamarul Hazani Yusof 2011. “Pelaksanaan Program Amali Solat Dalam Perkara Asas Fardu Ain (PAFA): Kajian Di Sekolah Menengah Daerah Hulu Langat.” Kuala Lumpur: Universiti Malaya. Mohd Majid Konting 2005. Kaedah Penyelidikan Pendidikan. Kuala Lumpur: Dewan Bahasa dan Pustaka. Mohd Sharani Ahmad 2004. Psikologi Kanak-Kanak. Batu Caves: PTS Publications & Distributors Sdn. Bhd. Mok Soon Sang 1996. Pendidikan di Malaysia. Kuala Lumpur: Kumpulan Budiman. Sidek Mohd Noah 2005. Reka Bentuk Penyelidikan: Falsafah, Teori Dan Praktis (Selangor: Penerbitan Universiti Putra Malaysia). 560 Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Suseela Malakolunthu 2001. Analisis Data Kualitatif: Satu imbasan, dalam Marohaini Yusoff. Zainuddin Sharif & Norazmah Mohamad Roslan 2011. Faktor-faktor yang mempengaruhi remaja terlibat dalam masalah sosial di Sekolah Tunas Bakti, Sungai lereh, Melaka. Journal of Education Psychology & Counseling. 1(115- 140). Proceedings of the International Conference on Special Education Vol.4 (2021) / e-ISSN 2948-4731 (553-561) SEAMEO Regional Centre for Special Educational Needs Suseela Malakolunthu 2001. Analisis Data Kualitatif: Satu imbasan, dalam Marohaini Yusoff. Suseela Malakolunthu 2001. Analisis Data Kualitatif: Satu imbasan, dalam Marohaini Yusoff. Zainuddin Sharif & Norazmah Mohamad Roslan 2011. Faktor-faktor yang mempengaruhi remaja terlibat dalam masalah sosial di Sekolah Tunas Bakti, Sungai lereh, Melaka. Journal of Education Psychology & Counseling. 1(115- 140). Zainuddin Sharif & Norazmah Mohamad Roslan 2011. Faktor-faktor yang mempengaruhi remaja terlibat dalam masalah sosial di Sekolah Tunas Bakti, Sungai lereh, Melaka. Journal of Education Psychology & Counseling. 1(115- 140). 561
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miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy
Scientific reports
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To cite this version: Mathilde Sanson, Ai Vu Hong, Emmanuelle Massourides, Nathalie Bourg, Laurence Suel, et al.. miR- 379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy. Scientific Reports, 2020, 10 (1), pp.9139. ￿10.1038/s41598-020-66016-7￿. ￿inserm-02881214￿ miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy Mathilde Sanson, Ai Vu Hong, Emmanuelle Massourides, Nathalie Bourg, Laurence Suel, Fatima Amor, Guillaume Corre, Paule Bénit, Inès Barthélémy, Stéphane Blot, et al. miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy M. Sanson1, A. Vu Hong1, E. Massourides2, N. Bourg1, L. Suel1, F. Amor1, G. Corre1, P. Bé I. Barthélémy3, S. Blot3, A. Bigot4, C. Pinset2, P. Rustin5, L. Servais6,7, T. Voit8, I. Richard D. Israeli1 ✉ Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000–6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD. Duchenne muscular dystrophy (DMD) is a lethal muscle disorder with a prevalence of 4.78 100,000 male1. DMD is caused by mostly out of frame mutations in the DMD gene, located on the X chromosome, leading to a lack or drastically reduced expression of the DMD gene product, dystrophin. Deficiency of the dystrophin protein leads to the degeneration of skeletal and cardiac muscle. Whilst severely affecting motor ability, DMD also leads to premature death by respiratory or cardiac failure2,3. Synthetic glucocorticoids (GC) are currently the standard medication for DMD patients4,5. GC treatment is associated with a delayed disease progression and a reduced risk of death6. The therapeutic effect of GC in DMD is thought to act principally through the reduc- tion of inflammation7, yet the complete mechanism of action is still unknown. However, GC drugs elicit a wide range of biological responses, including undesired side effects in DMD patients undergoing long-term treatment, such as obesity, bone fragility, pubertal delay, and short stature4,6. The loss of dystrophin initiates a complex pathological cascade, which includes sarcolemma destabilization, calcium influx, activation of proteases, and mitochondrial dysfunction, leading ultimately to fiber degeneration, inflammatory reaction and repeated cycles of myofiber degeneration and regeneration. HAL Id: inserm-02881214 https://inserm.hal.science/inserm-02881214v1 Submitted on 25 Jun 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. www.nature.com/scientificreports www.nature.com/scientificreports miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy M. Sanson1, A. Vu Hong1, E. Massourides2, N. Bourg1, L. Suel1, F. Amor1, G. Corre1, P. Bé I. Barthélémy3, S. Blot3, A. Bigot4, C. Pinset2, P. Rustin5, L. Servais6,7, T. Voit8, I. Richard D. Israeli1 ✉ The dystrophic process leads to a gradual replacement of contractile cells by fibro-adipogenic tissue and a loss of regenerative capacity. The mitochondrial defect associated with DMD pathology is characterized by reduction in expression of components in the electron transfer chain, accompanied 1Généthon INSERM, UMR_S951, INTEGRARE research unit, Evry, 91000, France. 2ISTEM, Inserm UMR 861, Evry, France. 3Inserm U955-E10, IMRB, Université Paris Est, Ecole nationale vétérinaire d’Alfort, 94700, Maisons-Alfort, France. 4Center for Research in Myology UMRS974, Sorbonne Université, INSERM, Myology Institute, Paris, France. 5INSERM, UMR S1141, Hôpital Robert Debré, Paris, France. 6MDUK Oxford Neuromuscular Centre, Department of Paediatrics, University of Oxford, Oxford, UK. 7Division of Child Neurology, Centre de Références des Maladies Neuromusculaires, Department of Pediatrics, University Hospital Liège & University of Liège, Liège, Belgium. 8NIHR Great Ormond Street Hospital Biomedical Research Centre and Great Ormond Street Institute of Child Health, University College London, London, UK. ✉e-mail: israeli@genethon.fr Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ by reduced ATP production (reviewed in8. The molecular mechanism(s) of mitochondrial dysfunction in DMD are, however, not entirely understood. It is thought that the calcium influx may lead to a Ca2+ overload within the mitochondrial matrix that may in turn result in the opening of the mitochondrial permeability transition pore (mPTP) and subsequently in mitochondrial swelling and degeneration9,10. However, there is an alterna- tive hypothesis suggesting that the mitochondria dysfunction precedes the increased Ca2+ load within the orga- nelle11,12. MicroRNAs (miRNAs) are short RNA molecules with a major influence on gene expression, due to their capacity to bind to target mRNAs. By profiling miRNA in muscle biopsies, Eisenberg and colleagues demon- strated disease-specific and distinct muscle miRNA profiles13. Similarly, by profiling circulating miRNA in mouse models for muscular dystrophies, we demonstrated more recently disease-specific miRNA profiles in the blood serum of these disease models14. Specifically for DMD, most prominent is the dysregulation of the myomiRs, which are a small number of miRNAs particularly abundant in myofibers14–22. However, the myomiRs are not the only dysregulated miRNA in DMD. Indeed, we recently reported such observation in a study performed in the canine GRMD (Golden retriever muscular dystrophy) model of DMD, which reproduces the human disease in many aspects23. Results i Screening for miRNA dysregulation in muscle biopsy of the GRMD dog model. During our pre- vious investigations on the profiling of circulating miRNAs in animal models of muscular dystrophies14,20,26, we identified the dysregulation of the myomiRs, the cardiomiRs and a large number of the Dlk1-Dio3 clustered miRNAs. However, whether or not these miRNAs are also dysregulated in the dystrophic skeletal muscle remains unknown. In the absence of adequate DMD muscle biopsies, we used the GRMD dog (a Golden Retriever DMD dog model), which is a highly useful animal model for DMD disease27. The GRMD model is characterized by a large phenotypic variability. In order to take advantage of this variability, the studied cohort was composed of phenotypically distinct 2-month old GRMD dogs: severely affected GRMD dogs (n = 10), characterized by a loss of ambulation before the age of 6 months, and moderately affected GRMD dogs (n = 9), still ambulant at 6 months of age. Healthy littermates (n = 9) were used as controls (Fig. 1a). In addition, we screened two distinct skeletal muscles of the thigh: the Biceps femoris (BF) and the Sartorius cranialis (SC). Initial molecular characteri- zation of the samples was performed through analysis of the expression of a set of muscular dystrophy indicators: myosin heavy chain-8 (MYH8) for muscle regeneration, Col6A3 (a collagen isoform) for muscle fibrosis, and CD11b for muscle inflammation26 (Fig. 1b). Notably, the dysregulation of mRNA transcripts in these four sample sets correlated with the global severity of the dogs but not with the level of impairment of the two studied muscles.ih g y g p We profiled in the GRMD muscles the expression of 34 miRNAs, of which three are the myomiRs. The others 31 miRNAs were previously identified as dysregulated in the serum of the mdx mouse14 of GRMD dogs20 or DMD patients (Supplemental Table 1), and were considered by us as candidates for the regulation of muscle metabolic and mitochondrial functions. Twenty-six out of the 34 miRNAs were dysregulated (FC > 1.5; p < 0.05) in at least one of the cohort groups (Table 1). g A heat map analysis of miRNA expression clustered muscle biopsies by their GRMD phenotype (severe BF with severe SC and moderate BF with moderate SC), while samples of the same muscle from the severe and mod- erate phenotype were separated (Fig. 1c). miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy M. Sanson1, A. Vu Hong1, E. Massourides2, N. Bourg1, L. Suel1, F. Amor1, G. Corre1, P. Bé I. Barthélémy3, S. Blot3, A. Bigot4, C. Pinset2, P. Rustin5, L. Servais6,7, T. Voit8, I. Richard D. Israeli1 ✉ The dysregulation of a large number of miRNAs in the serum of the GRMD dog model was shown, including miRNAs which are abundant in the heart (cardiomiRs) and a large number of miRNAs residing in two clusters on the genomic imprinted Dlk1-Dio3 locus20.i g p In the present study, we identified one particular member of this cluster, miR-379, as highly dysregulated in muscle biopsies of the GRMD model. We further identified a downstream GC-responding signaling pathway involving the translational factor EIF4G2 and DAPIT, a peptide associated to the mitochondrial ATP synthase. It is of note that a higher expression of DAPIT mRNA correlated with a later loss of ambulation in DMD patients24. More recently, DAPIT has been identified as mutated in Leigh syndrome25, a neurological disorder with per- turbed mitochondrial functions. Thus, the signaling pathway of miR-379, EIF4G2, and DAPIT could be part of the mitochondrial dysfunction, and a target of the GC treatment effect in DMD. Results i Interestingly, in the cluster of the most highly dysregulated miRNAs, we found the Dlk1-Dio3 rmiRNAs, miR-379, miR-410, miR-431, miR-433 (Table 1 and Fig. 1c), that were previously shown to be elevated in the serum of the GRMD20. In contrast, the myomiRs miR-133a, miR-133b and miR-206 clustered together at a reduced dysregulation level (Fig. 1c). A PCA analysis, based on the expression data of all tested miRNAs, separated the healthy control from the GRMD dogs, and to a lesser extent the severe from the moderate GRMD phenotypes (Fig. 1d). In contrast, the Sartorius and Biceps muscle profiles remained mostly indistinguishable (Fig. 1e). Taken together, these results demonstrated that (1) many dysregulated miRNAs in the plasma in condition of dystrophin mutations are also dysregulated in muscles; (2) globally, the miRNA upregu- lation is stronger in moderate versus severe GRMD muscle and (3) the Dlk1-Dio3 miRNAs (miRs −379, −410, −431, −433) are among the most highly dysregulated miRNAs in the dystrophic GRMD muscle. MiR-379 represses the expression of EIF4G2 and DAPIT in human myoblasts. To our knowledge, the Dlk1-Dio3 miRNAs dysregulation is yet to be investigated in the context of muscular dystrophy. We therefore sought to select one Dlk1-Dio3 miRNAs for a detailed investigation. We decided to focus on miR-379, which was reported to be GC responsive in the liver28. This point is of interest considering the influence of GC treatment on the DMD phenotype. In addition, miR-379 is reported to be a muscle-specific stem-cell miRNA29, thus poten- tially being involved in muscle regeneration, which is an important aspect of muscular dystrophies. A graphical representation of miR-379 expression in the GRMD BF and SC muscles is shown in Fig. 2a, demonstrating high dysregulation (except in the SC muscle, severe group, FC = 2.1, p = 0.052). In addition, the upregulation of miR- 379 was found in the tibialis cranial muscle of 3-month old GRMD (non-significance due to the small number of biopsies), while, in cardiac left ventricles, miR-379 was not dysregulated (supplemental Fig. 1). Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ Figure 1. GRMD cohort and miRNA expression. 1a. Phenotype variations through ages of GRMD dogs. 1b. Fold change of normalized mRNA abundance in muscle biopsies of 2-month old dogs (n = 9–10). CD11b: CD11 Antigen-Like Family Member, Col6a: collagen 6a3; MYH8: myosin heavy chain 8; BF: Biceps Femoris; SC: Sartorius Cranialis. 1c. Results i Heat map classification of miRNA dysregulation in GRMD muscle biopsies, Bicep Femoris (BF) and Sartorius Cranialis (SC) moderate and severe phenotypes. The myomiRs and miRNAs from the Dlk1-Dio3 locus are marked by black arrows. 1d. Principal component analysis of muscle (combined SC and BF) miRNA expression in dog groups, wild type (WT), GRMD severe (SEV), and GRMD moderate (MOD). 1e. Principal component analysis of miRNA expression in GRMD muscles (combined moderate and severe group) Sartorius cranialis (SC) and biceps femoral (BF). Circles mark the aggregation of the individuals according to their group. Small circles denoted 95% confident interval of the gravity center, while the larger discontinued-line circles surrounds 95% of the dots. Fi 1 GRMD h t d iRNA i 1 Ph t i ti th h f GRMD d 1b Figure 1. GRMD cohort and miRNA expression. 1a. Phenotype variations through ages of GRMD dogs. 1b. Fold change of normalized mRNA abundance in muscle biopsies of 2-month old dogs (n = 9–10). CD11b: CD11 Antigen-Like Family Member, Col6a: collagen 6a3; MYH8: myosin heavy chain 8; BF: Biceps Femoris; SC: Sartorius Cranialis. 1c. Heat map classification of miRNA dysregulation in GRMD muscle biopsies, Biceps Femoris (BF) and Sartorius Cranialis (SC) moderate and severe phenotypes. The myomiRs and miRNAs from the Dlk1-Dio3 locus are marked by black arrows. 1d. Principal component analysis of muscle (combined SC and BF) miRNA expression in dog groups, wild type (WT), GRMD severe (SEV), and GRMD moderate (MOD). 1e. Principal component analysis of miRNA expression in GRMD muscles (combined moderate and severe group) Sartorius cranialis (SC) and biceps femoral (BF). Circles mark the aggregation of the individuals according to their group. Small circles denoted 95% confident interval of the gravity center, while the larger discontinued-line circles surrounds 95% of the dots. Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ miR name Chr. Hs Fold-change (GRMD vs WT) p-value (GRMD vs WT) Severe GRMD moderate GRMD Severe GRMD moderate GRMD Biceps f. Sartorius c. Biceps f. Sartorius c. Biceps f. Sartorius c. Biceps f. Sartorius c. Results i miR-30e-3p 1 −1.46 −1.84 −1.09 −1.18 * miR-30e-5p 1 −1.29 −2.24 1.08 1.15 miR-128–3p 2 1.19 −1.67 1.98 1.3 ** miR-149-5p 2 1.37 1.39 2.45 2.61 *** *** let-7g-5p 3 −1.02 −1.25 2.01 2.02 ** ** miR-103a-3p 5 1.36 1.9 1.63 2.48 * ** **** miR-378a-5p 5 1.03 −1.92 2.13 1.41 ** miR-133b 6 −1.27 −1.68 1.53 1.47 * * miR-206 6 −1.02 −1.42 1.58 1.55 miR-106b-3p 7 1.07 −1.07 1.71 2.23 ** miR-106b-5p 7 1.13 1.12 1.93 1.84 ** * miR-320a 8 1.06 −1.18 1.32 1.34 let-7d-5p 9 1.54 1.46 2.01 2 ** ** miR-1307-3p 10 1.42 1.44 1.96 2.54 ** ** miR-139-5p 11 1.5 −1.23 3.26 3.01 *** * miR-342-3p 14 1.3 1.14 1.93 1.89 ** * miR-379-5p 14 2.41 2.07 3.3 2.92 ** ** *** miR-410-3p 14 5.05 5.3 8.37 7.21 ** ** ** *** miR-431-5p 14 12.41 14.89 22.09 17.61 *** ** ** *** miR-433 14 4.87 4.77 7.43 6.39 ** * *** ** miR-487b-3p 14 1.29 1.2 1.37 1.42 miR-193b-3p 16 −1.2 −1.61 1.9 1.72 ** miR-484 16 −1.41 −1.3 −1.14 1.65 miR-142-3p 17 3.1 4.15 3.09 5.12 ** * ** ** miR-21-5p 17 4.83 10.18 2.63 8.28 ** ** ** **** miR-451a 17 −1.13 1.58 1 1.9 * miR-133a 18 −1.28 −1.91 1.69 1.51 * * miR-23a 19 1.32 1.55 2.21 2.51 ** ** miR-199a-3p 19 1.04 1.1 −1.05 1.42 miR-185-5p 22 −1.65 −1.36 −1.18 1.3 miR-361-3p X 1.77 1.79 2.31 2.32 * ** * miR-361-5p X 1.23 1.02 2.25 2.13 ** ** miR-98-5p X −1.05 −1.47 1.16 1.55 miR-223-3p X 3.15 8.86 1.97 6.34 * * ** Table 1. MiRNA expression in GRMD muscle biopsies. MiRNAs are classified according chromosomal location order. Fold change GRMD/control is indicated in the middle part of the table. Respective p values are indicated *p < 0.05, **p < 0.001, ***p < 0.0005, ****p < 0.0001. Table 1. MiRNA expression in GRMD muscle biopsies. MiRNAs are classified according chromosomal location order. Fold change GRMD/control is indicated in the middle part of the table. Respective p values are indicated *p < 0.05, **p < 0.001, ***p < 0.0005, ****p < 0.0001. Table 1. MiRNA expression in GRMD muscle biopsies. MiRNAs are classified according chromosomal location order. Fold change GRMD/control is indicated in the middle part of the table. Respective p values are indicated *p < 0.05, **p < 0.001, ***p < 0.0005, ****p < 0.0001. Results i We used Diana-TarBase30 and TargetScanHuman31 for a bioinformatics prediction of miR-379 targets. Among the highest bioinformatics scores, we noticed EIF4G2, which has a binding site for miR-379-5p in its 3’ UTR (Fig. 2b). EIF4G2 (also designated  DAP5 or NAT1), is a translation factor proposed to promote cap-independent IRES-driven protein translation under cellular stress conditions32. In addition, EIF4G2 has been shown to be required for early mouse embryo development33, and in a recent study, also shown to promote a mitochon- drial shift of glycolytic to oxidative phosphorylation metabolism and subsequently the differentiation capacity of human ES cells34. Interestingly, we noted also that miR-139-5p, which is another GRMD dysregulated miRNA (Table 1) was predicted to target EIF4G2 (Supplemental Fig. 2). h h h h l d bl ll 35 To investigate whether this protein is a miR-379 target, human immortalized myoblast AB1190 cells35 were transfected with miR-379 and miR-139. A western blot analysis confirmed the prediction by demonstrating the downregulation of EIF4G2 in the transfected myoblasts, not only by miR-379 but also, to a very similar level, by miR-139 (Fig. 2c,d). We then used immunoprecipitation of the Argonaut protein (IP-Ago) method (described schematically in Fig. 2e) in order to validate the direct targeting of EIF4G2 by miR-379. After transfection of AB1190 myoblasts by miR-379, an anti-Argonaut antibody was used for the isolation of the RISC complex. The immunoprecipitated samples were analyzed by an RT-qPCR analysis using the PDK1 gene as a positive control, because it is an experimentally confirmed bona fide miR-379 target36, and the UQCR10 gene as a negative control, since it has no miR-379 recognition site. Enrichment for PDK1 and EIF4G2, but not for UQCR10, confirmed the targeting in myoblasts of EIF4G2 by miR-379-5p (Fig. 2f). Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ Figure 2. miR 379 target validation, EIF4G2 and DAPIT regulation by miR-379. 2a. miR-379 e GRMD muscle biopsy (a graphical presentation of miR-379 data of Table 1, (n = 9 or 10)); BF B Sartorius cranialis. (b) Bioinformatics prediction of the binding site of miR-379 on EIF4G2 in h mouse. MiR-379-5p seed and complementary targets sequences are in respectively in blue and r expression in miR-379 and miR-139 transfected AB1190 human myoblasts, shown representati three experiments. (d) Graphical presentation of (c). (e) Schematic description of the IP-Ago m miRNA target validation. Results i Unexpectedly, in the freshly isolated satellite cells, miR-379 expression was lower in mdx compared to that in the wt mouse. We then hypothesized that differential expression pattern of miR-379 between mdx and control may evolve through differentiation. The overall morphology of the day 2 and 4 differentiated myotubes was similar between mdx and control mice-derived cultures (Supplemental Fig. 3). However, upon in vitro differentiation, we detected a significantly higher level of miR-379 in mdx than in the wt control (Fig. 3a,b). Taken together, these results demonstrate that miR-379 is expressed in the myogenic lineage, at a high level in satellite cells (lower in mdx than in the control), downregulated upon satellite cells activation and proliferation, and increasingly expressed upon myogenic differentiation, to a higher level in mdx than in the control mouse. EIF4G2 was reported to promote differentiation of ES cells34 and during embryonic development33. Accordingly, we hypothesized that EIF4G2 and DAPIT may promote myogenic differentiation. To explore this aspect, we investigated the expression patterns of EIF4G2 and DAPIT in human AB1190 myoblasts during proliferation and differentiation. RNA levels of both genes were unchanged during these two stages (Fig. 3c). By contrast, the protein levels of both genes rose sharply on proliferation phase and over 3-differentiation days (Fig. 3d,e). EIF4G2 and DAPIT protein expressions were highly correlated over this period (Fig. 3f), indicating a possible translation control of DAPIT by EIF4G2. We used muscle biopsy of the adult mouse for the detection of EIF4G2 and DAPIT expression in situ, and identified in transversal sections a mosaic expression pattern for both proteins. Importantly, enriched expression of the two proteins was detected in the same myofibers (Fig. 3g), which is in agreement with translation control of DAPIT by EIF4G2. EIF4G2 and DAPIT are required for myogenic differentiation. To explore further the participation of EIF4G2 and DAPIT in myogenic differentiation we investigated the effect of their inhibition. AB1190 myoblasts were transfected by shRNA to knockdown the expression of EIF4G2, or DAPIT, and then shifted to differenti- ation medium. It is of significance that the knockdown of either gene resulted in an inhibition of myotube for- mation in the cultured myoblasts (Fig. 4a,b). To understand the molecular basis of this inhibition, we quantified the expression of myogenic factors. We detected only moderately reduced MyoD expression in the proliferating AB1190 myoblasts when EIF4G2 was knocked down (Fig. 4c). Results i (f) IP-Ago enrichment for miR-379 targets sequences in AB1190 hum shown result of one out of two independent experiments. (g) and (h) EIF4G2 and DAPIT mRN protein expressions (2 h) in miR-379 overexpressing AB1190 myoblasts. (i,j) EIF4G2 and DAPI and protein expressions (2j) in shEIF4G2 overexpressing AB1190 myoblasts. d DAPIT regulation by miR-379. 2a. miR-37 Figure 2. miR 379 target validation, EIF4G2 and DAPIT regulation by miR-379. 2a. miR-379 expression in GRMD muscle biopsy (a graphical presentation of miR-379 data of Table 1, (n = 9 or 10)); BF Biceps femoris; SC Sartorius cranialis. (b) Bioinformatics prediction of the binding site of miR-379 on EIF4G2 in human, dog and mouse. MiR-379-5p seed and complementary targets sequences are in respectively in blue and red. (c) EIF4G2 expression in miR-379 and miR-139 transfected AB1190 human myoblasts, shown representative results of three experiments. (d) Graphical presentation of (c). (e) Schematic description of the IP-Ago method for miRNA target validation. (f) IP-Ago enrichment for miR-379 targets sequences in AB1190 human myoblasts, shown result of one out of two independent experiments. (g) and (h) EIF4G2 and DAPIT mRNA (2 g) and protein expressions (2 h) in miR-379 overexpressing AB1190 myoblasts. (i,j) EIF4G2 and DAPIT mRNA (2i) and protein expressions (2j) in shEIF4G2 overexpressing AB1190 myoblasts. Figure 2. miR 379 target validation, EIF4G2 and DAPIT regulation by miR-379. 2a. miR-379 expression in GRMD muscle biopsy (a graphical presentation of miR-379 data of Table 1, (n = 9 or 10)); BF Biceps femoris; SC Sartorius cranialis. (b) Bioinformatics prediction of the binding site of miR-379 on EIF4G2 in human, dog and mouse. MiR-379-5p seed and complementary targets sequences are in respectively in blue and red. (c) EIF4G2 expression in miR-379 and miR-139 transfected AB1190 human myoblasts, shown representative results of three experiments. (d) Graphical presentation of (c). (e) Schematic description of the IP-Ago method for miRNA target validation. (f) IP-Ago enrichment for miR-379 targets sequences in AB1190 human myoblasts, shown result of one out of two independent experiments. (g) and (h) EIF4G2 and DAPIT mRNA (2 g) and protein expressions (2 h) in miR-379 overexpressing AB1190 myoblasts. (i,j) EIF4G2 and DAPIT mRNA (2i) and protein expressions (2j) in shEIF4G2 overexpressing AB1190 myoblasts. Results i Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ After survey of the literature, we noticed that DAPIT, a mitochondrial protein (encoded by the USMG5 gene), was identified amongst potential translational targets of EIF4G2 in human ES cells (Supplemental material34). Importantly, DAPIT was shown to be expressed nearly 5 fold higher in DMD patients with late, as opposed to early loss of ambulation24. We hypothesized thus that miR-379, EIF4G2, and DAPIT may constitute a GC-responsive dysregulated pathway in DMD. We investigated whether the expression of DAPIT could be affected by miR-379. AB1190 human myoblasts were transfected by miR-379, and the mRNA and protein levels EIF4G2 and DAPIT quantified. Despite a slight upregulation of DAPIT mRNA expression in miR-379 transfected myoblasts (Fig. 2g, FC = 1.20), the protein level of DAPIT was significantly downregulated (Fig. 2h, FC = −1.69), supporting thus the hypothesis that within myoblasts DAPIT might be a translational target of EIF4G2. A miR- 379-5p binding site could not be identify in the DAPIT UTRs and our bioinformatics analysis did not predict the possibility of a direct targeting of DAPIT transcript by miR-379. Yet, it could be hypothesized that DAPIT expres- sion is somehow affected directly by miR-379-5p, rather than indirectly via translational control of EIF4G2. Yet, to validate the hypothesis that in myogenic cells DAPIT is a translational target of EIF4G2, rather than a direct target of miR-379, we transfected AB1190 cells by shRNA against EIF4G2. The mRNA level of DAPIT was slightly downregulated (Fig. 2i, FC = −1.22), and more clearly downregulated was its protein level (Fig. 2j, FC = −1.34). Myogenic expression of miR-379 EIF4G2 and DAPIT. The skeletal muscle is a complex tissue, com- posed of a wide array of cell types. The origin of miRNA dysregulation in the dystrophic muscle could be of a myogenic or of a non-myogenic linage. To investigate miR-379 expression pattern in the myogenic lineage, muscle satellite cells were sorted from the hind limb muscles of the mdx and wt control mice. Satellite cells were cultured for 4 days in proliferative media (D4 proliferation) and then for additional 5 days in differentiation condition (D5 differentiation). Mir-379 expression was quantified by quantitative PCR (Fig. 3a,b). In agreement with a recent publication37, we detected a high-level of miR-379 expression in freshly isolated satellite cells from the control wt mouse, which was sharply downregulated upon myoblast proliferation (Fig. 3b). Results i In contrast, knocking down DAPIT expression resulted in a strong induction of MyoD1, Pax3 and Myf5 (Fig. 4d). A higher modification of gene expression was detected under differentiation conditions. The expression of the myogenic differentiation-associated factors myogenin, miR-1 and miR-133 was down regulated sharply upon knocking down of EIF4G2 (Fig. 4e) and DAPIT (Fig. 4f) while MyoD expression was unchanged by knocking down EIF4G2, and upregulated by knocking down DAPIT expression. This last observation would indicate a stronger requirement of EIF4G2 rather than that of DAPIT on myotube formation. Thus, knocking down the expression of either EIF4G2 or DAPIT during myo- blast differentiation repressed myotube formation, as demonstrated by the phenotype and the downregulation of differentiation-associated gene expression. These results demonstrate that the expression of DAPIT is increased in correlation to the level of EIF4G2, both proteins are co-expressed in the same myofibers in the adult muscle and are required for myogenic differentiation. DAPIT expression and biological activity in the skeletal muscle. DAPIT is reported to be a mito- chondrial complex 5 ATP synthase associated peptide38–40. In C2C12 myoblasts, DAPIT is localized on the mito- chondria, and a higher DAPIT expression can be observed in mouse oxidative myofibers, as opposed to that found in glycolytic myofibers41. In agreement, we confirmed a mosaic expression pattern in the mouse gastroc- nemius muscle, with enriched DAPIT expression in oxidative fibers where it co-localized with the mitochondrial ATP synthase ATP5a subunit, and the oxidative enzyme Nicotinamide adenine dinucleotide (NADH) (Fig. 5a). The co-localization of DAPIT with ATP5a was confirmed in single fibers that were isolated from the gastrocne- mius muscle (Fig. 5b). Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports entificreports/ Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42 In addition mutations in DAPIT were identified in Leigh syndrome a severe neuro Figure 3.  Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. In contrast, normal mitochondrial physiology is essential for the maintenance of Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ pluripotency and the differentiation potential of pluripotent stem cells45,46, which makes iPS cells a better choice Figure 4. Myogenic effect of inhibition of EIF4G2 and DAPIT. (a,b) Knocking down of EIF4G2 and DAPIT expression delays differentiation of AB1190 myoblast. AB1190 myoblast were treated by shRNA (shScramble, shEIF4G2, shDAPIT). Cells were differentiated for 4 days. Myotubes containing 6 or more nuclei (yellow arrows in representative images in 3 h) were counted (presented graphically in b). (c,d) Muscle regulatory factors (MRF) expression in AB1190 myoblast with knocked-down expression by shRNA of EIF4G2 (c) and DAPIT (d). (e,f) Transcriptional expression of genes associated with myogenic differentiation in shRNA knock-down EIF4G2 (e) and DAPIT (f). Figure 4. Myogenic effect of inhibition of EIF4G2 and DAPIT. (a,b) Knocking down of EIF4G2 and DAPIT expression delays differentiation of AB1190 myoblast. AB1190 myoblast were treated by shRNA (shScramble, shEIF4G2, shDAPIT). Cells were differentiated for 4 days. Myotubes containing 6 or more nuclei (yellow arrows in representative images in 3 h) were counted (presented graphically in b). (c,d) Muscle regulatory factors (MRF) expression in AB1190 myoblast with knocked-down expression by shRNA of EIF4G2 (c) and DAPIT (d). (e,f) Transcriptional expression of genes associated with myogenic differentiation in shRNA knock-down EIF4G2 (e) and DAPIT (f). Figure 4. Myogenic effect of inhibition of EIF4G2 and DAPIT. (a,b) Knocking down of EIF4G2 and DAPIT expression delays differentiation of AB1190 myoblast. AB1190 myoblast were treated by shRNA (shScramble, shEIF4G2, shDAPIT). Cells were differentiated for 4 days. Myotubes containing 6 or more nuclei (yellow arrows in representative images in 3 h) were counted (presented graphically in b). (c,d) Muscle regulatory factors (MRF) expression in AB1190 myoblast with knocked-down expression by shRNA of EIF4G2 (c) and DAPIT (d). (e,f) Transcriptional expression of genes associated with myogenic differentiation in shRNA knock-down EIF4G2 (e) and DAPIT (f). pluripotency and the differentiation potential of pluripotent stem cells45,46, which makes iPS cells a better choice for mitochondrial functional analyses. We therefore selected an iPS-based experimental system for the investi- gation of mitochondrial functions. The iPS M180 cells were derived from human muscle biopsies and have the potential of mesodermal engagement and myogenic differentiation47. M180 iPS cells were transfected by shRNA against EIF4G2 (Fig. 5c,d). www.nature.com/scientificreports entificreports/ Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42 In addition mutations in DAPIT were identified in Leigh syndrome a severe neuro Figure 3.  Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. www.nature.com/scientificreports/ atu e co /sc e t c epo ts www.nature.com/scientificreports/ Figure 3. Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200µm. (b) miR-379 relative abundance in freshly Figure 3. Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. Figure 3. Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42. In addition, mutations in DAPIT were identified in Leigh syndrome, a severe neuro- logical disorder characterized by a mitochondrial dysfunction25,43. We thus hypothesized a mitochondrial mal- function in muscle cells with reduced DAPIT expression. In transformed cell lines, mitochondrial physiology often changes into a glycolytic metabolism44,45. Thus, transformed cell lines might be unsuitable for mitochondria physiological investigations. www.nature.com/scientificreports entificreports/ Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42 In addition mutations in DAPIT were identified in Leigh syndrome a severe neuro Figure 3.  Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. Following downregulation of EIF4G2, a significant downregulation of DAPIT was detected only at the protein level (DAPIT FC = −1.87, Fig. 5d) but not at the RNA level. This result confirms that, similarly to the AB1190 myoblasts, EIF4G2 controls the translation of DAPIT in M180 cells as well. pluripotency and the differentiation potential of pluripotent stem cells45,46, which makes iPS cells a better choice for mitochondrial functional analyses. We therefore selected an iPS-based experimental system for the investi- gation of mitochondrial functions. The iPS M180 cells were derived from human muscle biopsies and have the potential of mesodermal engagement and myogenic differentiation47. M180 iPS cells were transfected by shRNA against EIF4G2 (Fig. 5c,d). Following downregulation of EIF4G2, a significant downregulation of DAPIT was detected only at the protein level (DAPIT FC = −1.87, Fig. 5d) but not at the RNA level. This result confirms that, similarly to the AB1190 myoblasts, EIF4G2 controls the translation of DAPIT in M180 cells as well. y y We then tested the activity of the mitochondrial complexes 1 and 5, and found that knocking-down of either IF4G2 or DAPIT in skeletal muscle precursor cells derived from the M180 iPS cells (skMPC), resulted in a Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ Figure 5. DAPIT IHC localization, mitochondrial functional studies in M180 iPS cell DAPIT with ATP5a (mitochondrial complex 5) into oxidative fibers (NADH positive) quadriceps muscle from adult C57Bl/10 mouse. (b) Co-localization DAPIT and ATP5 from the gastrocnemius of adult C57Bl/10 mouse. (c,d) EIF4G2 and DAPIT mRNA (c (d) in EIF4G2 and DAPIT knocked-down M180 iPS-derived skeletal myoblasts (skMP complex 5 to 1 activity ratio (e) and absolute activity (f,g) in EIF4G2 and DAPIT knoc TFAM (h) and PGC-1α (i) expression in knocked-down skMPC. (j) ATP to ADP ratio (sh-DAPIT) versus control (sh-Scramble) and untreated AB1190 (n = 4, in 3 independ differentiated myotubes. Figure 5. DAPIT IHC localization, mitochondrial functional studies in M180 iPS cells. (a) Co-localization of DAPIT with ATP5a (mitochondrial complex 5) into oxidative fibers (NADH positive) in transversal section of quadriceps muscle from adult C57Bl/10 mouse. (b) Co-localization DAPIT and ATP5a in single fibers isolated from the gastrocnemius of adult C57Bl/10 mouse. (c,d) EIF4G2 and DAPIT mRNA (c) and protein expressions (d) in EIF4G2 and DAPIT knocked-down M180 iPS-derived skeletal myoblasts (skMPC). Discussion Following the identification of a large number of dysregulated miRNAs in plasma samples from DMD patients and GRMD dogs, we have now demonstrated the dysregulation of many of these miRNAs, including Dlk1-Dio3 miRNAs, within the skeletal muscle, using the GRMD dog as a model of DMD. Furthermore, we have shown that the Dlk1-Dio3 miRNAs are not only dysregulated, but are in fact among the most highly dysregulated miRNAs in the GRMD dystrophic muscle, to a higher level than the myomiRs. This observation suggests an as yet unrec- ognized significant participation of these miRNAs in the dystrophic process. Of these Dlk1-Dio3 miRNAs, we selected miR-379 for further investigation. We showed subsequently that this miR is expressed within the myo- genic lineage. Further investigation demonstrated an axis where miR-379 controls the expression of the EIF4G2 translation factor, which itself controls the translation of the mitochondrial protein DAPIT. We then demon- strated that this signaling pathway is not only dysregulated in DMD but also reversed toward normalization by a GC treatment. Importantly, we found that miR-379 and miR-139 are GC responsive following the determination of expression level in plasma samples of DMD patients. Thus, the data suggesting that miR-379 may participate in the GC response in the dystrophic muscle, linking therefore a glucocorticoid effect to DAPIT-related mitochon- drial dysfunction and highlighting a new aspect of the DMD pathophysiology. The involvement of Dlk1-Dio3 miRNAs in muscle pathophysiology. The Dlk1-Dio3 miRNAs are processed from long noncoding RNA (lncRNA) genes expressed from the maternal chromosome of the highly conserved Dlk1-Dio3 imprinted genomic locus. A critical involvement of these lncRNAs in mammalian striated muscle development was demonstrated in mice, where the deletion of the Gtl2 lncRNA resulted in skeletal mus- cle developmental defect and perinatal death48. Another study demonstrated that the Dlk1-Dio3 lncRNAs and the processed miRNAs are upregulated in the myogenic lineage by the MEF2A transcription factor and that this pathway is involved in muscle differentiation and regeneration49. In a third example, Gao et al.50 created a mouse model with deletion of the miR-379-544 cluster that presented skeletal muscle hypertrophy. Two recent inves- tigations, focused on muscle precursor cells, identified a large number of Dlk1-Dio3 miRNAs which are highly expressed in quiescent muscle satellite cells37, and participate in the control of their metabolic maturation and mitochondrial functions51. Taken together, these studies provide compelling evidence for the critical involvement of the Dlk1-Dio3 clustered miRNAs in muscle function. www.nature.com/scientificreports entificreports/ Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42 In addition mutations in DAPIT were identified in Leigh syndrome a severe neuro Figure 3.  Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. (e,f,g) Mitochondrial complex 5 to 1 activity ratio (e) and absolute activity (f,g) in EIF4G2 and DAPIT knocked-down skMPC. (h,i) TFAM (h) and PGC-1α (i) expression in knocked-down skMPC. (j) ATP to ADP ration in DAPIT knockdown (sh-DAPIT) versus control (sh-Scramble) and untreated AB1190 (n = 4, in 3 independent experiments) in vitro differentiated myotubes. Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ decreased ratio of complex 5 to complex 1 (C5/C1) activity (Fig. 5e). This decreased ratio C5/C1 was caused by conjoint reduced complex-5 and increased complex 1 activities (Fig. 5f,g respectively). Increased complex 1 activ- ity could be explained by the increased density of mitochondria in the treated cells, possibly due to compensatory mitochondria biosynthesis. In fact, we identified the upregulation of the mitochondrial transcription regulators TFAM and PGC1α (Fig. 5h,i respectively) in the shDAPIT-treated skMPCs. Such increased of TFAM and PGC1α levels was not detected in the shEIF4G2 treated skMPCs. This divergence between the mitochondrial outcomes of knocking down EIF4G2 versus DAPIT may result from the effect of EIF4G2 on the translation of other mitochon- drial Oxphos-related proteins in addition to DAPIT35. Taken together, it is possible that knocking down DAPIT expression in the skMPCs resulted in reduced complex 5 activity and compensatory mitochondrial biosynthesis.f p p y p y y Next, we investigated the effect of reduced DAPIT expression in skeletal myotubes on ATP production. DAPIT expression was knocked down in AB1190 myotubes. ATP to ADP ratio (ATP/ADP) was significantly reduced in sh-DAPIT as compared to sh-Scramble (p < 0.043, Fig. 5j), indicating that DAPIT knockdown low- ered the myotubes ATP production. This data support the hypothesis that reduced DAPIT expression in skeletal myoblasts results in reduced ATP synthesis. The pathway of miR-379, EIF4G2 and DAPIT is glucocorticoid responsive. Because miR-379 was previously demonstrated to be glucocorticoid-responsive28, and considering the effect of GC on the phenotype of DMD, we investigated whether GC can have an effect on the muscle expression of miR-379, EIF4G2 and DAPIT. For this purpose, we treated mice with a subcutaneous injection of dexamethasone (2 mg /kg, n = 11) for 5 days. The upregulation of Foxo1 indicated the expected GC response. The expression of miR-379 was significantly reduced, while the mRNA levels EIF4G2 and DAPIT were significantly increased (Fig. 6a). In agreement, the protein levels of Foxo1, EIF4G2 and DAPIT were significantly upregulated (Fig. 6b,c). www.nature.com/scientificreports entificreports/ Previous investigation demonstrated that in vitro knockdown of DAPIT expression in Hela cells resulted in reduced ATP production42 In addition mutations in DAPIT were identified in Leigh syndrome a severe neuro Figure 3.  Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse. Lastly, we evaluated the GC response of miR-139 and miR-379 in DMD patients. In the absence of adequate muscle biopsies, we turned our attention to the circulating miRNAs. Blood plasma samples of 54 DMD and 27 healthy control patients were divided into three age groups. Of the DMD patients, half were treated by glucocorticoid. Both miR-139 and miR- 379 were found upregulated in DMD plasma and partially normalized by GC treatment (FC = 0.625, p < 0.047 and FC = 0.710, p < 0.041, respectively for miR-139 and miR-379) (Fig. 6d). Thus, in the mouse, GC treatment correlated with the reduced expression of miR-379 and with the increased levels of EIF4G2 and DAPIT in the muscle. In DMD patients, GC treatment correlated with normalized expression of miR-139 and 379 in the blood plasma. Discussion DAPIT is a mitochondrial ATP synthase non-catalytic subunit38,39, which is required for the dimerization of the ATP Synthase40. Thus, whereas DAPIT has no role in the production of ATP per se, the dimerization of mitochondrial ATP synthase is required for the shaping of the mitochondrial cristae43,56–58 and the subsequent optimal ATP production25,59. In agreement, it was shown that knocking down DAPIT in Hela cells resulted in approximately 40% reduced ATP production42. It is of particular interest that most recently a mutation in the DAPIT gene was found in Leigh syndrome, characterized by a severe neurodevelopmental regression and early childhood death, and at the cellular level by disorganized skeletal mus- cle mitochondria, reduced crista density and ATP production25,43. Important in the context of DMD disease, a 2011 paper identified higher DAPIT expression in muscle biopsies of DMD patients with a late, as compared to early, loss of walking capacity24. Taken all together, it is reasonable to hypothesize that reduced DAPIT expression may participate in mitochondrial dysfunction in the dystrophic muscle. Our data support the hypothesis that one cause of the mito- chondrial defect in DMD is the upregulation of miR-379. Accordingly, in addition to mechanical destabilization and elevated cytosolic Ca2+, an increased expression of miR-379, results in reduced expression of EIF4G2, reduced expres- sion of DAPIT, reduced ATP synthase dimerization and reduced ATP production. Thus, in agreement with a previous proposition11, our data supports a revised mitochondrial dysfunction model in DMD, in which a mitochondrial inter- nal defect in DAPIT expression and ATP production may precede and synergize with an extra-mitochondrial damage cascade (Fig. 7). reported to be expressed higher in relatively moderate as compared severe DMD patients24. DAPIT is a mitochondrial ATP synthase non-catalytic subunit38,39, which is required for the dimerization of the ATP Synthase40. Thus, whereas DAPIT has no role in the production of ATP per se, the dimerization of mitochondrial ATP synthase is required for the shaping of the mitochondrial cristae43,56–58 and the subsequent optimal ATP production25,59. In agreement, it was shown that knocking down DAPIT in Hela cells resulted in approximately 40% reduced ATP production42. It is of particular interest that most recently a mutation in the DAPIT gene was found in Leigh syndrome, characterized by a severe neurodevelopmental regression and early childhood death, and at the cellular level by disorganized skeletal mus- cle mitochondria, reduced crista density and ATP production25,43. Discussion Relatively few studies have focused on the muscle func- tions of individual miRNAs of this locus. Of these, miR-431 has been proposed as promoting the regeneration of old and of dystrophic muscles, due to the inhibition respectively of SMAD452, and Pax753. miR-410 has been proposed as a participant in diverse cardiomyopathies54. As for miR-379, apart from the observations of its dys- regulation in the serum and muscles in muscular dystrophies13,20, no previous publications have identified a role for this microRNA in muscle pathology. Dysregulation of the miR-379 EIF4G2 DAPIT mitochondria pathway in DMD disease. The mito- chondrial Oxphos respiration, which is required for myoblast differentiation55, is dysfunctional in the muscles in DMD8, which suggested the possible involvement of EIF4G2 in muscular dystrophy. Of the potential mitochondrial targets of EIF4G2, we decided to study DAPIT, because it is associated to the ATP synthase complex and has been Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 10 www.nature.com/scientificreports ntificreports/ Figure 6. Glucocorticoid response of miR-379, EIF4G2 and DAPIT. (a) Foxo1, EIF4G2 and USMG5 mRNAs (normalized to p0 (RPLP0)), and miR-379-5p (normalized to U6/miR-93/miR-16), relative abundance in the gastrocnemius muscle of C57Bl/10 (n = 11), treated by dexamethasone (D), and untreated control (C). (b) and (c), Protein expression levels of Foxo1, EIF4G2 and DAPIT (normalized to total protein) in the gastrocnemius muscle of C57Bl/10 treated by dexamethasone (D) and untreated (C). (d) miR-139-5p and miR-379-5p expression in the plasma in a human cohort (each dot represents one patient, n = 9). Healthy control (Cont), DMD patients, untreated (DMD UT), and treated by glucocorticoid (DMD GC), of the age groups 4–8, 8–12 and 12–20 years old. www.nature.com/scientificreports/ Figure 6. Glucocorticoid response of miR-379, EIF4G2 and DAPIT. (a) Foxo1, EIF4G2 and USMG5 mRNAs (normalized to p0 (RPLP0)), and miR-379-5p (normalized to U6/miR-93/miR-16), relative abundance in the gastrocnemius muscle of C57Bl/10 (n = 11), treated by dexamethasone (D), and untreated control (C). (b) and (c), Protein expression levels of Foxo1, EIF4G2 and DAPIT (normalized to total protein) in the gastrocnemius muscle of C57Bl/10 treated by dexamethasone (D) and untreated (C). (d) miR-139-5p and miR-379-5p expression in the plasma in a human cohort (each dot represents one patient, n = 9). Healthy control (Cont), DMD patients, untreated (DMD UT), and treated by glucocorticoid (DMD GC), of the age groups 4–8, 8–12 and 12–20 years old. reported to be expressed higher in relatively moderate as compared severe DMD patients24. Discussion In the modified model, presented on the image lower part, it is proposed that the absence of dystrophin induces gene-expression changes, activating miR-379 expression, reducing EIF4G2 and DAPIT expression, reducing ATP synthase activity and ATP production, (see main text for details), which might synergizes with the external Ca2+ overload, to produce mitochondrial damage. The effect of glucocorticoid treatment in DMD. The mechanism by which the GCs protect the dystrophic muscle in DMD patients is yet not completely understood. It has been suggested that immunosuppres- sive and anti-inflammatory activities may mediate the beneficial GC effects. Additionally, it was shown recently that GCs are involved in the regulation of metabolic pathways60 and accelerated sarcolemma repair61. Here, we are proposing a link between GCs and the modulation of a critical mitochondrial function. Accordingly, the GC treatment would reduce the expression of both miR-379 and miR-139, and ultimately provide a positive effect on the mitochondrial function in DMD through the cascade identified here. A previous investigation has identified the upregulation of miR-379 in the liver of a mouse model for hyperglucocorticoidemia28. It is however well documented that the glucocorticoid transcription response can vary for a given target between up and down reg- ulation in a cell type and physiological context dependent manner62,63. Of interest, a recent paper suggested that in tuberculosis patients, dexamethasone treatment provides protection not by immunosuppression (as previously proposed), but rather by reversing a signaling pathway that involves mitochondrial ATP depletion64, providing thus another example for GC reversion of mitochondrial dysfunction.i p y In summary, in the present study we identified a signaling pathway, which (1) links miR-379 to mitochondrial function, (2) is dysregulated in the dystrophic muscle, and (3) partially normalized by glucocorticoids. These findings supporting an updated model of mitochondrial dysfunction and glucocorticoid effect in DMD. Discussion Important in the context of DMD disease, a 2011 paper identified higher DAPIT expression in muscle biopsies of DMD patients with a late, as compared to early, loss of walking capacity24. Taken all together, it is reasonable to hypothesize that reduced DAPIT expression may participate in mitochondrial dysfunction in the dystrophic muscle. Our data support the hypothesis that one cause of the mito- chondrial defect in DMD is the upregulation of miR-379. Accordingly, in addition to mechanical destabilization and elevated cytosolic Ca2+, an increased expression of miR-379, results in reduced expression of EIF4G2, reduced expres- sion of DAPIT, reduced ATP synthase dimerization and reduced ATP production. Thus, in agreement with a previous proposition11, our data supports a revised mitochondrial dysfunction model in DMD, in which a mitochondrial inter- nal defect in DAPIT expression and ATP production may precede and synergize with an extra-mitochondrial damage cascade (Fig. 7). 11 Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ Figure 7. Revised model for mitochondria dysfunction in the DMD disease. Graphical model for mechanisms of mitochondrial dysfunction in the DMD disease. The Ca2+ hypothesis, which is presented on the model upper part, was proposed initially by Wrogemann and Pena at 197675. Accordingly, in the absence of dystrophin, Ca2+ entry via sarcolemma leakiness and/or activated ion channels is initiating a cytoplasmic pathological cascade (see main text for details) that culminate by the opening of the mitochondrial permeability transition pore (mPTP) and mitochondrial degeneration. In the modified model, presented on the image lower part, it is proposed that the absence of dystrophin induces gene-expression changes, activating miR-379 expression, reducing EIF4G2 and DAPIT expression, reducing ATP synthase activity and ATP production, (see main text for details), which might synergizes with the external Ca2+ overload, to produce mitochondrial damage. Figure 7. Revised model for mitochondria dysfunction in the DMD disease. Graphical model for mechanisms of mitochondrial dysfunction in the DMD disease. The Ca2+ hypothesis, which is presented on the model upper part, was proposed initially by Wrogemann and Pena at 197675. Accordingly, in the absence of dystrophin, Ca2+ entry via sarcolemma leakiness and/or activated ion channels is initiating a cytoplasmic pathological cascade (see main text for details) that culminate by the opening of the mitochondrial permeability transition pore (mPTP) and mitochondrial degeneration. Materials and Methods Animal care and use. All animals were handled according to French and European guidelines for human care and use of experimental animals. Procedures on GRMD dogs were approved by the ethical Committee Afssa/EnvA/UPEC n°CNRE-EA-16f under the number n° 12-095 / notice n° 20/12/12-19. Experiments on mice were approved by the ethical committee n°C2AE-51 of Evry and the regulatory affairs of the French Ministry of Research (MESRI) under the number APAFIS#3519. Dogs were bred and housed at the facility of the National Veterinary School of Alfort (France). Canine biopsies from the Biceps femoris and Sartorius cranialis were sam- pled at the age of 2 months on normal (n = 9), severely (n = 10) and moderately (n = 9) affected dogs. GRMD clinical stratification was based on the ambulatory status of the dogs at the age of 6 months: severe cases being characterized by a loss of ambulation before this age, and moderately affected dogs, still ambulant at the age of 6 months and usually keeping the ambulation ability65. Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ C57Bl10 (WT) and Mdx (C57BL/10ScSn-Dmdmdx/J) mice were obtained from Charles River laboratories. Mice were housed in a SPF barrier facility with 12-h light, 12-h dark cycles, and were provided with food and water ad libitum. In vivo mice experiments. Subcutaneous injections of 2 mg/kg/dose of dexamethasone (Sigma Aldrich, D2915) were performed over 5 consecutive days at 7–8 weeks of age in C57Bl/10 (n = 11) and mdx (n = 6). Mice were sacrificed the day after the last injection and skeletal muscles were frozen. RNA Expression analysis in the dog study. Total RNA was extracted from frozen muscles or cells using Trizol (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer protocol. Total RNA was quantified using a Nanodrop 8000 spectrophotometer (Labtech, Wilmington Delaware). To measure microRNA expression, 10 ng of total RNA were reverse-transcribed using Exiqon Universal cDNA Synthesis Kit II (Exiqon). Real-time PCR was performed using LightCycler 480 system (Roche, Bâle, Switzerland), with the Exiqon miRNAs assays and miRCURY LNA SYBR Green Master Mix (Exiqon) for the 34 miRNAs indi- cated in Table 1 and three normalizers. Results obtained with hsa-miR-93-5p, hsa-miR-16-5p and the U6 small nuclear RNA were used to normalize data across samples. Each experiment was performed in duplicate. Materials and Methods To measure gene expression, 1 µg of total RNA was reverse-transcribed using a mixture of random oligonucleotides and oligo-dT and RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed using LightCycler 480 system (Roche, Bâle, Switzerland) with the following Taqman Gene Expression assays (Thermo Fisher Scientific): Col6A3 (Cf02702942_m1), Cd11b (Cf02663762_m1), Myh8 (Cf02730386_m1), EIF4G2 (Hs00154952_m1), PDK1 (Hs01561847_m1), UQCR10 (Hs00912476_m1), USMG5/DAPIT (Hs00910071_g1), Myogenin (Hs01072232_m1) and MYOD1 (Hs00159528_m1) according to the manufacturer’s protocol. Results obtained with the ubiquitous acidic ribosomal phosphoprotein gene (P0/ RPLP0) were used to normalize the data across samples. The primers and Taqman probe used for P0 ampli- fication were: P0.F (5′-CTCCAAGCAGATGCAGCAGA-3′), P0.R (5′-ACCATGATGCGCAAGGCCAT-3′) and P0.P (5′-CCGTGGTGCTGATGGGCAAGAA-3′). Amplification of human Pax3 and Myf5 was per- formed using primers AGTTCCATCAGCCGCATC and TTCTTCTCGCTTTCCTCTGC for Pax3, AACCCTCAAGAGGTGTACCAC and AGGACTGTTACATTCGGGCAT for Myf5. Satellite cell isolation from mouse muscles. Satellite cells were isolated from limb muscles as follows. All hind limb muscles of 5-weeks old mice were collected and minced together in a Petri dish using scissors. The samples were put in a digestion medium [DMEM, Dispase II (3U/ml), Collagenase A (0.5 U/ml), 0.2% BSA, Pen-Strep; 10 ml for one mouse] for two hours at 37 °C with gentle shaking. Mononucleated cells were collected after passing through successive strainers (mesh diameters of 100, 70 and 40 µm) in ice-cold DMEM to eliminate fibers and debris. After washing in ice-cold PBS 0.1% BSA and centrifugation at 2000 rpm for 2 min at 4 °C, the samples were treated using 1 ml Red Blood Cell Lysis (Versalyse – A09777 Beckman Coulter) for 5–10 min at RT to eliminate the blood cells. After a final centrifugation at 2000 rpm for 2 min at 4 °C, the cells were resuspended in 1 ml ice-cold PBS 0.1% BSA before FACS sorting for CD45- (BD Biosciences 559864), Sca1- (BD Biosciences 553108), CD31-(BD Biosciences 551262), and Vcam1 + (BD Biosciences 553331) cells. Cell culture and differentiation. Myogenic cell line (AB1190, paravertebral muscle, 16 years old healthy male)35, were grown in proliferation medium (Promocell, Ref. C-23060) supplemented with 15% of fetal calf serum and 1% GlutaMAX (Thermo Scientific, Ref. 35050061). Differentiation into myotubes was performed at 80% confluence using differentiation medium (Promocell, Ref. C-23061) supplemented with the according Supplemental Mix provided by the manufacturer. Differentiated myotubes were blindly counted (n = 3, average myotubes for 5 images per transfection) on phase microscopy images. Materials and Methods The proteins were separated using a NuPAGE pre-cast 4–12% Bis-Tris gel and then transferred to nitrocellulose membrane with iBlot2 Dry Blotting system (ThermoFisher) using the 8 min30/20 V program in the case of detection of proteins with a size higher than 10 kDA. In the case of proteins with lower size, the proteins were separated using a NuPAGE pre-cast 10–20% Tricine gel and then transferred to PVDF mem- brane with iBlot2 Dry Blotting system using the 7 minutes/10 V program. Detection of proteins was performed using standard Odyssey protocol by incubation with primary specific antibodies: EIF4G2 (BD Biosciences, 610742), PDK1 (Santa Cruz, sc-293160), USMG5/DAPIT (Abcam, ab108225), Actin (Sigma Aldrich, A2066) and α-actinin (Santa Cruz, sc-15335). All antibodies were diluted between 1:2000 and 1:1000 in PBS/Odyssey 1:1 buffer. Membranes were incubated with antibodies either 2 h RT or 4 °C overnight. Western blot were revealed using Odyssey secondary antibodies donkey anti mouse (DAM) 680, donkey anti goat (DAG) 680 and don- key anti rabbit (DAR) 800 diluted 1:1000, 1H30 hour at room temperature and then scanned with the Odyssey machine. Band density was quantified using the Image Studio Lite 4.0 software (LI-COR Biosciences, Lincoln, NE). The integrated density of assayed proteins were normalized by the Alpha-actinin or alpha actin values. Ago 1/2/3 immunoprecipitation. The Ago 1/2/3 immunoprecipitation was performed using the miRNA Target IP (Active Motif, n°25500) following the manufacturer’s protocol. After immunoprecipitation, RT-qPCR was performed with Taqman Gene Expression primers of target genes of interest (PDK1, EIF4G2 and UQCR10) with P0 as a normalizer. The IP-Ago/IP-Negative control ratio was calculated for each sample, and this ratio was compared between hsa-miR-379-5p-treated samples and scramble-treated samples. A ratio higher in hsa-miR- 379-5p-treated samples for an mRNA was considered positive enrichment of the IP-Ago fraction. A ratio at least as high as the positive control one indicated that the mRNA was a target of miR-379-5p. Measure of miRNA level in human plasma. The human study (DMD patients and controls) was con- ducted according the principles of the declaration of Helsinki “ethical principles for medical research”, and was specifically approved by the ethical committee CPP Ile de France VI, on July 20, 2010, and the Comité d’Ethique (412) du CHR La Citadelle (Liège, Belgium) January 26, 2011. Samples were collected from individuals under a written informed consent of parents or legal guardians. Materials and Methods The muscle was washed twice in PBS and incubated in 1% w/v Collagenase A (Sigma, 11088793001)/DMEM for 90 minutes on an orbital shaker at 25 rpm, in 37 °C incubator. Single myofibers were released, washed in PBS, fixed immediately in 3.7% PFA for immuno-histofluorescence staining. Myofibers were permeabilised for 15 minutes in 0.3% Triton X-100 (Sigma Aldrich) and blocked in Blocking Buffer, of 5% Goat Serum (Gibco) + 10% Fetal Bovine Serum in 1X PBS, for 1 hour at room temperature. Fibers were then incubated with primary antibodies diluted in 1/10 Blocking Buffer (Usmg5, ProteIntech 17716-1-AP, 1:100; ATP5a, Abcam sc-58613, 1:100) overnight at 4 °C. Samples were washed with 1X PBS twice and incubated with Alexa-conjugated secondary antibodies diluted in 1/10 Blocking Buffer (Life Technologies, 1/1000) for 45 min at room temperature. After washing twice in 1X PBS, samples were mounted in DAPI Fluoromount-G (Southern Biotech, O100-20). Confocal microscopy images were acquired with Leica TCS-SP8, using a 63x oil objective. Mitochondrial activity measurement. The measure of activity of complexes I and V of the mitochondria respiratory chain was performed by spectrophotometry using a Cary 60 apparatus (Agilent Technologies), follow- ing a published protocol67. Each sample contained approximatively 2 million cells. For normalization, the level of protein was measured by BCA Pierce Assay (Thermo Scientific) at the end of the experiment. Complex activities were calculated according to the Beer-Lambert formula, then a ratio between the 2 activities was calculated. ATP/ADP of in vitro differentiated myotubes. ATP/ADP ratio was assayed with the ATP/ADP assay kit of Sigma (MAK135) following manufacturer’s instructions. AB1190 myoblast were grown in a low glucose (1 g/L) DMDM (Thermo-Fisher, 22320-022), 15% fetal calf serum. Confluent myoblasts were shifted to a low glucose/ low horse-serum (5%), DMEM differentiation medium, for 5 days until the appearance of myotubes. In vitro differentiated myotubes were transfected (as described in the transfections section). ATP/ADP ratio was assayed with the ATP/ADP assay kit of Sigma (MAK135) following manufacturer’s instructions. Protein analysis. Proteins were extracted from tissues or cells by cell lysis buffer (RIPA Buffer, Thermo Scientific), Protease Inhibitor Cocktails (Complete PIC, Roche) and benzonase 1:10 000 (Millipore) after homog- enization using pellet pestles in the case of tissues. The samples were prepared following the NuPAGE Gel protocol (ThermoFisher). Materials and Methods The M180 human iPS cell line, from a healthy donor (14-year-old female)47, were grown in mTESR1 medium (Stemcell, Ref. 85850) supplemented by Rock Inhibitor (Stemcell, Ref.10,72304 µM final and using Matrigel as coating (Corning, Ref. 354234).f i g g g g M180 iPS cells were differentiated toward skeletal muscle lineage (skMPC) using commercial media designed from Caron’s work66. (Skeletal Muscle Induction medium SKM01, Myoblast Cell Culture Medium SKM02, Myotube Cell Culture Medium SKM03, (AMSbio, Milton Park, Abingdon, OX14 4SE, UK)). This protocol is a 2D directed differentiation that uses 3 consecutive defined media (SKM01 from day 0 to 10, SKM02 from day 10 to 17 and SKM03 from day 17 to d25) and cell passages at day 7 and day 10. Cells were seeded at 3,500 cells/cm2 at day 0, 13,000 cells/cm2 at day 7 and 5000 cells/cm2 at day 10 on BioCoat Collagen I cultureware (356485, Corning Incorporated). Skeletal Muscle Precursor Cells M180 were frozen at day 17 of differentiation until further use. Transfection. For miR-379-5p overexpression, immortalized myoblasts were plated (80 000 cells) in 6-well plates. The day after the cells were transfected by hsa-miR-379-5p mimics (miRCURY LNA, Exiqon, Ref. 470847- 001) using Lipofectamine RNAiMAX (Thermo Scientific, Ref. 13778075) at 0.5, 1 or 5 nM final concentrations, for 48 hours until analysis. A scramble LNA was used as negative control (Negative Control 5, miRCURY LNA, Exiqon, Ref. 479904-001). For gene inhibition experiments, cells were transfected by MISSIONesiRNA for human EIF4G2 and USMG5 (DAPIT) (Sigma Aldrich, Ref. EHU032391-50UG and EHU103831-50UG, respectively) at 10, 15 or 30 nM, using Lipofectamine RNAiMAX (Thermofisher 13778150). The negative control was the universal-1 negative control (MISSION = , Sigma Aldrich, Ref. SIC001-5×1NMOL). For the transfection of in vitro differen- tiated myotubes (for the ATP/ADP quantification experiment), five-day in vitro differentiated AB1190 myotubes were transfected (Lipofectamine RNAiMAX Thermofisher 13778150) with either human Si-USMG5 (Dapit) or Si-scramble, at a final concentration 30 nM, for 48 hours. Dapit knockdown (60%) was confirmed by RT-qPCR. Histological staining and labelling of muscle sections. Mouse muscles (psoas, tibialis anterior, quadri- ceps, gastrocnemius and diaphragm) were sampled and frozen in isopentane cooled in liquid nitrogen. Transverse Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 13 www.nature.com/scientificreports/ cryosections (8–10 µm) were prepared from frozen muscles and were processed for hematoxylin-phloxine-saffron (HPS) histological staining. Immunostaining of single myofibers. Gastrocnemius muscles from 10-week old C57Bl/10 mice were dissected from tendon to tendon. Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 Materials and Methods DMD patients and healthy controls were admitted from 10 European medical centers, two of them from Belgium, one from Romania, and seven from France. The entire studied miRNA-sequencing cohort was composed of 54 DMD patients and 27 healthy controls (N = 81) divided into 9 groups of 9 subjects, composed of three age groups (4–8, 8–12, and 12–20 years old), of DMD patients treated by GCs, of untreated DMD patients, and of age matching healthy controls. In the younger age group (4–8 years old), the same donors contributed to the GC-treated and untreated samples, with untreated samples obtained before and treated samples after their first GC treatment, with less than 6-month interval. Peripheral blood samples were collected into 5 ml K3EDTA tubes (Greiner Bio-One). Plasma was separated from buffy coat and red blood cells after 10 minutes centrifugation at 1800 g and stored at −80 °C. Three hundred µl human plasma were used for RNA extractions, employing the mirVana PARIS kit (ThermoFisher). MiRNA sequencing was performed by Integragen (Evry, France). Library cloning was modified from68. Libraries were quantified by qPCR, to load precisely 7pM pool per line of HiSeq Flow-Cell. The HiSeq. 36b and index (barcode) sequenc- ing was done as instructed (Illumina) with a SBS V3 kit leading on 150 million passing filter clones. All clean Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 www.nature.com/scientificreports/ reads were compared to the Rfam database (http://rfam.xfam.org/), repeatmasker (http://www.repeatmasker. org/, UCSC download 01/04/2014), and the NCBI RefSeq.69, download 10/04/2014). Unique miR reads and their copy numbers were aligned with miRanalyzer online software70, using Ensembl human gene browser (genome assembly GRCh38) and (miRbase v20, June 201371). MiR count Raw data were processed for differential expres- sion analysis with the Deseq. 2 R and ggplot2 packages72. The significance of miRNAs differential expression was ranked by T-test, with a false discovery rate (FDR) correction according73. P values and fold change values were used for the classification of differential expression. reads were compared to the Rfam database (http://rfam.xfam.org/), repeatmasker (http://www.repeatmasker. org/, UCSC download 01/04/2014), and the NCBI RefSeq.69, download 10/04/2014). Unique miR reads and their copy numbers were aligned with miRanalyzer online software70, using Ensembl human gene browser (genome assembly GRCh38) and (miRbase v20, June 201371). MiR count Raw data were processed for differential expres- sion analysis with the Deseq. 2 R and ggplot2 packages72. Materials and Methods The significance of miRNAs differential expression was ranked by T-test, with a false discovery rate (FDR) correction according73. P values and fold change values were used for the classification of differential expression. Statistical analysis. Expression results were processed using R version 3.6.0 (2019-04-26) and PCA analysis using the FactoMineR packages74. Data for each group are represented as the means plus standard error of the mean (SEM). P-values were calculated by the Student test. Statistical significance is represented by stars: (*) for P < 0.05, (**) for P < 0.01 (***), for P < 0.001 and (****) for P < 0.0001. Received: 23 December 2019; Accepted: 11 May 2020; Published: xx xx xxxx References & Cooke, M. B. Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscle May Be Caused by Complex I Insufficiency. 1–16, https://doi.org/10.1371/journal.pone.0115763 (2014). fi 3. Eisenberg, I. et al. Distinctive patterns of microRNA expression in primary muscular disorders. Proc Natl Acad Sci USA 104 17016–17021 (2007).i et al. 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Nucleic Acids Res. 40, D130–5 (2012). 0. Acknowledgements g We are grateful to the In vivo evaluation, Imaging and Cytometry and Histology services of Genethon, Nicolas Sandoval Villegas for technical support, Umut Cagin, for stimulating discussions, and to Siân Cronin for critical reading of the manuscript. The human study was financially supported by ADNA (Advanced Diagnostics for New Therapeutic Approaches). p g The authors declare no competing interests. p g The authors declare no competing interests. References Hackenberg, M., Rodríguez-Ezpeleta, N., Aransay, A. M., Rodriguez-Ezpeleta, N. & Aransay, A. M. miRanalyzer: an update on the d d l f A h h h h l d ( ) 0. Hackenberg, M., Rodríguez-Ezpeleta, N., Aransay, A. M., Rodriguez-Ezpeleta, N. & Aransay, A. M. miRanalyzer: an update on the detection and analysis of microRNAs in high-throughput sequencing experiments. 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Lê, S., Josse, J. & Husson, F. FactoMineR: An R Package for Multivariate Analysis. J. Stat. Softw. 25, 1–18 (2008). 75. Wrogemann, K. & Pena, S. D. Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases. Lancet ( d l d) ( ) 74. Lê, S., Josse, J. & Husson, F. FactoMineR: An R Package for Multivariate Analysis. J. Stat. Softw. 25, 1–18 (2008). 75 Wrogemann K & Pena S D Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases Lancet 74. Lê, S., Josse, J. & Husson, F. FactoMineR: An R Package for Mul J J g y J ft ( ) Wrogemann, K. & Pena, S. D. Mitochondrial calcium overload: A general mechanism for cell-necrosis in muscle diseases. Lancet London, England) 1, 672–4 (1976). Scientific Reports | (2020) 10:9139 | https://doi.org/10.1038/s41598-020-66016-7 16 www.nature.com/scientificreports/ Author contributions I.R. and D.I. were responsible for experimental design and project management. M.S., A.V.H. and E.M. designed and performed experiments, and analyzed results. N.B., L.S. and F.A. performed experiments and analyzed results. G.C. analyzed results. A.B. contributed reagents. P.B., I.B., S.B., C.P., P.R., L.S. and T.V. contributed reagents, designed experiments and analyze results. M.S., I.R. and D.I. wrote the manuscript. I.R. and D.I. were responsible for experimental design and project management. M.S., A.V.H. and E.M. designed and performed experiments, and analyzed results. N.B., L.S. and F.A. performed experiments and analyzed results. G.C. analyzed results. A.B. contributed reagents. P.B., I.B., S.B., C.P., P.R., L.S. and T.V. contributed reagents, designed experiments and analyze results. M.S., I.R. and D.I. wrote the manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66016 Correspondence and requests for materials should be addressed to D.I. 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Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway
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Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway Juan Liu  Zhejiang University Min Liao  (  liaomin4545@zju.edu.cn ) Yan Yan  Zhejiang University Hui Yang  Zhejiang University Hailong Wang  Zhejiang University Jiyong Zhou  Zhejiang University Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway Juan Liu  Zhejiang University Min Liao  (  liaomin4545@zju.edu.cn ) Yan Yan  Zhejiang University Hui Yang  Zhejiang University Hailong Wang  Zhejiang University Jiyong Zhou  Zhejiang University Research Keywords: rabies virus phosphoprotein P5, beclin1, binding domain, incomplete autophagy, viral replication Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-21394/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Version of Record: A version of this preprint was published on September 18th, 2020. See the published version at https://doi.org/10.1186/s12964-020-00644-4. Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway Juan Liu  Zhejiang University Min Liao  (  liaomin4545@zju.edu.cn ) Yan Yan  Zhejiang University Hui Yang  Zhejiang University Hailong Wang  Zhejiang University Jiyong Zhou  Zhejiang University Research Keywords: rabies virus phosphoprotein P5, beclin1, binding domain, incomplete autophagy, viral replication Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-21394/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on September 18th, 2020. See the published version at https://doi.org/10.1186/s12964-020-00644-4. Research Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-21394/v3 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on September 18th, 2020. See the published version at https://doi.org/10.1186/s12964-020-00644-4. Page 1/22 Page 1/22 Abstract Background: Rabies virus (RABV) is reported to encode five phosphoproteins (P), which are involved in viral genomic replication, axonal transport, oxidative stress, interferon antagonism, and autophagy induction. However, the functions of the different P proteins are poorly understood. Methods: Immunofluorescence staining and western blot were performed to detect the autophagy activity, the form of ring-like structure, and the colocalization of BECN1 and P. Co-immunoprecipitation was performed to detect the interaction between P and BECN1. QRT-PCR and TCID50 assay were performed to detect the replication level of RABV. Small interfering RNA was used to detect the autophagy signaling pathway. Results: We found that P5 attaches to N-terminal residues 1–139 of BECN1 (beclin1) on the BECN1 ring- like structure through amino acid residues 173–222 of P5. Subsequently, we found that P5-induced autophagosomes did not fuse with lysosomes. Becn1 silencing did not recover P5 overexpression- induced promotion of RABV replication. Mechanistically, RABV protein PΔN82 (P5) induced incomplete autophagy via the BECN1-mediated signaling pathway. Conclusions: Our data indicate that P5 binding to the BECN1 ring benefits RABV replication by inducing BECN1 signaling pathway-dependent incomplete autophagy, which provides a potential target for antiviral drugs against RABV. Background Rabies is associated with severe neurological symptoms and a high mortality rate, causing over 59,000 human deaths worldwide each year [1]. Rabies virus (RABV), belonging to the Rhabdoviridae family, is a single nonsegmented negative-stranded RNA virus with genome of 12 kb. The RABV genome encodes a nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) [2]. The RABV M protein induces apoptosis by targeting mitochondria [3]. The viral protein P is a multifunctional protein that is involved in viral transcription and replication [4]. The P protein–dynein LC8 interaction is involved in the axonal transport of rabies virus along microtubules through neuronal cells [5]. The interaction of RABV P protein with complex I in mitochondria causes mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), and oxidative stress [6]. In addition, the interaction of RABV P protein with the focal adhesion kinase and nucleolin positively regulates viral replication [7]. However, RABV P protein also directly binds to the interferon-induced promyelocytic leukemia protein, which inhibits viral replication [8]. P protein’s interaction with both cell division cycle 37 (CDC37) and heat shock protein 90 (HSP90) promoted self-stability [9]. RABV P binding to beclin1 (BECN1) can induce incomplete autophagy through the caspase2 (CASP2)-mediated signaling pathways to promote viral genome replication [10]. RABV P also interacts with signal transducer and activator of transcription 1 (STAT1) to counteract interferon (IFN) signaling by creating both cytoplasmic and nuclear blocks for STAT1 [11]. RABV P protein, via an interferon antagonist interaction with activated STAT3, Page 2/22 Page 2/22 inhibits membrane glycoprotein 130 (GP130) receptor signaling to generate optimal cellular conditions for viral replication and spread [12]. The RABV P protein is phosphorylated by a RABV protein kinase and protein kinase C, forming different phosphorylated versions of the P protein [13]. The full length P and small P proteins P2, P3, P4, and P5, are translated from downstream in-frame AUG initiation codons by a leaky scanning mechanism [14]. These small P proteins have different subcellular localizations. The small P proteins P3, P4, and P5 are located in the nucleus because of the nuclear localization signal (NLS) located in the C-terminal part of the protein (amino acids 172–297), whereas the cytoplasmic distributions of P and P2 result from a CRM1 nuclear export signal (NES) in the N-terminal part of the protein [15]. Thus, the functions of these small P proteins have not been fully determined. Cells and viruses Mouse neuroblastoma N2a cells (CCL-131) and human embryonic kidney 293T cells (CRL-3216) from ATCC were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% heat-activated fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA), 100 U of penicillin mg/ml, and 100 mg of streptomycin/ml. RABV strains HEP-Flury and CVS‑11 were stored in our laboratory and were propagated in N2a cells. Briefly, N2a cells were infected with RABV at a multiplicity of infection (MOI) of 2. The infected cells were grown in fresh medium at 37°C and 5% CO2 for the indicated times. Background In the present study, we aimed to determine the function of the small P protein PΔN82 (P5). The results showed that P5 binding to the BECN1 ring-like structure induced incomplete autophagy via the BECN1 mediated signaling pathway, which promotes RABV replication. Our study highlights the role of viral protein P5 in modulating RABV replication. Confocal microscopy HEK293T or N2a cells were grown to 70% confluence on a confocal plate (In Vitro Scientific, Sunnyvale, CA, USA) and then transfected with the indicated plasmids or infected with RABV (MOI = 2) for 24 h. The cells were washed with phosphate-buffered saline (PBS), fixed, and permeabilized with 4% paraformaldehyde in PBS at 4°C for 20 min, and then incubated with the appropriate primary and secondary antibodies. The nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI). Fluorescence signals were scanned under a Zeiss LSM 780 laser confocal microscopy (Zeiss, Oberkochen, Baden- Württemberg, Germany). Plasmids construction and transfection The specific primers used to make the constructs generated in this study are listed in Table S1. The truncated P genes were amplified from the cDNA of HEP-Flury (Accession: AB085828.1) and cloned into pCMV-N-Flag (Clontech, Mountain View, CA, USA, 635688) or pCMV-N-Myc (Clontech, 635689). The truncated Becn1 genes were amplified from the cDNA of Mus musculus Becn1 (Accession: NM_019584.3) and cloned into pCMV-N-Myc. The other plasmids Flag-P (full-length) and Myc-BECN1 were stored in our laboratory. The siRNA targeting mouse Becn1 gene and shRNA targeting mouse Akt, Mtor, Ampk, Mapk genes were also synthesized by Genepharma (Suzhou, China). The siRNA or shRNA and all the plasmids were transfected into HEK293T or N2a cells using the jetPRIME kit according to the manufacturer’s instructions. Antibodies and reagents Rabbit anti-LC3A/B (4108), anti-p-AKT (Ser473) (4060), anti-AKT (4691), and anti-p-MTOR (Ser2448; 5536), anti-MTOR (2983), anti‑p‑AMPK (4185), anti- AMPK (2532), anti-p-ERK (4370), anti- ERK (4695), anti‑p-P38 (4511), and anti-P38 (9212) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-P62 (3340–1) antibodies were purchased from Epitomics (Burlingame, CA, USA). Rabbit anti-CASP2 (ab179520), anti-ULK1 (ab128859), and anti-ATG5 (ab108327) antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse anti-ATG7 (sc-376212) and anti-BECN1 (sc-48341) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-Flag (clone M2) (F1804) mAb, wortmannin (W1628), and 3-MA (M9281) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-MYC (R1208‑1) and anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (EM1101) antibodies were purchased from Huaan Biological Technology (Hangzhou, China). Mouse monoclonal antibodies (mAbs) recognizing the N/P proteins of RABV were produced in Page 3/22 Page 3/22 our laboratory [16]. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (62-6511) and Alexa Fluor 546-conjugated anti-rabbit IgG (A10036) were obtained from Thermo Scientific (Waltham, MA, USA). Secondary antibodies comprising horseradish peroxidase-labeled anti-mouse (074–1806) or anti- rabbit (074‑1506) IgG were purchased from Kirkegaard & Perry Laboratories (Millford, MA, USA). Adenovirus expressing mCherry-GFP-LC3B fusion protein (C3011), cell lysis buffer NP-40 (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40) (P0013F) and phenylmethyl sulfonylfluoride protease inhibitor (ST505) was purchased from Beyotime (Shanghai, China). Earle’s balanced salt solution (EBSS) (14155–063) was purchased from Gibco. The jetPRIME cell transfection kit (PT-114-07) was purchased from Polyplus Transfection (Sébastien Brant, Illkirch, France). QRT-PCR The qPCR conditions were as follows: 95°C for 300 s; 40 cycles of 95°C for 10 s and 60°C for 30 s; 95°C for 10 s, 65°C for 60 s and 97°C for 1 s; 37°C for 30 s. Quantitative analysis was performed using the LightCycler96 software with a relative quantification method (ΔΔCt) to analyze the levels of viral N mRNA and anti-genomic RNA. Western blotting Cells were harvested and lysed with NP-40 buffer containing phenylmethyl sulfonyl fluoride at 4°C for 2 h. Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide-gel electrophoresis and were transferred onto nitrocellulose membranes. 5% nonfat dry milk containing 0.1% Tween 20 was used to block the nonspecific binding sites for 1 h at room temperature, and then the membranes were incubated with primary antibody at 4°C overnight, followed by the appropriate secondary antibody for 1 h at room temperature. The blots were developed using a SuperSignal West Femto Substrate Trial Kit (Thermo Scientific, 34096) according to the manufacturer’s protocol. Images were captured using FluorChemm M chemiluminescent imaging system (Protein Simple, Santa Clara, CA, USA) and ImageJ Page 4/22 Page 4/22 software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the intensity of the immunoreactive protein bands. software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the intensity of the immunoreactive protein bands. Statistical analysis Statistically significant differences between groups were determined using one-way analysis of variance (ANOVA) with the Tukey Multiple Comparison Test and using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). A P value of less than 0.05 was considered statistically significant. Co-immunoprecipitation Transfected HEK293T cells were lysed for 2 h with NP-40 buffer containing 1 mM phenylmethyl sulfonylfluoride protease inhibitor at 4°C. The insoluble fractions were removed after the cell lysates were centrifuged at 12,000 g for 10 min at 4°C. The soluble fractions were incubated with the indicated antibodies overnight at 4°C. Fresh protein A/G agarose (Santa Cruz Biotechnology, sc-2003) was then added at 4°C for 8 h before washing with PBS. The eluted proteins were subjected to western blotting analysis. QRT-PCR Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses of the mRNA transcripts and anti-genomic RNA were performed as previously described [17]. Briefly, Total RNA was extracted from N2a cells using the TRIZOL Reagent (SuperfecTRITM, 3101-100; Shanghai Pufei Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions. The total RNA was reverse transcribed into cDNAs using a RevertAid RT Reverse Transcription Kit (Thermo Scientific, K1691). The qRT-PCR specific primers used are as follows: upstream primer 5′-AAGGAGTTGAATGACAGGGTGCCA-3′ and downstream primer 5′-ACT TGGGATGGTTCGAAAGGAGGA-3′ for the RABV anti-genome (115 bp in length), upstream primer 5′-AGCAGCAATGCAGTTCTTTGAGGG-3′ and downstream primer 5′- Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses of the mRNA transcripts and anti-genomic RNA were performed as previously described [17]. Briefly, Total RNA was extracted from N2a cells using the TRIZOL Reagent (SuperfecTRITM, 3101-100; Shanghai Pufei Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s instructions. The total RNA was reverse transcribed into cDNAs using a RevertAid RT Reverse Transcription Kit (Thermo Scientific, K1691). The qRT-PCR specific primers used are as follows: upstream primer 5′-AAGGAGTTGAATGACAGGGTGCCA-3′ and downstream primer 5′-ACT TGGGATGGTTCGAAAGGAGGA-3′ for the RABV anti-genome (115 bp in length), upstream primer 5′-AGCAGCAATGCAGTTCTTTGAGGG-3′ and downstream primer 5′- TTGTCAGTTCCATGCCTCCTGT CA-3′ for the RABV N gene (164 bp in length), and upstream primer 5′ - TCAACAGCAACTCCCACTCTTCCA-3′ and downstream primer5′-ACCCTGTTGCTGTAGCCGTATTCA-3′ for Gapdh gene (92 bp in length). The qPCR reaction was performed using ChamQTM Universal SYBR® qPCR Master Mix (Vazyme Biotechnology, Q711-02, Nanjing, China) and a LightCycler96 real-time PCR system (Roche, Basel, Switzerland). The qPCR conditions were as follows: 95°C for 300 s; 40 cycles of 95°C for 10 s and 60°C for 30 s; 95°C for 10 s, 65°C for 60 s and 97°C for 1 s; 37°C for 30 s. Quantitative analysis was performed using the LightCycler96 software with a relative quantification method (ΔΔCt) to analyze the levels of viral N mRNA and anti-genomic RNA. TTGTCAGTTCCATGCCTCCTGT CA-3′ for the RABV N gene (164 bp in length), and upstream primer 5′ - TCAACAGCAACTCCCACTCTTCCA-3′ and downstream primer5′-ACCCTGTTGCTGTAGCCGTATTCA-3′ for Gapdh gene (92 bp in length). The qPCR reaction was performed using ChamQTM Universal SYBR® qPCR Master Mix (Vazyme Biotechnology, Q711-02, Nanjing, China) and a LightCycler96 real-time PCR system (Roche, Basel, Switzerland). Phosphoprotein P5 forming ring-like structures induces autophagosomes accumulation Small phosphoprotein P5 contains residues aa 83-172, 173-222, and 223-297 of full-length P protein. Interestingly, a ring circle-like structure was observed in N2a cells transfected only with the P5 mutant but not in N2a cells either transfected individually with other P protein mutants or co-transfected with P5 and other P protein mutants (Fig. 1C, 2A and S2). Similarly, the ring circle-like structure was observed in N2a cells co‑transfected with both Flag-P5 and Myc-P5 (Fig. 2B), indicating that only P5 could form the ring- like structure. Moreover, the number of GFP-LC3B puncta autophagosomes surrounded by the P5 ring-like structure increased significantly in N2a cells cotransfected with GFP-LC3B and Flag-P5 in comparison with N2a cells cotransfected with Flag-vector and GFP-LC3B (Fig. 2C). Collectively, these data demonstrated that the P5 ring-like structure induced autophagosomes accumulation. Residues 173–222 of the RABV P protein form an autophagy-inducing domain Residues 173–222 of the RABV P protein form an autophagy-inducing domain Page 5/22 Our previous report discovers that the RABV P protein induces incomplete autophagy [10], however, the domain responsible for this incomplete autophagy induction is unknown. To identify the functional domain of the P protein required for autophagy induction, we constructed six truncated P protein mutants (Fig. 1A). 293T cells were transfected with the plasmids pCMV-N-Flag-tagged P, PΔC75, PΔC125, PΔN19 (P2), PΔN52 (P3), PΔN68 (P4), and PΔN82 (P5), respectively. Western blotting assay revealed that the level of endogenous LC3‑phosphatidylethanolamine conjugate (LC3-II) was dramatically increased in cells transfected with all truncated P mutants except for those transfected with PΔC125 compared with the empty vector transfected cells, and chloroquine (CQ), a lysosomal proteolysis inhibitor, as a control for autophagic flux (Fig. 1B; P < 0.05, P < 0.001). Moreover, all truncated P proteins caused no significant increases in autophagy associated proteins autophagy related (ATG)5, ATG7, Unc-51 like autophagy activating kinase 1 (ULK1), BECN1, and autophagic degradation substrate sequestome 1 (also known as P62) levels (Fig. 1B). Consistently, confocal observation also revealed green fluorescent protein (GFP)- LC3B punctas were dramatically increased in cells transfected with all truncated P mutants except for those transfected with PΔC125 compared with the empty vector transfected cells (Fig. 1C and D; P < 0.001), however, there were no significant differences in GFP-LC3B punctas caused by all truncated P proteins after these cells were treated with CQ (Fig. S1). Therefore, these data showed that a P protein containing C-terminal residues aa 173-222 was responsible for autophagic activity. The protein P5 attaches to the BECN1 ring-like structure by interaction with BECN1 We previously demonstrated that the RABV P protein could interact with BECN1 [10]. To identify whether BECN1 binding to the P protein involves P5, N2a cells were cotransfected with Myc-BECN1 and Flag-P5 or Flag-PΔC75, Flag-PΔC125, Flag‑PΔN19, Flag-PΔN52, and Flag-PΔN68, respectively. Confocal microscopy showed that the BECN1 colocalized with the full-length P and the P mutants except for PΔC125P5 mutant, notably, the P5 formed ring-like structure had stronger localization with BECN1 ring-like structure compared with P ring-like structure (Fig. 4A, and S4). Similarly, endogenous P protein colocalized with BECN1 to form the ring-like structure in RABV infected cells, and interestingly, the ring-like structure was not observed after Becn1 gene was knocked down, suggesting that BECN1 was necessary for RABV infection to form the ring-like structure (Fig. 4B). Subsequently, a co-immunoprecipitation assay (Co-IP) was performed to further analyze whether the colocalization involves protein-protein interactions. The Co- IP data demonstrated that full-length and all truncated P proteins except PΔC125 could We previously demonstrated that the RABV P protein could interact with BECN1 [10]. To identify whether BECN1 binding to the P protein involves P5, N2a cells were cotransfected with Myc-BECN1 and Flag-P5 or Flag-PΔC75, Flag-PΔC125, Flag‑PΔN19, Flag-PΔN52, and Flag-PΔN68, respectively. Confocal microscopy showed that the BECN1 colocalized with the full-length P and the P mutants except for PΔC125P5 mutant, notably, the P5 formed ring-like structure had stronger localization with BECN1 ring-like structure compared with P ring-like structure (Fig. 4A, and S4). Similarly, endogenous P protein colocalized with BECN1 to form the ring-like structure in RABV infected cells, and interestingly, the ring-like structure was not observed after Becn1 gene was knocked down, suggesting that BECN1 was necessary for RABV infection to form the ring-like structure (Fig. 4B). Subsequently, a co-immunoprecipitation assay (Co-IP) was performed to further analyze whether the colocalization involves protein-protein interactions. The Co- IP data demonstrated that full-length and all truncated P proteins except PΔC125 could immunoprecipitate BECN1, and that P5 showed stronger binding to BECN1 than the other truncated P mutants (Fig. 4C). In addition, surprisingly, P5’s binding ability to BECN1 was stronger than that of the full-length protein (Fig. 4C). To identify the P protein binding domain of BECN1, Myc-tagged truncation mutants of BECN1 (1–351aa, 139–351aa, and 139–448aa) were constructed and transfected into 293T cells (Fig. 4D). The protein P5 attaches to the BECN1 ring-like structure by interaction with BECN1 Confocal microscopy analysis showed that only the 1–351aa BECN1 mutant formed the ring-like structure, and the P protein colocalized with the ring-like structure and the 1–351aa BECN1 mutant (Fig. 4E). Further co-IP experiments showed that only 1–351aa BECN1, but not 139–351aa BECN1 and 139–448aa BECN1, interacted with P protein (Fig. 4F), revealing that first 139 N-terminal residues of BECN1 are responsible for interacting with P. Collectively, these data confirmed that RABV protein P attached to the BECN1 ring-like structure by residues 173–222 of P binding to N-terminal residues 1–139 of BECN1. Incomplete autophagic vesicles are induced by P5 protein To further investigate the relationship between autophagosomes and P5, N2a cells were cotransfected with Flag-P5 and GFP-LC3B, and labeled with LysoTracker Red. As expected, the number of GFP-LC3B puncta autophagosomes increased markedly and did not colocalize with LysoTracker Red in Flag-P5- transfected N2a cells compared with EBSS-treated N2a cells (Fig. 3A), indicating that the autophagosomes did not fuse with acidic compartments after P5 transfection. To rule out the possibility that autophagosomes fused with lysosomes but were not efficiently acidified in the transfected cells, we investigated the colocalization of GFP-LC3B with lysosomal associated membrane protein 1 (LAMP1) in Flag-P5-transfected N2a cells. GFP-LC3B puncta did not colocalize with LAMP1 in Flag-P5-transfected Page 6/22 N2a (Fig. S3). These data suggested that autophagosomes did not efficiently fuse with lysosomes in Flag‑P5‑transfected cells. In addition, we used adenovirus that expressed mCherry-GFP-LC3B, which was used to discriminate autophagosomes (expressing both mCherry and GFP fluorescent) from acidified autolysosomes (expressing red fluorescentonly) to determine the function of P5 in autophagosome maturation. N2a cells were transfected with Flag-P5 plasmids for 12 h, and infected with the adenovirus. In Flag vector transfected cells, few yellow puncta autophagosomes could be detected after adenovirus infection (Fig. 3B). In contrast, in Flag-P5 transfected cells, we observed the accumulation of yellow puncta autophagosomes but a low number of mcherry puncta autophagosomes, suggesting impaired autophagosome fusion with lysosomes. These results implied that P5 protein was responsible for the observed incomplete autophagic induction. P5 binding to the BECN1 ring-like structure regulates RABV replication via the BECN1-mediated signaling pathway To further investigate the BECN1-dependent signaling pathway through which P5 regulates RABV replication, we examined whether BECN1, AMP-activated protein kinase (AMPK), CASP2, protein kinase B (AKT), mammalian target of rapamycin (MTOR), and mitogen activated protein kinases MAPKs [extracellular signal-regulated kinase (ERK), P38] levels changed during overexpression of P5. Western blotting analysis showed that P5 dramatically upregulated the phosphorylation (p) level of AKT, MTOR, AMPK, ERK1/2, and P38, and reduced the CASP2 level; however, it did not affect the total amount of these proteins nor BECN1 levels (Fig. 6A). Moreover, we knocked down cellular Becn1 using siBecn1 to further show whether the P5 protein affected the expression of BECN1, AMPK, CASP2, AKT, MTOR, and MAPK (ERK, P38). The results showed that there was a significant downregulation of CASP2 and p-AMPK, p- AKT, p-MTOR, and p-MAPK (ERK1/2, P38) levels, and an insignificant alteration of the total amount of these proteins in Becn1-knockdown cells with viral gene P5 transfection (Fig. 6B). To investigate whether P5 regulated RABV replication depends on the downstream of BECN1-dependent signaling pathway, next we knocked down cellular Akt, Mtor, Ampk, Mapk respectively, and examined the NP expression levels in the absence or presence of P5. The results showed that there was a significant decrease of NP in absence or presence of P5, suggesting that the RABV replication was dependent of the AKT, MTOR, AMPK, MAPK proteins (Fig. 6C). Collectively, these data demonstrated that the BECN1 binding to P5 was responsible for regulating RABV replication via a BECN1-mediated signaling pathway. P5 binding to BECN1 ring-like structure promoted RABV replication Page 7/22 To determine the effect on RABV replication of P5 binding to the BECN1 ring-like structure, we investigated the dynamics of RABV infection under condition of P5 overexpression. N2a cells transfected Page 7/22 Page 7/22 with Flag-P5 for 12 h were infected with RABV. We found that the level of viral N protein, viral N mRNA, viral anti-genomic RNA, and infectious RABV progeny were all significantly increased; however, when Becn1 was knocked down using two short interfering RNAs (siBecn1), there was a detectable downregulation of viral N protein, viral N mRNA, viral anti-genomic RNA, and infectious RABV progeny in the absence or presence of P5 (Fig. 5A-E, P < 0.05, 0.01, or 0.001), suggesting a positive role of P5 in regulating RABV infection dependent of BECN1. In addition, to further confirm whether the effect of the ring-like structure on RABV replication was dependent of autophagy induction, we also detected the level of viral N protein in presence of protein P5 together with the autophagy inhibitor 3-methyladenine (3-MA), or wortmannin treatment. The results showed 3-MA or wortmannin treatment significantly inhibited the level of viral N protein compared with that in non-treated P5 group (Fig. 5F and G, P < 0.01 or 0.001). Collectively, these data demonstrated that RABV replication hijacked BECN1 by P5 binding to the BECN1 ring-like structure. Discussion Previous studies showed that full-length P protein and at least four additional shorter products P2 (PΔN19), P3 (PΔN52), P4 (PΔN68), and P5 were detected in RABV-infected cells, viral gene P transfected cells, and purified RABV virions [14]. BECN1 plays an interacting partner role for the mammalian phosphatidylinositol 3‑kinase catalytic subunit type 3 (PIK3C3) involving macroautophagy, in which it is Page 8/22 Page 8/22 an essential chaperone or adaptor [18-20]. However, in the relationship between the virus and autophagy, although it has been reported that BECN1 interacts with a virus protein to regulate autophagy, the specific domain responsible for the BECN1 interaction is not clear [21, 22]. The present study showed that BECN1 exists in a ring-like structure, and identified that among five truncated P proteins (PΔC75, PΔN19, PΔN52, PΔN68, and P5), residues 173–222 induced autophagy by interacting with N-terminal residues 1–139 of BECN1. Meanwhile, only the full-length P protein and P5 were visibly colocalized with the BECN1 ring-like structure (Fig. 4). Notably, in co-IP experiments, P5 showed stronger binding than full length P protein. Therefore, we concluded that RABV small phosphoprotein P5 is responsible for binding to the BECN1 ring‑like structure. As an essential cofactor of RABV RNA polymerase, P may participate in additional physiological processes [23]. Our previous research reports incomplete autophagy induced by the RABV phosphoprotein [10]. In this study, we demonstrated that the P proteins with, but not without, amino acid segment 173–222 are involved in increasing the level of endogenous lipidated LC3-II. In particular, the LC3-II was surrounded by the P5 protein (Figs. 1 and 2). However, P5 did not change the levels of autophagy associated proteins ATG5, ATG7, ULK1, BECN1, and P62, markedly. In addition, the P5-induced autophagosome did not colocalize with lysosomes (Figs. 1, 3 and S2). Nonetheless, we observed that P5 upregulated the phosphorylation of AMPK, MAPK (P38, ERK1/2), AKT, and MTOR, and decreased BECN1- dependent CASP2 levels (Fig. 6). Collectively, our data demonstrated that amino acid residues 173–222 of the viral P protein are responsible for inducing incomplete autophagy, and the binding of P5 to the BECN1 ring-like structure induced this incomplete autophagy by activating the BECN1 signaling pathway. Conclusions In conclusion, we identified the binding domain between the RABV phosphoprotein and beclin1, and found that RABV P5 protein interacted with the BECN1 ring-like structure to induce incomplete autophagy through a BECN1 signaling pathway. P5 attached to the BECN1 ring-like structure promoted RABV self- replication (Fig. 6D). Thus, the results of the present study identified potential antiviral drug targets against RABV. Discussion Autophagy can remove intracellular pathogens, including bacteria and viruses, by activating various cellular defense responses, including direct digestion of intracytoplasmic virions [21, 24], recognition of pathogen-associated molecular patterns through the delivery of viral genomes to endosomal toll-like receptors [25], activation of innate immune signaling [26], and regulation of the inflammatory response [27-31]. However, many viruses also subvert the autophagic machinery to enhance viral replication [32- 37]. In this study, we demonstrated that P5 overexpression increased the level of viral N protein, viral N mRNA, viral anti-genomic RNA, and infectious RABV progeny, and these indexes were significantly inhibited in the absence or presence of P5 and knockdown of Becn1 (Fig. 5). In addition, we also demonstrated that the P5 still increased the RABV replication when autophagy was inhibited (Fig. 5). These results suggested that RABV replication was regulated by the binding of P5 to the BECN1 ring-like structure. In our previous study, RABV P binding to BECN1 can induce incomplete autophagy through the pathways BECN1-CASP2-AMPK-MAPK and BECN1-CASP2-AMPK-AKT-MTOR and RABV-induced incomplete autophagy provides the scaffolds for the replication of RABV genome. However, in this study, we found that small phosphoprotein P5 binding to BECN1 formed a ring-like structure to induce incomplete autophagy through a BECN1 signaling pathway. The ring-like structure wrapped the immature autophagic vesicles and might prevent the fusion of autophagic vesicles and lysosomes from degrading the RABV, and thus benefited self-replication. Page 9/22 Funding This work was supported by grants from National Key Technology R&D Program of China (2016YFD0500400), and the National Special Fund for Public Welfare Industry (Project No. 201103032) of China. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Ethics approval and consent to participate Consent for publication All authors have read this manuscript and approved for the submission. The authors declare no competing interests. Acknowledgments wledge Ms. Yunqin Li for technical assistance on laser confocal microscopy. We gratefully acknowledge Ms. Yunqin Li for technical assistance on laser confocal microscopy. Funding Corresponding authors Corresponding authors Correspondence to Min Liao Abbreviations AKT, protein kinase B; AMPK, AMP-activated protein kinase; ANOVA, analysis of variance; ATG, autophagy related; BECN1, beclin1; CASP2, caspase2; Co-IP, co-immunoprecipitation; DAPI, 6-diamidino-2- phenylindole; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal associated membrane protein 1; LC3, microtubule associated protein 1 light chain 3 alpha; mAbs, Mouse monoclonal antibodies; MAPKs, mitogen activated protein kinases; MOI, multiplicity of infection; MTOR, mammalian target of rapamycin; P, phosphoprotein; RABV, Rabies virus; siRNA, small interfering RNA. Collaborative innovation center and State Key laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou 310058, China Jiyong Zhou Contributions Jiyong Zhou Contributions JZ, JL and ML designed the research. JL performed the experiments, analyzed and interpreted data. JL, ML and JZ wrote the manuscript. ML, YY, HW and HY provide the experimental materials. All authors read and approved the final manuscript. Author information Affiliations MOA Key Laboratory of Animal Virology, Center of Veterinary Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China. Juan Liu, Min Liao, Yan Yan, Hui Yang, Hailong Wang, Jiyong Zhou Page 10/22 Page 10/22 Collaborative innovation center and State Key laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou 310058, China References 1. 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Zeng X, Overmeyer JH, and Maltese WA. Functional specificity of the mammalian Beclin-Vps34 PI 3- kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking. Journal of cell science. 2006; 119: 259-270 Page 12/22 20. Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM, et al. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. The EMBO journal. 2011; 30: 1195-1208 Page 12/22 20. Strappazzon F, Vietri-Rudan M, Campello S, Nazio F, Florenzano F, Fimia GM, et al. Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy. The EMBO journal. 2011; 30: 1195-1208 21. Orvedahl A, Alexander D, Talloczy Z, Sun Q, Wei Y, Zhang W, et al. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell host & microbe. 2007; 1: 23-35 22. Gannage M, Dormann D, Albrecht R, Dengjel J, Torossi T, Ramer PC, et al. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes. Cell host & microbe. 2009; 6: 367- 380 23. Marschalek A, Drechsel L, and Conzelmann KK. The importance of being short: the role of rabies virus phosphoprotein isoforms assessed by differential IRES translation initiation. European journal of cell biology. 2012; 91: 17-23 24. Talloczy Z, Virgin HWt, and Levine B. PKR-dependent autophagic degradation of herpes simplex virus type 1. Autophagy. 2006; 2: 24-29 25. Delgado MA, Elmaoued RA, Davis AS, Kyei G, and Deretic V. Toll-like receptors control autophagy. The EMBO journal. 2008; 27: 1110-1121 26. Shi CS, and Kehrl JH. MyD88 and Trif target Beclin 1 to trigger autophagy in macrophages. The Journal of biological chemistry. 2008; 283: 33175-33182 27. Lupfer C, Thomas PG, Anand PK, Vogel P, Milasta S, Martinez J, et al. References Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection. Nature immunology. 2013; 14: 480-488 28. Zhang M, Covar J, Zhang NY, Chen W, Marshall B, Mo J, et al. Virus spread and immune response following anterior chamber inoculation of HSV-1 lacking the Beclin-binding domain (BBD). Journal of neuroimmunology. 2013; 260: 82-91 29. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008; 456: 264-268 30. Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, et al. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science (New York, N.Y.). 2005; 307: 593-596 31. English L, Chemali M, Duron J, Rondeau C, Laplante A, Gingras D, et al. Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection. Nature immunology. 2009; 10: 480-487 32. Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, et al. Autophagosome supports coxsackievirus B3 replication in host cells. Journal of virology. 2008; 82: 9143-9153 32. Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, et al. Autophagosome supports coxsackievirus B3 replication in host cells. Journal of virology. 2008; 82: 9143-9153 33. Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, et al. Autophagic machinery activated by dengue virus enhances virus replication. Virology. 2008; 374: 240-248 33. Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, et al. Autophagic machinery activated by dengue virus enhances virus replication. Virology. 2008; 374: 240-248 34. Panyasrivanit M, Khakpoor A, Wikan N, and Smith DR. Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes. The Journal of general virology. 2009; 90: 448-456 34. Panyasrivanit M, Khakpoor A, Wikan N, and Smith DR. Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes. The Journal of general virology. 2009; 90: 448-456 35. Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, et al. Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. The Journal of cell biology. 2009; 186: 255-268 35. Kyei GB, Dinkins C, Davis AS, Roberts E, Singh SB, Dong C, et al. Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. The Journal of cell biology. References 2009; 186: 255-268 Page 13/22 Page 13/22 enizot M, Varbanov M, Espert L, Robert-Hebmann V, Sagnier S, Garcia E, et al. HIV-1 gp41 fusogenic nction triggers autophagy in uninfected cells. Autophagy. 2008; 4: 998-1008 hou Z, Jiang X, Liu D, Fan Z, Hu X, Yan J, et al. Autophagy is involved in influenza A virus plication. Autophagy. 2009; 5: 321-328 36. Denizot M, Varbanov M, Espert L, Robert-Hebmann V, Sagnier S, Garcia E, et al. HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells. Autophagy. 2008; 4: 998-1008 36. Denizot M, Varbanov M, Espert L, Robert-Hebmann V, Sagnier S, Garcia E, et al. HIV-1 gp41 fusoge function triggers autophagy in uninfected cells. Autophagy. 2008; 4: 998-1008 37. Zhou Z, Jiang X, Liu D, Fan Z, Hu X, Yan J, et al. Autophagy is involved in influenza A virus replication. Autophagy. 2009; 5: 321-328 37. Zhou Z, Jiang X, Liu D, Fan Z, Hu X, Yan J, et al. Autophagy is involved in influenza A virus replication. Autophagy. 2009; 5: 321-328 Figures Figure 1 Figure 1 Truncated P proteins induce autophagy. A Schematic diagram of the full length and truncated P proteins. B HEK293T Cells were transfected with the plasmids containing the truncated P genes for 48 h, harvested, and analyzed using western blotting using mouse anti-ATG7, anti-Flag mAb, rabbit anti- LC3A/B, anti-ATG5, anti-BECN1, anti-P62, anti-ULK1, and anti-GAPDH antibodies. C HEK293T cells were cotransfected with GFP-LC3B and the plasmids containing the truncated P genes for 24 h, fixed, and Page 14/22 Page 14/22 immunostained with mouse anti-Flag antibodies (red), and then visualized using confocal microscopy. DAPI (blue) was used to stain nuclear DNA. Scale bar: 10 μm. D The graph shows the quantification of autophagosomes by taking the average number of dots in 50 cells. Means and SD (error bars) of three independent experiments are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). independent experiments are indicated ( , P < 0.05; , P < 0.01; , P < 0.001). Page 15/22 Figure 2 Figure 2 Page 15/22 Accumulated autophagosomes are surrounded by a ring-like structure comprising P5. A-C N2a cells were transfected with Flag-P or Flag-P5, or cotransfected with Flag-P5 and Myc-P5 or GFP-LC3B plasmids for 24 h, fixed, and immunostained with mouse anti-Flag antibodies and rabbit anti-Myc antibodies, and then visualized using confocal microscopy. DAPI (blue) was used to stain nuclear DNA. Scale bar: 10 or 20 μm. The graph shows the quantification of autophagosomes by taking the average number of dots in 50 cells. Means and SD (error bars) of three independent experiments are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Page 16/22 ***, P < 0.001). Page 16/22 Figure 3 Autophagosomes fail to fuse with lysosomes in Flag-P5-transfected cells. A N2a cells were cotransfected with Flag-P5 and GFP-LC3B for 24 h, and were treated with EBSS or CQ for 4 h. Cells were incubated with LysoTracker Red (50 nM) for 15 min, and then were fixed, and immunostained with mouse anti-Flag mAb (blue), and observed using confocal microscopy to analyze fusion of autophagosomes with lysosomes. Scale bars: 10 μm. The graph shows the quantification of autolysosomes by taking the average number of dots in 50 cells. B N2a cells were transfected with Flag-P5 for 8 h, and infected with the adenovirus expressing mCherry-GFP-LC3B protein for 24 hr, and were treated with EBSS or CQ for 4 h. Cells were fixed, and immunostained with mouse anti-Flag mAb (blue), and observed using confocal microscopy to analyze fusion of autophagosomes with lysosomes. Scale bars: 10 μm. The graph shows the quantification of autophagosomes by taking the average numberof dots in 50 cells. Means and SD (error bars) of three independent experiments are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Page 17/22 Figure 4 Truncated protein P5 interacted with BECN1. A, B N2a cells were cotransfected with Fla BECN1 (A) or singly transfected with siBecn1 RNA for 12 h and infected with RABV stra MOI = 2 for 24 h (B), and the viral P/P5 protein (green), BECN1 (red), and DAPI (blue) w the indicated antibodies via confocal microscopy. White arrows indicate the colocaliza structure. Scale bar: 10 μm. The graph shows the quantification of the percentage of B Page 18/22 Figure 4 Truncated protein P5 interacted with BECN1. A, B N2a cells were cotransfected with Flag-P5 and Myc- BECN1 (A) or singly transfected with siBecn1 RNA for 12 h and infected with RABV strain HEP Flury at MOI = 2 for 24 h (B), and the viral P/P5 protein (green), BECN1 (red), and DAPI (blue) were detected using the indicated antibodies via confocal microscopy. White arrows indicate the colocalization of the ring-like structure. Scale bar: 10 μm. The graph shows the quantification of the percentage of BECN1 localization Figure 4 Figure 4 Page 18/22 Truncated protein P5 interacted with BECN1. A, B N2a cells were cotransfected with Flag-P5 and Myc- BECN1 (A) or singly transfected with siBecn1 RNA for 12 h and infected with RABV strain HEP Flury at MOI = 2 for 24 h (B), and the viral P/P5 protein (green), BECN1 (red), and DAPI (blue) were detected using the indicated antibodies via confocal microscopy. White arrows indicate the colocalization of the ring-like structure. Scale bar: 10 μm. The graph shows the quantification of the percentage of BECN1 localization Page 18/22 Page 18/22 with P5 ring-like structure. C HEK 293T cells were cotransfected with the plasmids containing the truncated P genes and Myc-BECN1 for 48 h, and the interactions between the truncated P protein and BECN1 were determined using the indicated antibodies. IP, immunoprecipitation. The asterisk indicates the heavy chains. D Schematic diagram of the full-length and truncated BECN1 proteins. E N2a cells were singly transfected or cotransfected with the plasmids encoding the truncated BECN1 genes and Flag-P for 24 h, and the viral P protein (red), BECN1 (green), and DAPI (blue) were detected using the indicated antibodies via confocal microscopy. White arrows indicate the colocalization of the ring-like structure. Scale bar: 10 μm. The graph shows the quantification of the ring-like structures formed with BECN1 deletion mutants. F HEK 293T cells were cotransfected with the plasmids containing the truncated Becn1 genes and Flag-P for 48 h, and the interactions between the truncated BECN1 proteins and P were determined using the indicated antibodies. IP, immunoprecipitation. The asterisk indicates the heavy chains. Means and SD (error bars) of three independent experiments are indicated (***, P < 0.001). Page 19/22 Figure 5 A ring-like structure containing P5 was beneficial to virus replication. A, B N2a cells were (A) transfected with Flag-P5 (B) or cotransfected with Flag-P5 and two siRNAs targeting Becn1 for 12 h, and infected with RABV strain HEP-Flury or CVS-11 at an MOI = 2 for 36 h, and then harvested for western blotting analysis with mouse anti-NP, anti-BECN1, anti-Flag mAbs, and rabbit anti-GAPDH antibodies. C The ratio of viral protein N to GAPDH was normalized to that of the control conditions. D N2a cells were treated as described for panels A and B. qRT-PCR analysis of cellular viral N mRNA, and the anti-genomic RNA level. Figure 4 All qRT PCR data were normalized to the expression of Gapdh and the control group was used as a Figure 6 The protein P5 binding to the BECN1 ring-like structure regulates RABV replication via the BECN1- mediated signaling pathway. A, B N2a cells were transfected with Flag-P5 (A) or cotransfected with Flag- P5 and an siRNA targeting Becn1 (B) for 48 h, and then harvested for western blotting analysis with the indicated antibodies. C N2a cells were cotransfected with Flag-P5 and the shRNA/siRNA targeting Akt or Mtor or Ampk or Mapk for 12 h, and infected with RABV HEP-Flury strain at an MOI = 2 for 36 h. Cells were harvested for western blotting analysis with the indicated antibodies. D Proposed model showing how P5 protein regulates RABV replication. Data are are representative of 3 independent experiments. The protein P5 binding to the BECN1 ring-like structure regulates RABV replication via the BECN1- mediated signaling pathway. A, B N2a cells were transfected with Flag-P5 (A) or cotransfected with Flag- P5 and an siRNA targeting Becn1 (B) for 48 h, and then harvested for western blotting analysis with the indicated antibodies. C N2a cells were cotransfected with Flag-P5 and the shRNA/siRNA targeting Akt or Mtor or Ampk or Mapk for 12 h, and infected with RABV HEP-Flury strain at an MOI = 2 for 36 h. Cells were harvested for western blotting analysis with the indicated antibodies. D Proposed model showing how P5 protein regulates RABV replication. Data are are representative of 3 independent experiments. Figure 5 A ring-like structure containing P5 was beneficial to virus replication. A, B N2a cells were (A) transfected with Flag-P5 (B) or cotransfected with Flag-P5 and two siRNAs targeting Becn1 for 12 h, and infected with RABV strain HEP-Flury or CVS-11 at an MOI = 2 for 36 h, and then harvested for western blotting analysis with mouse anti-NP, anti-BECN1, anti-Flag mAbs, and rabbit anti-GAPDH antibodies. C The ratio of viral protein N to GAPDH was normalized to that of the control conditions. D N2a cells were treated as described for panels A and B. qRT-PCR analysis of cellular viral N mRNA, and the anti-genomic RNA level. All qRT-PCR data were normalized to the expression of Gapdh and the control group was used as a reference. E Cellular supernatant in panels A and B were harvested for virus titer detection. Virus titers in N2a cells were determined using a TCID50 assay. F N2a cells were transfected with Flag-P5 for 12 h and were pretreated with 5 mM 3-MA or 1 mM wortmannin for 2 h, and then infected with RABV HEP-Flury strain at an MOI = 2 and incubated in the absence or presence of 5 mM 3-MA or 1 mM wortmannin for 24 h, and the cells were harvested for western blotting analysis using the indicated antibodies. G The ratio of viral protein N to GAPDH was normalized to that of the control conditions. Means and SD (error bars) of three independent experiments are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). reference. E Cellular supernatant in panels A and B were harvested for virus titer detection. Virus titers in N2a cells were determined using a TCID50 assay. F N2a cells were transfected with Flag-P5 for 12 h and were pretreated with 5 mM 3-MA or 1 mM wortmannin for 2 h, and then infected with RABV HEP-Flury strain at an MOI = 2 and incubated in the absence or presence of 5 mM 3-MA or 1 mM wortmannin for 24 h, and the cells were harvested for western blotting analysis using the indicated antibodies. G The ratio of viral protein N to GAPDH was normalized to that of the control conditions. Means and SD (error bars) of three independent experiments are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Page 21/22 Page 21/22 Page 21/22 Page 21/22 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. suppdata.rar TableS1.docx suppFigs.pptx Page 22/22
https://openalex.org/W4378881639
https://journal.upnvj.ac.id/index.php/esensihukum/article/download/154/76
Indonesian
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URGENSI PENGAWASAN PASCA ADOPSI GUNA MENCEGAH MOTIF ADOPSI SEBAGAI MODUS OPERANDI TINDAK PIDANA PENJUALAN ANAK
Jurnal Esensi Hukum
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cc-by-sa
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Abstrak Anak telah memiliki hak asasi yang melekat sejak masih di dalam kandungan berupa penghidupan yang layak. Sayangnya, hak tersebut seringkali tidak dapat dipenuhi karena kondisi keluarga yang tidak memungkinkan, contohnya banyak terjadi penelantaran anak karena faktor ekonomi oleh suatu keluarga. Untuk melindungi Hak anak tersebut diaturlah pengadopsian anak oleh Undang-Undang Perlindungan Anak. Namun, tata pengawasan yang dinilai kurang mampu dilakukan telah menimbulkan fenomena pengadopsian anak yang tidak sesuai peraturan perundang-undangan atau disebut adopsi ilegal. Hal ini dinilai dapat memunculkan permasalahan baru yakni penjualan anak dengan modus adopsi. Penelitian ini menggunakan metode yuridis-normatif, yaitu penelitian hukum yang didasarkan pada penelitian terhadap bahan hukum yang ada. Tujuan penelitian ini adalah untuk menunjukkan urgensi pengawasan terhadap praktik adopsi anak guna meminimalisasi praktik penjualan anak dengan modus adopsi anak. Hasil Penelitian ini adalah dengan melegitimasi pemberlakuan Post Adoption Report (PAR) terhadap domestic adoption di Indonesia yang diawasi oleh Dinas Sosial. Saran dari penulis adalah dengan diadakan perubahan pada Permensos Nomor 3 Tahun 2018 guna memberlakukan PAR dalam domestic adoption. Kata kunci: Adopsi ilegal; Penjualan anak; Post Adoption Report Tiara Alfarissa1, Syalaisha Amani Puspitasari2 Tiara Alfarissa1, Syalaisha Amani Puspitasari2 1Fakultas Hukum Universitas Pembangunan Nasional “Veteran” Jakarta, E-mail: 2110611109@mahasiswa.upnvj.ac.id 2Fakultas Hukum Universitas Pembangunan Nasional “Veteran” Jakarta, E-mail: 2110611120@mahasiswa.upnvj.ac.id Abstract Children have had basic human rights inherent in the womb of decent living. Unfortunately, such rights are often inadequate because of poor family conditions, for example, many are abandoned by the economic factors of a family. To protect the rights of the child, the adoption of children by Undang-Undang Perlindungan Anak was arranged. However, inadequate surveillance measures have led to a phenomenon of adoption that ignores legislation or calls illegal adoption. This has been suggested that it may lead to a new problem of child sales in adoption. The study used a juridicy-normative-law study based on the study of available legal material. The purpose of this study is to demonstrate the urgency of the supervision of child adoption practices to minimize the practice of child trafficking with child adoption mode. The result of this research is the legitimacy of the Post Adoption Report (PAR) enforcement of domestic adoption in Indonesia supervised by Dinas Sosial.. The authors' advice is that changes to Permensos number 3 in the year 2018 to apply Post Adoption Report (PAR) within the domestic adoption. Keywords: illegal adoption; Child trafficking; Post Adoption Report 4 Viva Budy Kusnandar. 2022. Kasus Perdagangan dan Eksploitasi Anak (2016-2021). databoks. akses<https://databoks.katadata.co.id/datapublish/2022/10/07/perdagangan-anak-ala-suhendra-ayah-sejuta -anak-terbongkar-berikut-jumlah-korban-trafficking-anak-di-2021> pukul 11.08 3 Ghasani, Annisa Nuridha. 2016. Pengaturan Tindak Pidana Adopsi Ilegal yang Dapat Dikualifikasikan sebagai Perdagangan Orang. UII 2  Lisa Novita, Adi Hermansyah. 2018. PENERAPAN SANKSI PIDANA TERHADAP PELAKU TINDAK PIDANA PENELANTARAN ANAK (Suatu Penelitian Di Wilayah Hukum Kepolisian Resor Aceh Besar). Jurnal Ilmiah Mahasiswa: Universitas Syiah Kuala 1  Cenderawasih Pos, Tanggal 26 Februari 2015 I. Pendahuluan Pada pasal 28B ayat 2 Undang-Undang Dasar Negara Republik Indonesia Tahun 1945 menyatakan bahwa “Setiap anak berhak atas kelangsungan hidup, tumbuh dan berkembang serta berhak atas perlindungan dari kekerasan dan diskriminasi.” Pada hakikatnya, di dalam sebuah keluarga anak memiliki hak. Namun, seringkali hak 79 79 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 tersebut tidak dapat dipenuhi karena kondisi keluarga yang tidak memungkinkan. Hal ini dapat ditemui terjadinya fenomena anak terlantar di suatu negara yang masih memiliki permasalahan ekonomi dan tingginya angka pengangguran. Sayangnya permasalahan anak dinilai sebagai masalah secondary bila dibandingkan dengan permasalahan ekonomi suatu negara. Padahal permasalahan ini berkaitan erat dengan kemanusiaan, anak dinilai memiliki kepekaan psikologis yang tinggi dan masih dalam tahap membutuhkan bimbingan orang dewasa1. g g Bentuk penelantaran anak oleh orang tuanya bermacam-macam, salah satunya adalah penelantaran anak yang dilakukan oleh ibu kandung sang anak. Walaupun kasus tersebut jarang terjadi, tetapi kasus penelantaran anak yang dilakukan oleh ibu kandung lebih cenderung pada masalah kejiwaan, adanya tekanan atau beban psikologis yang ditanggung oleh seorang ibu terhadap anak yang dilahirkannya2. Berdasarkan pasal 305 KUHP yang berbunyi “barang siapa menaruhkan anak yang dibawah umur tujuh tahun di suatu tempat supaya dipungut oleh orang lain, atau dengan maksud akan terbebas dari pada pemeliharaan anak itu, meninggalkannya, dihukum penjara sebanyak-banyaknya lima tahun 6 bulan” sayangnya dalam pengimplementasiannya, kasus penelantaran anak kurang diperhatikan di Indonesia.Tak jarang kasus penelantaran oleh ibu kandung memberikan peluang tindak pidana baru yaitu dengan memberikan anak kepada pihak lain tanpa memperhatikan tata cara yang diatur di dalam peraturan perundang-undang lalu pihak lain tersebut memberikan kepada pihak ketiga dengan modus adopsi sebagaimana yang terjadi pada kasus Penjualan Anak, dapat dikatakan tindakan ini termasuk tindak pidana penjualan anak. p p p j Adopsi anak yang tidak dilakukan sesuai dengan peraturan perundang-undangan yang selanjutnya disebut adopsi ilegal. Adopsi ilegal merupakan bentuk dari klasifikasi penjualan anak, jika dilakukan demi mendapatkan keuntungan. Modus adopsi biasanya digunakan untuk perdagangan bayi3. Mengacu pada data dari Komisi Perlindungan Anak Indonesia (KPAI) terdapat 147 korban trafficking (perdagangan anak) dan eksploitasi anak sepanjang 2021 dan pada 2020 sebanyak 149 anak. Kasus penjualan anak tertinggi adalah pada tahun 2019 yakni 347 anak. Sedangkan KPAI sendiri telah menerima 1.358 laporan Perlindungan Anak sepanjang bulan Januari-Juni pada tahun 20224. 6  Ibid, hlm.141.  5  Jonaedi Efendi, Johnny Ibrahim, 2018, Metode Penelitian Hukum Normatif dan Empiris, Depok: Prenadamedia Group. I. Pendahuluan Pada proses “pengadopsian” tersebut, Terdakwa A memasang tarif Rp. 14.000.000 (empat belas juta rupiah) sebagai keuntungannya dengan dalih “uang persalinan saya”, padahal bayi tersebut bukanlah anak yang ia lahirkan sendiri, melainkan anak dari Terdakwa R. Ditakutkannya terjadi tindak pidana penjualan anak dengan menggunakan adopsi sebagai motif terselebung sebelumnya pada uraian kasus di atas sejatinya dapat terjadi jikalau tidak ada pengawasan yang ketat dalam prosesnya. Namun, melihat hukum positif yang ada di Indonesia saat ini, memang tidak ada aturan mengenai pengawasan pasca adopsi anak oleh warga negara Indonesia layaknya pengawasan pasca adopsi oleh warga negara asing. Oleh karenanya, penulis tertarik untuk meneliti lebih lanjut terkait pengawasan terhadap anak pasca adopsi oleh warga negara Indonesia. Berdasarkan uraian latar belakang diatas, penulis tertarik untuk meneliti lebih lanjut mengenai bagaimana pengadopsian sebagai modus operandi penjualan anak & urgensi pengawasan pasca adopsi anak oleh dinas sosial setempat yang dituangkan dalam judul “Urgensi Pengawasan Pasca Adopsi Anak Oleh Dinas Sosial Setempat Guna Mencegah Motif Adopsi Sebagai Modus Operandi Tindak Pidana Penjualan Anak”. 2. Metode Penelitian Pada penelitian ini, penulis menggunakan metode yuridis-normatif, yaitu penelitian hukum yang didasarkan pada penelitian terhadap bahan hukum yang ada5. Penulis menggunakan pendekatan peraturan perundang-undangan sesuai dengan topik yang sedang dikaji yang dinilai masih terdapat kekurangan dan pendekatan komparasi, yakni membandingkan dengan konsep praktik di negara lain guna mengidentifikasi kondisi sosial dan menentukan bentuk penormaannya6. Objek kajian studi kepustakaan berupa peraturan perundang-undangan dan literatur lain yang berhubungan dengan penelitian ini. Jenis penelitian yang digunakan merupakan deskriptif analitis yang bersumber dari sumber primer, yakni peraturan perundang-undangan seperti PP Nomor 54 Tahun 2007 dan Permensos Nomor 3 Tahun 2018, serta sumber sekunder berupa jurnal atau artikel ilmiah yang sesuai dengan topik yang sedang penulis angkat. 3.1 Pengadopsian Anak Sebagai Modus Operandi Penjualan Anak I. Pendahuluan Banyaknya kasus penjualan anak, perlu difokuskan oleh pemerintah terkait usaha pemberantasan Penjualan Anak sebagai bentuk perlindungan anak yang telah dituangkan dalam Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak. g g Fenomena penjualan anak dengan modus adopsi anak yang pada faktanya dilakukan secara ilegal telah penulis temukan secara langsung pada sebuah persidangan di Pengadilan Negeri Jakarta Selatan (yang sampai penulisan saat ini masih dalam proses pemeriksaan saksi). Penulis menyaksikan fakta-fakta persidangan selama pemeriksaan berlangsung dan mendapatkan fakta bahwa Terdakwa R yang merupakan seorang single mom dari tiga anak yang telah ditinggal oleh suaminya tanpa tanggung 80 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 jawab dan merasa tidak sanggup untuk mengasuh ketiga anaknya dengan keterbatasan ekonomi yang dialaminya memutuskan untuk memberikan anak ketiganya yang masih bayi untuk diadopsi oleh Terdakwa A, karena Terdakwa A mengunggah kesedihannya di laman sosial media Facebooknya bahwa Terdakwa A ingin sekali mempunyai anak. Namun, proses adopsi yang dilakukan oleh Terdakwa A terhadap bayi dari Terdakwa R tidaklah sesuai dengan PP No. 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak. Tidak berhenti di sana, pada fakta-fakta persidangan yang dikemukakan saksi lainnya, Terdakwa A hanya mengasuh bayi yang diadopsinya dari Terdakwa R selama tiga bulan saja dan setelahnya diberikan kepada Terdakwa RN dengan modus yang sama, yaitu adopsi, tetapi tidak sesuai dengan PP Nomor 54 Tahun 2007. Pada proses “pengadopsian” tersebut, Terdakwa A memasang tarif Rp. 14.000.000 (empat belas juta rupiah) sebagai keuntungannya dengan dalih “uang persalinan saya”, padahal bayi tersebut bukanlah anak yang ia lahirkan sendiri, melainkan anak dari Terdakwa R. jawab dan merasa tidak sanggup untuk mengasuh ketiga anaknya dengan keterbatasan ekonomi yang dialaminya memutuskan untuk memberikan anak ketiganya yang masih bayi untuk diadopsi oleh Terdakwa A, karena Terdakwa A mengunggah kesedihannya di laman sosial media Facebooknya bahwa Terdakwa A ingin sekali mempunyai anak. Namun, proses adopsi yang dilakukan oleh Terdakwa A terhadap bayi dari Terdakwa R tidaklah sesuai dengan PP No. 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak. Tidak berhenti di sana, pada fakta-fakta persidangan yang dikemukakan saksi lainnya, Terdakwa A hanya mengasuh bayi yang diadopsinya dari Terdakwa R selama tiga bulan saja dan setelahnya diberikan kepada Terdakwa RN dengan modus yang sama, yaitu adopsi, tetapi tidak sesuai dengan PP Nomor 54 Tahun 2007. 9  Pedoman Pelaksanaan Pengangkatan Anak terbitan Departemen Sosial Republik Indonesia, Direktorat Jenderal Pelayanan dan Rehabilitasi Sosial Direktorat Bina Pelayanan Sosial Anak, hal 7-17 8  Peraturan Pemerintah Nomor 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak 7  Keizerina Devi Azwar, Rita Armelia, Sri Muktiningsih. . KEDUDUKAN PENGANGKATAN ANAK DALAM SYSTEM HUKUM NASIONAL. Jurnal Universitas Sumatera Utara. hlm 6 3. Hasil Dan Pembahasan 3.1 Pengadopsian Anak Sebagai Modus Operandi Penjualan Anak 81 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 Anak merupakan karunia yang diberikan oleh Tuhan kepada hambanya. Sebagai individu, anak memiliki hak yang melekat sejak di dalam kandungan yaitu hak asasi termasuk untuk mendapatkan perlindungan. Maka menurut Peraturan Pemerintah Nomor 54 Tahun 2007 menjelaskan bahwa tujuan dari pengangkatan anak adalah memberikan kesejahteraan dan perlindungan anak, demi mewujudkan kepentingan anak berdasarkan adat kebiasaan setempat dan ketentuan peraturan perundang-undangan. Adapun di dalam Undang-Undang Nomor 23 Tahun 2002 tentang Perlindungan Anak menjelaskan bahwa motivasi pengangkatan anak semata-mata demi kepentingan yang terbaik untuk anak dengan menjalankan sesuai kebiasaan setempat dan hukum positif yang berlaku di daerah tersebut. Jadi, pengangkatan anak harus didasari rasa untuk menolong dan melindungi anak supaya masa depan mereka lebih cemerlang. Pengangkatan anak dilakukan dengan cara adopsi anak yang pengaturannya telah termaktub di dalam Undang-Undang Perlindungan Anak serta tata cara pelaksanaannya di dalam Peraturan Pemerintah Nomor 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak selanjutnya dirincikan di dalam Peraturan Menteri Sosial Nomor 110 Tahun 2009 (PERMEN) tentang Persyaratan Pengangkatan Anak7. Dalam pasal 1 PP Tentang Pelaksanaan Pengangkatan Anak memberikan definisi Adopsi Anak yaitu pengalihan hak anak dari suatu lingkungan keluarga yang sah atau dari seseorang yang memiliki tanggung jawab atas perawatan, pendidikan, dan membesarkan anak tersebut, kepada keluarga angkatnya sesuai penetapan pengadilan8. Terdapat empat jenis pengangkatan anak9, yaitu: 1. Pengangkatan Anak antar warga negara Indonesia (Domestic Adoption) 1. Pengangkatan Anak antar warga negara Indonesia (Domestic Adoption) g g g g ( p ) 3. Pengangkatan Anak oleh Orang Tua Tunggal (Single Parent) 3. Pengangkatan Anak oleh Orang Tua Tunggal (Single 4. Pengangkatan Anak menurut Hukum Adat. Pengangkatan anak tidak dapat dilakukan secara asal, melainkan harus melewati beberapa tahap sesuai dengan peraturan perundang-undangan. Sebelum secara resmi hak anak tersebut dialihkan, para calon orang tua asuh perlu melakukan permohonan pengangkatan anak yang ditujukan kepada Instansi Sosial Kabupaten/Kota. Selanjutnya dapat dilakukan proses penilaian kelayakan calon orang tua asuh dengan melakukan sidang Tim Pertimbangan Izin Pengangkatan Anak (PIPA) Daerah, sampai dikeluarkan Surat Keputusan Kepala Dinas Sosial yang telah ditetapkan Pengadilan Negeri setempat. 3. Hasil Dan Pembahasan Ada pula segala hal yang wajib dipenuhi calon orang tua asuh dalam melakukan adopsi anak adalah dengan mewujudkan kepentingan terbaik bagi anak, tidak memutuskan hubungan darah antara anak yang diangkat dengan orang tua asuhnya, calon orang tua asuh harus memiliki agama yang sama dengan calon anak angkatnya jika asal-usul anak tidak diketahui maka agama anak berasal dari agama 82 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 mayoritas penduduk tersebut, dan pengangkatan anak oleh warga negara asing merupakan upaya terakhir10. mayoritas penduduk tersebut, dan pengangkatan anak oleh warga negara asing merupakan upaya terakhir10. Sedangkan pengangkatan anak yang ilegal telah diatur pula di dalam pasal 39 Undang-Undang Perlindungan Anak yakni, adopsi anak dilakukan demi kepentingan pribadi atau tidak sesuai dengan adat setempat dan peraturan perundang-undangan; memutuskan hubungan nasab dengan orang tua kandungnya; calon orang tua angkat tidak seagama dengan anak; jika anak diasuh oleh warga negara asing tetapi bukan dikategorikan upaya terakhir. Jika mengacu pada pasal 79 Undang-Undang Perlindungan Anak maka terdapat sanksi bagi pengadopsian ilegal yaitu di penjara paling lama lima tahun dan/atau denda paling banyak Rp 100.000.000,00 (seratus juta rupiah). Selain itu terdapat tindak pidana lain yang memperbarengi adopsi ilegal antara lain perdagangan orang atau penjualan anak. Adopsi ilegal bisa dikatakan sebagai salah satu motif dari usaha perbuatan perdagangan orang jika telah memenuhi unsur-unsur tindak pidana perdagangan orang11. Selain data yang sudah dicantumkan di bagian latar belakang, KPAI pun juga mencatat adanya motif adopsi terhadap penjualan anak, sekurang-kurangnya ada 20% kasus perdagangan orang pada anak di negara Indonesia dari 20.000 kasus perdagangan orang berkedok adopsi12. Dalam tindak pidana perdagangan orang, ada tiga unsur yang harus dipenuhi yaitu13: 1. Unsur dari rangkaian perbuatan atau tindakan berupa perekrutan, pengangkutan, penampungan, pengiriman, pemindahan, atau penerimaan seseorang; 2. Unsur yang berupa usaha dari rangkaian tindakan tersebut dapat terpenuhi seperti adanya ancaman, penggunaan kekerasan, penculikan, penyekapan, pemalsuan, penipuan, penyalahgunaan kekuasaan atau posisi rentan, penjeratan utang atau memberi bayaran atau manfaat sehingga memperoleh persetujuan dari orang yang memegang kendali atas orang lain tersebut. p j g y g g g g 3. 13  Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdagangan Orang 12  Situs Komisi Perlindungan Anak Indonesia 11  Anisa N Ghasani. 2017. Pengaturan Tindak Pidana Adopsi Ilegal Yang Dapat Dikualifikasikan Sebagai Perdagangan Orang. UII DSPACE Fakultas Hukum. hlm 83 10  Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak 11 10  Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak 13  Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdagangan Orang  Situs Komisi Perlindungan Anak Indonesia 13  Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdagangan Orang 12  Situs Komisi Perlindungan Anak Indonesia Sebagai Perdagangan Orang. UII DSPACE Fakultas Hukum. hlm 83 3. Hasil Dan Pembahasan Unsur maksud atau tuntutan yang akan dicapai atau tujuan yaitu meliputi pemerasaan guna keuntungan diri sendiri seperti eksploitasi orang yang dapat mengakibatkan orang tereksploitasi sesuai tata cara yang dijelaskan di dalam Pasal 1 angka 1 dan Pasal 2 ayat (1) (UU TPPO) Jika korban di bawah delapan belas tahun maka unsur yang harus diperhatikan adalah proses atau pergerakan dari upaya direkrut, di transportasi, dipindahkan dan eksploitasi berupa tindakan diancam, dipaksa dengan cara lain, diculik, korban pemalsuan, ditipu bahkan korban penyalahgunaan kekuasaan. Jika korban di bawah delapan belas tahun maka unsur yang harus diperhatikan adalah proses atau pergerakan dari upaya direkrut, di transportasi, dipindahkan dan eksploitasi berupa tindakan diancam, dipaksa dengan cara lain, diculik, korban pemalsuan, ditipu bahkan korban penyalahgunaan kekuasaan. Adapun pengaturan tentang tindak pidana perdagangan orang dengan modus adopsi ilegal yang diatur di dalam Pasal 76F Undang-Undang Nomor 35 tahun 2014 tentang Perlindungan Anak yang berbunyi, “Setiap orang dilarang menempatkan, 83 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 membiarkan, melakukan, menyuruh melakukan, atau turut serta melakukan penculikan, penjualan, atau perdagangan anak” dengan sanksi yang diatur di pasal 83 UU Perlindungan Anak yang berbunyi, “Setiap orang yang melanggar ketentuan sebagaimana dimaksud dalam Pasal 76F dipidana dengan pidana penjara paling singkat 3 (tiga) tahun dan paling lama 15 (lima belas) tahun dan denda paling sedikit Rp 60.000.000,00 (enam puluh juta rupiah) dan paling banyak Rp 300.000.000,00 (tiga ratus juta rupiah)”. Selain itu, diatur pula di dalam Undang-undang No. 21 Tahun 2007 tentang Pemberantasan Tindak Pidana Perdagangan Orang. Tindakan adopsi ilegal telah menimbulkan peluang tindak pidana lainnya yaitu perdagangan atau penjualan anak karena dilakukan dengan tujuan menguntungkan diri sendiri tanpa memperhatikan kepentingan anak sehingga telah terjadi eksploitasi anak. Oleh karena itu pelaku pengadopsian anak secara ilegal harus mendapatkan sanksi sesuai resiko dan dampak dari perbuatannya. 3.2 Urgensi Pengawasan Pasca Adopsi Anak Oleh Dinas Sosial Setempat Setelah melihat aturan hukum terkait penjualan anak yang dilatar belakangi dengan motif adopsi, maka hal ini dapat ditinjau dari teori negara sebagai welfare state yang berarti keadaan di mana negara berkedudukan sebagai penjamin kesejahteraan bagi rakyatnya, maka Indonesia harus dapat menjadi wadah untuk memfasilitasi lalu lintas per-adopsian anak yang aman bagi kehidupan anak di masa depan dengan orang tua angkatnya. Apabila negara tidak dapat memfasilitasi keamanan tersebut, maka ditakutkan akan tercipta fenomena adopsi ilegal yang dapat berujung pada praktik perdagangan/penelantaran/kekerasan/eksploitasi anak akan merebak tak terkendali14. 16  Maharani dan Irit Suseno, 2018, Pengangkatan Anak Warga Negara Indonesia Oleh Warga Negara Asing, Mimbar Keadilan: Jurnal Ilmu Hukum. 15  Jumlah Korban Trafficking Anak di 2021, https://databoks.katadata.co.id/datapublish/2022/10/07/perdagangan-anak-ala-suhendra-ayah-sejuta-anak- terbongkar-berikut-jumlah-korban-trafficking-anak-di-2021, diakses pada 25 November 2022. 14  Dinas Sosial Daerah Istimewa Yogyakarta, 2022, Peningkatan Kapasitas Petugas Urusan Adopsi Anak DIY, https://dinsos.jogjaprov.go.id/wp-content/cache/all/peningkatan-kapasitas-petugas-urusan-adopsi-anak-di y%EF%BF%BC/index.html, diakses pada 25 November 2022. 3. Hasil Dan Pembahasan Bahkan seperti yang tertera dalam latar belakang, di Indonesia sendiri menurut data yang dikeluarkan oleh Komisi Perlindungan Anak Indonesia (KPAI), korban dari perdagangan dan eksploitasi anak pada tahun 2021 mencapai angka 14715. Saat ini, instrumen hukum di Indonesia menurut hemat penulis hanyalah ketat untuk pengadopsian oleh Warga Negara Asing saja (intercountry adoption) dan tidak kepada ranah domestic adoption atau adopsi oleh yang berkewarganegaraan sama16. Dalam proses intercountry adoption, Pasal 16 ayat (2) Permensos Nomor 3 Tahun 2018 mengatur bahwa pengawasan pelaksanaan pengangkatan anak dilakukan sampai anak berusia 18 tahun. Lebih lanjut dalam Pasal 18 ayat (2), pengawasan perkembangan anak angkat juga dilakukan sampai anak berusia 18 tahun selama minimal satu kali dalam satu tahun oleh Kementerian Luar Negeri melalui Perwakilan di negara orang tua angkatnya. Laporan perkembangan anak yang dimaksud salah satunya adalah laporan mengenai keadaan biologis, psikologis anak atau sebagainya yang dilakukan secara berkala selama minimal satu tahun sekali yang dikenal sebagai post adoption reports (selanjutnya disebut sebagai PAR). 84 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 Dilansir dari situs travel.state.gov, banyak negara yang menerapkan PAR setelah dilakukan proses adopsi anak, tetapi lagi dan lagi hanya berlaku bagi pengadopsian anak oleh warga negara asing. Praktik ini diterapkan di negara Ethiopia yang harus melaporkan secara berkala perkembangan anaknya sampai berumur 18 tahun, Madagascar yaitu selama enam bulan dalam satu tahun pertama dan setelahnya setiap tahun sampai anak berumur 18, bahkan di Mongolia terdapat ketentuan berbeda, ada yang dilaporkan selama satu tahun dua kali (anak usia satu bulan-tiga tahun), satu tahun sekali (anak umur empat sampai delapan tahun), dan dua tahun sekali (anak umur delapan sampai enam belas tahun). Namun, ketentuan ini menurut penulis tidaklah benar, karena dalam konteks domestic adoption, pemerintah tidak boleh menutup mata terhadap kemungkinan-kemungkinan buruk yang akan terjadi, seperti yang berujung pada praktik penjualan anak. Di India, prosedur adopsi anak yang dilengkapi dengan PAR berlaku tidak hanya untuk intercountry adoption, tetapi juga bagi warga negara India itu sendiri. Dikutip dari website cara.nic.in, terdapat prosedur adopsi anak dalam tahap pre-adoption, adoption, dan follow-up progress of adopted child yang dalam hal ini berwujud pada PAR. PAR disiapkan oleh badan yang melakukan home-study pada tahap pre-adoption yang disusun pasca adopsi setiap enam bulan sekali selama dua tahun17. 17  Piyush Kakkar, 2019, Adoption in India- The Past, Present, and Future Trends, Journal Issue Vol.6. 3. Hasil Dan Pembahasan Berkaca dari prosedur tindakan yang juga memperhatikan kondisi pasca diadopsinya anak oleh warga negara India dan tidak hanya berfokus pada intercountry adoption, sedangkan di Indonesia menurut ius constitutum saat ini hanya menerapkan PAR untuk orang tua angkat yang berkewarganegaraan asing, maka penulis dapat mengkonsepkan hal tersebut untuk diberlakukan di Indonesia. Argumentasi tersebut penulis tarik berdasarkan Pasal 2 PP Nomor 54 Tahun 2007 yang menyatakan bahwa pengangkatan anak dilangsungkan untuk kepentingan anak berupa kesejahteraan dan perlindungan anak, tetapi bagaimana negara dapat menjamin terwujudnya kesejahteraan anak apabila negara yang berkedudukan sebagai penjamin kesejahteraan masyarakatnya tidak memperhatikan kondisi anak setelah diadopsi? Memenuhi syarat administratif sebagai calon orang tua angkat tidak cukup untuk menentukan bahwa pengadopsi akan mewujudkan kehidupan yang lebih baik untuk masa depan calon anak angkat, maka dari itu penulis menganggap bahwa hal ini dapat diukur melalui parameter penilaian pasca adopsi dalam kurun waktu tertentu sampai anak berusia 18 tahun layaknya PAR yang dilakukan bagi pengadopsi warga negara asing. Pada konsep ini, penulis menilai bahwa lembaga yang berhak untuk menindaklanjuti perihal PAR adalah Dinas Sosial Provinsi dan/atau Kabupaten/Kota selaku yang telah mendampingi dan menilai kelayakan calon orang tua angkat hingga dapat memberikan izin hingga sah melalui penetapan pengadilan sebagai perpanjangan tangan dari pemerintah pusat, yakni Kementrian Sosial dan/atau kementrian terkait. Dengan ini, apabila terdapat pelanggaran dalam hal perlakuan yang tidak baik terhadap anak angkat oleh orang tua angkat, maka sesuai Pasal 14 ayat (1) Permensos Nomor 3 Tahun 2018, dapat dilaporkan ke pihak kepolisian, KPAI, dinas sosial setempat atau melalui kementerian sosial, sehingga dapat meminimalisir lahirnya tindak pidana penjualan anak karena anak yang diadopsi tetap diawasi oleh lembaga yang berwenang dengan mengetahui perkembangan anak selama minimal 85 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 satu tahun sekali berkaitan dengan kondisi biologis, psikologis, sosial, spiritual, pendidikan, dan hak-hak anak lainnya sampai anak berumur 18 tahun. satu tahun sekali berkaitan dengan kondisi biologis, psikologis, sosial, spiritual, pendidikan, dan hak-hak anak lainnya sampai anak berumur 18 tahun. 4. Kesimpulan Anak adalah karunia dari Tuhan. Sejak di dalam kandungan, anak memiliki Hak Asasi yang harus dilindungi, yaitu berupa penghidupan yang layak baik dari sisi ekonomi, pendidikan maupun lingkungan sosialnya. Salah satu wujud perlindungan terhadap kepentingan terbaik untuk anak adalah dengan dilakukan pengangkatan anak atau adopsi yang telah diatur di dalam Undang-Undang Perlindungan Anak serta tata cara pelaksanaannya di dalam Peraturan Pemerintah Nomor 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak. Namun, jika pelaksanaannya tidak sesuai dengan adat kebiasaan setempat serta peraturan perundang-undangan maka adopsi dapat dikatakan ilegal. Adopsi ilegal dinilai dapat menyertai tindak pidana yang baru antara lain tindak pidana penjualan anak. Hal ini dapat dilihat dari data kasus penjualan anak dengan modus adopsi anak yang marak di Indonesia yaitu setidaknya ada 20 persen dari data kasus perdagangan terhadap anak. Maka dari itu, sebagai bentuk hadirnya negara sebagai penjamin kesejahteraan rakyatnya dalam rangka meminimalisir lahirnya tindak pidana penjualan anak, perlu diberlakukan Post Adoption Report (PAR) terhadap domestic adoption di Indonesia seperti yang berlaku di India yang berisi tentang perkembangan kondisi biologis, psikologis, sosial, spiritual, pendidikan, dan hak-hak anak lainnya sampai anak berumur 18 tahun oleh Dinas Sosial setempat seperti yang diberlakukan bagi orang tua angkat yang berkewarganegaraan asing dalam Permensos Nomor 3 Tahun 2018. Pemerintah perlu meninjau ulang terkait ketentuan akan urgensi untuk menilai terjaminnya kehidupan kesejahteraan anak yang telah diadopsi oleh orang tua angkatnya melalui Post Adoption Report (PAR) sebagaimana pengadopsian oleh warga negara asing terhadap anak berkewarganegaraan Indonesia yang diatur dalam Pasal 18 Permensos Nomor 3 Tahun 2018. Oleh karena itu, penulis menyarankan perlu diadakan perubahan pada Permensos Nomor 3 Tahun 2018 guna memberlakukan PAR dalam domestic adoption yang diawasi langsung oleh Dinas Sosial setempat, sehingga lebih lanjut dapat disisipkan Pasal di dalamnya untuk memberi wewenang kepada Dinas Sosial setempat untuk melakukan pengawasan PAR terhadap domestic adoption. Ucapan terima Kasih (Acknowledgments) Selama penelitian ini berlangsung, penulis ingin menghaturkan ucapan terima kasih kepada Allah SWT, Tuhan Yang Maha Esa atas karunia-Nya kami telah menyelesaikan jurnal ini. Tak lupa kami berterima kasih kepada Dr. Beniharmoni Harefa, S.H., LLM. selaku dosen mata kuliah Hukum Acara Pidana FH UPN Veteran Jakarta, karena telah memberikan kesempatan bagi kami selaku penulis untuk meneliti jurnal ini. Daftar Pustaka / Daftar Referensi Azwar, Devi, Keizerina, Armelia, Rita, Muktiningsih, Sri. KEDUDUKAN PENGANGKATAN ANAK DALAM SYSTEM HUKUM NASIONAL. Jurnal Universitas Sumatera Utara. Peraturan Perundang-undangan Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak Undang-undang Republik Indonesia Nomor 35 Tahun 2014 tentang Perubahan atas Undang-undang Nomor 23 Tahun 2002 tentang Perlindungan Anak Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdagangan Orang g g g g Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdaganga Orang Undang-undang (UU) No. 21 Tahun 2007 Pemberantasan Tindak Pidana Perdagangan Orang eraturan Pemerintah Nomor 54 Tahun 2007 tentang Pelaksanaan Pengangkatan Anak Buku Efendi, Jonaedi, Ibrahim, Johnny. (2018). Metode Penelitian Hukum Normatif da Empiris. Depok: Prenadamedia Group. Efendi, Jonaedi, Ibrahim, Johnny. (2018). Metode Penelitian Hukum Normatif da Empiris. Depok: Prenadamedia Group. Jurnal 86 Jurnal ESENSI HUKUM, Vol. 4 No. 1 Bulan Juni Tahun 2022, hlm 79-87 Ghasani, Anisa, N, (2017). Pengaturan Tindak Pidana Adopsi Ilegal Yang Dapat Dikualifikasikan Sebagai Perdagangan Orang. UII DSPACE Fakultas Hukum. hlm 83 Ghasani. Nuridha, Annisa Nuridha. (2016). Pengaturan Tindak Pidana Adopsi Ilegal yang Dapat Dikualifikasikan sebagai Perdagangan Orang. UII Maharani, dan Irit Susen,. (2018). Pengangkatan Anak Warga Negara Indonesia Oleh Warga Negara Asing, Mimbar Keadilan: Jurnal Ilmu Hukum. Novita, Lisa, Hermansyah, Adi. (2018). PENERAPAN SANKSI PIDANA TERHADAP PELAKU TINDAK PIDANA PENELANTARAN ANAK (Suatu Penelitian Di Wilayah Hukum Kepolisian Resor Aceh Besar). Jurnal Ilmiah Mahasiswa: Universitas Syiah Kuala Piyush Kakkar, (2019). Adoption in India- The Past. Present. and Future Trends. Journal Issue Vol.6. Online/World Wide Web: Cenderawasih Pos, Tanggal 26 Februari 2015 Cenderawasih Pos, Tanggal 26 Februari 2015 Dinas Sosial Daerah Istimewa Yogyakarta, 2022, Peningkatan Kapasitas Petugas Urusan Adopsi Anak DIY, https://dinsos.jogjaprov.go.id/wp-content/cache/all/peningkatan-kapasitas-p etugas-urusan-adopsi-anak-diy%EF%BF%BC/index.html, diakses pada 25 November 2022. Jumlah Korban Trafficking Anak di 2021, https://databoks.katadata.co.id/datapublish/2022/10/07/perdagangan-anak- ala-suhendra-ayah-sejuta-anak-terbongkar-berikut-jumlah-korban-trafficking-a nak-di-2021, diakses pada 25 November 2022. Situs Komisi Perlindungan Anak Indonesia, diakses pada 2 Desember 2022 Pedoman Pelaksanaan Pengangkatan Anak terbitan Departemen Sosial Republik Indonesia, Direktorat Jenderal Pelayanan dan Rehabilitasi Sosial Direktorat Bina Pelayanan Sosial Anak, Diakses Pada 29 November 2022 87
https://openalex.org/W4383058277
https://link.springer.com/content/pdf/10.1007/s11538-023-01170-3.pdf
English
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Chemical Systems with Limit Cycles
Bulletin of mathematical biology
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cc-by
16,866
Abstract The dynamics of a chemical reaction network (CRN) is often modeled under the assumption of mass action kinetics by a system of ordinary differential equations (ODEs) with polynomial right-hand sides that describe the time evolution of concen- trations of chemical species involved. Given an arbitrarily large integer K ∈N, we show that there exists a CRN such that its ODE model has at least K stable limit cycles. Such a CRN can be constructed with reactions of at most second-order provided that the number of chemical species grows linearly with K. Bounds on the minimal number of chemical species and the minimal number of chemical reactions are presented for CRNs with K stable limit cycles and at most second order or seventh-order kinetics. We also show that CRNs with only two chemical species can have K stable limit cycles, when the order of chemical reactions grows linearly with K. Keywords Chemical reaction networks · Limit cycles · Mass action kinetics Bulletin of Mathematical Biology (2023) 85:76 https://doi.org/10.1007/s11538-023-01170-3 Bulletin of Mathematical Biology (2023) 85:76 https://doi.org/10.1007/s11538-023-01170-3 ORIGINAL ARTICLE 1 Mathematical Institute, University of Oxford, Radcliffe Observatory Quarter, Woodstock Road, Oxford OX2 6GG, UK 2 Department of Mathematics and Statistics, University of Maryland, Baltimore County, 1000 Hilltop Ci l B l i 21250 M l d USA B Radek Erban erban@maths.ox.ac.uk Hye-Won Kang hwkang@umbc.edu B Radek Erban erban@maths.ox.ac.uk 2 Department of Mathematics and Statistics, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore 21250, Maryland, USA Chemical Systems with Limit Cycles Radek Erban1 · Hye-Won Kang2 1 Mathematical Institute, University of Oxford, Radcliffe Observatory Quarter, Woodstock Road, Oxford OX2 6GG, UK B Radek Erban erban@maths.ox.ac.uk Hye-Won Kang hwkang@umbc.edu 1 Mathematical Institute, University of Oxford, Radcliffe Observatory Quarter, Woodstock Road, Oxford OX2 6GG, UK 2 Department of Mathematics and Statistics, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore 21250, Maryland, USA Radek Erban1 · Hye-Won Kang2 Received: 4 January 2023 / Accepted: 19 May 2023 / Published online: 4 July 2023 © The Author(s) 2023 Received: 4 January 2023 / Accepted: 19 May 2023 / Published online: 4 July 2023 © The Author(s) 2023 1 Introduction Chemical reaction networks (CRNs) are often modeled by reaction rate equations, which are systems of first-order, autonomous, ordinary differential equations (ODEs) describing the time evolution of the concentrations of chemical species involved. Considering CRNs which are subject to the law of mass action, their reaction rate equations have polynomials on their right-hand sides (Yu and Craciun 2018; Craciun et al. 2020). The mathematical investigation of ODEs with polynomial right-hand sides has a long history and includes a number of challenging open mathematical problems, 123 123 76 Page 2 of 29 76 Page 2 of 29 R. Erban, H.-W. Kang 76 for example, Hilbert’s 16th Problem (Ilyashenko 2002), which asks questions about the number and position of limit cycles of the planar ODE system of the form for example, Hilbert’s 16th Problem (Ilyashenko 2002), which asks questions about the number and position of limit cycles of the planar ODE system of the form dx dt = f (x, y), (1) dy dt = g(x, y), (2) (1) (2) where f (x, y) and g(x, y) are real polynomials of degree at most n. Denoting H(n) the maximum number of limit cycles for the system (1–2), neither the value of H(n) (for n ≥2) nor any upper bound on H(n) has yet been found (Ilyashenko 1991). Since a quadratic system with 4 limit cycles has been constructed (Shi 1980), we know that H(2) ≥4. Similarly, H(3) ≥13, because cubic systems with at least 13 limit cycles have been found (Li et al. 2009; Yang et al. 2010). g Considering CRNs with two chemical species undergoing chemical reactions of at most n-th order, their reaction rate equations are given in the form (1–2), where f (x, y) and g(x, y) are real polynomials of degree at most n. In particular, if we denote by C(n) the maximum number of stable limit cycles in such reaction rate equations, then we have C(n) ≤H(n). Considering CRNs with two chemical species undergoing chemical reactions of at most second order, it has been previously shown (Póta 1983; Schuman and Tóth 2003) that their reaction rate equations cannot have any limit cycles (i.e., C(2) = 0), while general ODEs with quadratic right-hand sides can have multiple limit cycles, with H(2) ≥4. 1 Introduction Then there exists a CRN with N(K chemical species which are subject to M(K) chemical reactions of at most sevent order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤K + 2 and M(K) ≤29 K. Theorem 1 provides a stronger result than finding K limit cycles in a polynomial ODE system of the form (3), because not every polynomial ODE system corresponds to a CRN and, therefore, the set of reaction rate equations is a proper subset of ODEs with polynomial right-hand sides. To make the existence of K limit cycles possible while restricting to polynomials of degree at most n = 7, we allow for more than two chemical species, replacing the ODE system (1–2) by a more general ODE system (3) with N(K) equations. In particular, the next important question is how small the CRN canbesothatithas K limitcycles.Ouranswerispartiallygiveninpart(ii)ofTheorem1 where we provide upper bounds on the number of chemical species involved and the number of chemical reactions (of at most seventh order). Another important parameter to consider is the maximum order of the chemical reactions involved, i.e., the degree n of the polynomials on the right-hand side of the ODE system (3). Since systems of at most second-order reactions (the case n = 2) are of special interest in the theory of CRNs and applications (Wilhelm 2000), we state our second main result as: Theorem 2 Let K be an arbitrary positive integer. Then there exists a CRN with N(K) chemical species which are subject to M(K) chemical reactions of at most second order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤7K + 14 and M(K) ≤42 K + 24. By restricting to second-order (bimolecular) reactions, we obtain CRNs with more realistic second-order kinetics, but our construction increases the number of species andchemical reactions involved, as it canbeseenbycomparingparts (ii) of Theorems 1 and 2. The precise definitions of CRNs, mass action kinetics, reaction rate equations and limit cycles in N-dimensional systems are given in Sect.2. 1 Introduction In particular, we observe that finding CRNs with two chemical species which have, under mass action kinetics, multiple stable limit cycles, is even more challenging than finding planar polynomial ODEs with multiple limit cycles. Considering cubic systems, we have H(3) ≥13, but most of the chemical systems (with at most third-order reactions) in the literature often have at most one limit cycle (Field and Noyes 1974; Schnakenberg 1979; Plesa et al. 2016). A chemical system with two stable limit cycles has been constructed (Plesa et al. 2017), giving C(3) ≥2, but this is still much less than 13 limit cycles which can be found in some ODE systems with cubic right-hand sides in the literature (Li et al. 2009; Yang et al. 2010). To obtain multiple stable limit cycles in chemical systems, we have to consider higher-order chemical reactions or systems with more than two chemical species (Boros and Hofbauer 2021, 2022). Considering CRNs with N chemical species undergoing chemical reactions of at most n-th order, their reaction rate equations are given as the following system of ODEs dx dt = f(x), (3) (3) (3) where x = (x1, x2, . . . , xN) ∈RN is the vector of concentrations of N chemical species and its right-hand side f : RN →RN is a vector of real polynomials of degree at most n. In this paper, we prove the following first main result: 123 Page 3 of 29 76 Chemical Systems with Limit Cycles… 76 Theorem 1 Let K be an arbitrary positive integer. Then there exists a CRN with N(K) chemical species which are subject to M(K) chemical reactions of at most seventh order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤K + 2 and M(K) ≤29 K Theorem 1 Let K be an arbitrary positive integer. Then there exists a CRN with N(K) chemical species which are subject to M(K) chemical reactions of at most seventh order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤K + 2 and M(K) ≤29 K. Theorem 1 Let K be an arbitrary positive integer. 1 Introduction In both Theorems 1 and 2, we restrict our considerations to systems described by polynomial ODEs where the degree of polynomials is bounded by a constant independent of K, i.e., we consider polynomials of the degree at most n = 7 (in Theorem 1) or at most n = 2 (in Theorem 2), and we increase the number of chemical species, N(K), as K increases, to get K stable limit cycles. Another approach is to restrict our considerations to chemical systems with only N = 2 chemical species. In Sect.8, we construct two-species CRNs with K stable limit cycles which include chemical reactions of at most n(K)-th order, where n(K) = 6K −2. This establishes our third main result: Theorem 3 Let C(n) be the maximum number of stable limit cycles of reaction rate equations (1–2) corresponding to CRNs with two chemical species undergoing chem- ical reactions of at most n-th order. Then we have C(n) ≥ n + 2 6  , (4) (4) 123 Page 4 of 29 R. Erban, H.-W. Kang 76 where the floor function ⌊.⌋denotes the integer part of a positive real number. To prove Theorems 1, 2 and 3, we first construct a planar system given by Eqs. (1–2), where f and g are continuous non-polynomial functions chosen in such a way that the ODE system (1–2) has K stable limit cycles in the positive quadrant [0, ∞)×[0, ∞). Such a planar non-polynomial ODE system is constructed in Sect.3. In Sect.4, we then increase the number of chemical species from 2 to N(K) to transform the non- polynomialODEsystemtoapolynomialone.InSect.5,wemodifythisconstructionby using an x-factorable transformation to arrive at reaction rate equations corresponding to a CRN (Samardzija et al. 1989). Theorem 1 is then proven in Sect.6 by showing that the reaction rate equations have at least K stable limit cycles. This is followed by our proofs of Theorems 2 and 3 in Sects.7 and 8, respectively. 2 Notation and Mathematical Terminology , N, (8) (8) where αi, j are real constants and νi, j are non-negative integers, for i = 1, 2, . . . , N and j = 1, 2, . . . , M. Then the polynomial ODE system (8) can be written as reaction rate equations (6) of a CRN if and only if where αi, j are real constants and νi, j are non-negative integers, for i = 1, 2, . . . , N and j = 1, 2, . . . , M. Then the polynomial ODE system (8) can be written as reaction rate equations (6) of a CRN if and only if νi, j = 0 implies αi, j ≥0 for any i = 1, 2, . . . , N and j = 1, 2, . . . , M. (9) νi, j = 0 implies αi, j ≥0 for any i = 1, 2, . . . , N and j = 1, 2, . . . , M. (9) Proof Reaction rate equations (6) are of the form (8). The non-negativity condition (9) follows from νi, j = 0 and the non-negativity of both k j and ν′ i, j in Eq. (6). Conversely, if an ODE is of the form (8) and αi, j > 0, then we can choose ν′ i, j = νi, j +1 in Eq. (6) and put the reaction rate as k j = αi, j. On the other hand, if αi, j < 0, then the condition (9) implies that νi, j ≥1, because νi, j are non-negative integers. Therefore, we can put ν′ i, j = νi, j −1 and k j = −αi, j > 0. ⊓⊔ 123 Proof Reaction rate equations (6) are of the form (8). The non-negativity condition (9) follows from νi, j = 0 and the non-negativity of both k j and ν′ i j in Eq. (6). Proof Reaction rate equations (6) are of the form (8). The non-negativity condition (9) follows from νi, j = 0 and the non-negativity of both k j and ν′ i, j in Eq. (6). j Conversely, if an ODE is of the form (8) and αi, j > 0, then we can choose ν′ i, j = νi, j +1 in Eq. (6) and put the reaction rate as k j = αi, j. 2 Notation and Mathematical Terminology Definition 1 A chemical reaction network (CRN) is defined as a collection (S, C, R) consisting of chemical species S, reaction complexes C and chemical reactions R. We denote by N the number of chemical species and by M the number of chemical reactions, i.e., |S| = N and |R| = M. Each chemical reaction is of the form N  i=1 νi, j Xi −→ N  i=1 ν′ i, j Xi, for j = 1, 2, . . . , M, (5) (5) (5) where Xi, i = 1, 2, . . . , N, are chemical species, and linear combinations N i=1 νi, j Xi and N i=1 ν′ i, j Xi of species with non-negative integers νi, j and ν′ i, j are reaction complexes. where Xi, i = 1, 2, . . . , N, are chemical species, and linear combinations N i=1 νi, j Xi and N i=1 ν′ i, j Xi of species with non-negative integers νi, j and ν′ i, j are reaction complexes. Definition 2 Let (S, C, R) be a CRN with N chemical species and M chemical reac- tions. Let xi(t) be the concentration of chemical species Xi ∈S, i = 1, 2, . . . , N. The time evolution of xi(t) is, under the assumption of the mass action kinetics, described by the reaction rate equations, which are written as a system of N ODEs in the form dxi dt (t) = M  j=1 k j (ν′ i, j −νi, j) N  ℓ=1 x νℓ, j ℓ , for i = 1, 2, . . . , N, (6) (6) where k j, j = 1, 2, . . . , M, is a positive constant called the reaction rate for the j-th reaction. where k j, j = 1, 2, . . . , M, is a positive constant called the reaction rate for the j-th reaction. To illustrate Definitions 1 and 2, we present a simple example system, which is known to have a stable limit cycle for certain parameter values (Schnakenberg 1979; Erban and Chapman 2020). Example Consider a chemical reaction network with two chemical species X1 and X2 which are subject to the following four chemical reactions 2X1 + X2 k1 −→3X1, ∅ k2 −→X1, X1 k3 −→∅, ∅ k4 −→X2. 2 Notation and Mathematical Terminology (7) (7) 123 Page 5 of 29 76 Chemical Systems with Limit Cycles… Then N = |S| = 2, M = |R| = 4 and the set of reaction complexes is given as C = {2X1 + X2, 3X1, X1, X2, ∅}. Denote ν and ν′ as matrices with elements νi, j and ν′ i, j. Then, both are N × M = 2 × 4 matrices given by ν = 2 0 1 0 1 0 0 0  , ν′ = 3 1 0 0 0 0 0 1  . The reaction rate equations (6) are given as the following system of two ODEs The reaction rate equations (6) are given as the following system of two ODEs dx1 dt (t) = k1x2 1x2 + k2 −k3x1, dx2 dt (t) = −k1x2 1x2 + k4, which describes the time evolution of the concentrations x1(t) and x2(t) of chemical species X1 and X2, respectively. I l h i i (6) i D fii i 2 ODE f h f (3) which describes the time evolution of the concentrations x1(t) and x2(t) of chemical species X1 and X2, respectively. In general, the reaction rate equations (6) in Definition 2 are ODEs of the form (3), where the right-hand side f : RN →RN is a vector of real polynomials. However, not every polynomial ODE system can be obtained as the reaction rate equations of a CRN as we formalize in Lemma 1, where we provide a necessary and sufficient condition (9) when a polynomial ODE system can be written as reaction rate equations of a CRN. The condition is that any polynomial right-hand side terms not proportional to xi in the equation for chemical species Xi are non-negative. The necessity of the condition (9) is shown based on the fact that any reaction rate equations of a CRN satisfy this condition. The sufficiency of the condition is provided by showing that each term satisfying the condition in a polynomial ODE system can correspond to a chemical reaction. Lemma 1 Consider a system of N ODEs with polynomial right-hand sides describing the time evolution of xi(t), i = 1, 2, . . . , N, in the form dxi dt (t) = M  j=1 αi, j N  ℓ=1 x νℓ, j ℓ , for i = 1, 2, . . . 2 Notation and Mathematical Terminology A stable limit cycle is a trajectory xc(t) for t ∈[0, ∞) such that (i) xc(t) is a solution of the ODE system (3), (i) xc(t) is a solution of the ODE system (3), (ii) There exists a positive constant T > 0 such that xc(T ) = xc(0) and xc(t) ̸= xc(0) for 0 < t < T , (iii) There exists ε > 0 such that (ii) There exists a positive constant T > 0 such that xc(T ) = xc(0) and ( ) ̸ (0) f 0 T (ii) There exists a positive constant T > 0 such that xc(T ) = xc(0) and xc(t) ̸= xc(0) for 0 < t < T , (iii) There exists ε > 0 such that (ii) There exists a positive constant T > 0 such that xc(T ) = xc(0) and xc(t) ̸= xc(0) for 0 < t < T , (iii) There exists ε > 0 such that xc(t) ̸= xc(0) for 0 < t < T , (iii) There exists ε > 0 such that xc(t) ̸= xc(0) for 0 < t < T , (iii) There exists ε > 0 such that dist{x(0), xc} < ε implies dist{x(t), xc} →0 as t →∞. dist{x(0), xc} < ε implies dist{x(t), xc} →0 as t →∞. In Definition 3, the constant T is the period of the limit cycle and the property (iii) states that the limit cycle attracts all trajectories which start sufficiently close to it. The distance in the property (iii) of Definition 3 is the Euclidean distance defined by dist{z, xc} = min t∈[0,T ] dist{z, xc(t)} = min t∈[0,T ] N  i=1 zi −xc,i(t) 2 1/2 for z = [z1, z2, . . . , zN] ∈RN and xc(t) = [xc,1(t), xc,2(t), . . . , xc,N(t)] ∈RN. for z = [z1, z2, . . . , zN] ∈RN and xc(t) = [xc,1(t), xc,2(t), . . . , xc,N(t)] ∈RN. for z = [z1, z2, . . . , zN] ∈RN and xc(t) = [xc,1(t), xc,2(t), . . . , xc,N(t)] ∈RN. 2 Notation and Mathematical Terminology On the other hand, if αi, j < 0, then the condition (9) implies that νi, j ≥1, because νi, j are non-negative integers. Therefore, we can put ν′ i, j = νi, j −1 and k j = −αi, j > 0. ⊓⊔ Page 6 of 29 R. Erban, H.-W. Kang 76 In this paper, we prove the existence of limit cycles in chemical systems in Sects.6, 7 and 8 by proving the existence of limit cycles in systems of ODEs (8) with polynomial right-hand sides satisfying the condition (9). Then the approach used in the proof of Lemma 1 can be used to construct the corresponding CRN. However, the construction of a CRN corresponding to reaction rate equations is not unique. For example, consider a term of the form −x3 1 on the right-hand side of Eq. (8). Using the construction in the proof of Lemma 1, it would correspond to the chemical reaction 3X −→2X with the rate constant equal to 1, but the same term can also correspond to the chemical reaction 3X −→X with the rate constant equal to 1/2. We conclude this section by a formal definition of a stable limit cycle. In this paper, we prove the existence of limit cycles in chemical systems in Sects.6, 7 and 8 by proving the existence of limit cycles in systems of ODEs (8) with polynomial right-hand sides satisfying the condition (9). Then the approach used in the proof of Lemma 1 can be used to construct the corresponding CRN. However, the construction of a CRN corresponding to reaction rate equations is not unique. For example, consider a term of the form −x3 1 on the right-hand side of Eq. (8). Using the construction in the proof of Lemma 1, it would correspond to the chemical reaction 3X −→2X with the rate constant equal to 1, but the same term can also correspond to the chemical reaction 3X −→X with the rate constant equal to 1/2. We conclude this section by a formal definition of a stable limit cycle. Definition 3 Consider a system of N ODEs given by (3), where their right-hand side f : RN →RN is continuous. 3 Planar ODE Systems with Arbitrary Number of Limit Cycles In this section, we construct a planar ODE system of the form (1–2) with K limit cycles in the positive quadrant. It is constructed as a function of 2K parameters denoted by a1, a2, . . . , aK and b1, b2, . . . , bK , as dx dt = K  k=1 (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk) 1 + (x −ak)6 + (y −bk)6 = f (x, y), (10) dy dt = K  k=1 (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak) 1 + (x −ak)6 + (y −bk)6 = g(x, y). (11) (10) An illustrative dynamics of the ODE system (10–11) is shown in Fig.1(a) for K = 4, An illustrative dynamics of the ODE system (10–11) is shown in Fig.1(a) for K = 4, 123 Page 7 of 29 76 Chemical Systems with Limit Cycles… (b) (a) 0 2 4 6 8 0 2 4 6 8 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Fig. 1 a Twenty illustrative trajectories of the ODE system (10–11) for K = 4 and the parameter choices a1 = b1 = a2 = b3 = 2 and a3 = b2 = a4 = b4 = 6. As t →∞, all presented trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. b Twenty illustrative trajectories of the ODE system (10–11) for K = 4 and the parameter choices a1 = b1 = a2 = b3 = 2 and a3 = b2 = a4 = b4 = 4. As t →∞, some trajectories converge to the stable limit cycle denoted by the black dashed line, while some trajectories, which started inside the limit cycle, converge to the stable fixed point denoted as the red dot (Color figure online) (b) 0 1 2 3 4 5 6 0 1 2 3 4 5 6 (a) 0 2 4 6 8 0 2 4 6 8 (b) (a) Fig. 1 a Twenty illustrative trajectories of the ODE system (10–11) for K = 4 and the parameter choices a1 = b1 = a2 = b3 = 2 and a3 = b2 = a4 = b4 = 6. As t →∞, all presented trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. 3 Planar ODE Systems with Arbitrary Number of Limit Cycles b Twenty illustrative trajectories of the ODE system (10–11) for K = 4 and the parameter choices a1 = b1 = a2 = b3 = 2 and a3 = b2 = a4 = b4 = 4. As t →∞, some trajectories converge to the stable limit cycle denoted by the black dashed line, while some trajectories, which started inside the limit cycle, converge to the stable fixed point denoted as the red dot (Color figure online) where the ODE system has four limit cycles, which is highlighted in Fig.1a by plotting some representative trajectories. The existence of K stable limit cycles for the ODE system (10–11) can also be proven analytically, as it is done in Lemma 2. Remark To construct the ODE system (10–11), one can first consider a planar ODE system written as dr/dt = r(1 −r2) in the polar coordinate system. It has one stable limit cycle (a unit circle) and can be rewritten in the Cartesian coordinate system as dx dt = x(1 −x2 −y2) −y, (12) dy dt = y(1 −x2 −y2) + x. (13) (12) (13) To obtain the ODE system (10–11), we translate the right-hand sides of ODEs (12– 13) by (x, y) →(x −ak, y −bk) so that the corresponding limit cycle is centered at the point (ak, bk). Then we add the K terms (corresponding to K limit cycles) together with suitable weights. Note that the denominator of the k-th term in (10–11) is approximately one when (x, y) is close to (ak, bk), while the k-th term in (10–11) is approximately zero when (x, y) is far away from the point (ak, bk). In Fig.1a, we have presented an example with K = 4 and parameter choices (a1, b1) = (2, 2), (a2, b2) = (2, 6), (a3, b3) = (6, 2) and (a4, b4) = (6, 6). In particular, the distance between points (ai, bi), i = 1, 2, 3, 4, is at least four. If we decrease this distance, then the ODE system (10–11) can have less limit cycles. This is highlighted in Fig.1b, where we present an example with K = 4 and parameter R. Erban, H.-W. Kang 76 Page 8 of 29 76 choices (a1, b1) = (2, 2), (a2, b2) = (2, 4), (a3, b3) = (4, 2) and (a4, b4) = (4, 4). 3 Planar ODE Systems with Arbitrary Number of Limit Cycles In Fig.1b, we observe that there is only one limit cycle, denoted as the black dashed line. This limit cycle is stable and a number of illustrative trajectories converge to this limit cycle as t →∞. However, there is also a stable equilibrium point at (3, 3), which attracts some of the trajectories. In particular, we can only expect that the ODE system (10–11) will have K stable limit cycles provided that points (ai, bi) are sufficiently separated. This is formally proven in Lemma 2, by defining K disjoint open sets (15), where each is an annulus with a center (ai, bi) for i = 1, 2, . . . , K. First, we show that each annulus does not contain any equilibrium points. Then, we show that a directional vector of the ODE system (10–11) on the boundary of each annulus points inside the domain. Applying the Poincaré-Bendixson theorem (Strogatz 2015), we conclude that each annulus contains at least one stable limit cycle; thus, the ODE system (10–11) has at least K stable limit cycles. Lemma 2 Let us assume that Lemma 2 Let us assume that (ai −a j)2 + (bi −b j)2 > 15  K 2/3 + 2  for all i ̸= j, (14) (14) where i, j = 1, 2, . . . , K. Then the ODE system (10–11) has at least K stable limit cycles. Proof We define the sets i =  (x, y) : 1/2 < (x −ai)2 + (y −bi)2 < 2  , for i = 1, 2, . . . , K. (15) Then the condition (14) implies that i ∩ j = ∅, for all i ̸= j, where i, j = 1, 2, . . . , K, i.e., the sets i are pairwise disjoint sets. We will show that each of them contains at least one stable limit cycle. The boundary of  consists of two parts: outer and inner circles defined by i.e., the sets i are pairwise disjoint sets. We will show that each of them contains at least one stable limit cycle. The boundary of  consists of two parts: outer and inner circles defined by ∂i1 =  (x, y) : (x −ai)2 + (y −bi)2 = 2  (16) (16) and ∂i2 =  (x, y) : (x −ai)2 + (y −bi)2 = 1/2  , (17) (17) respectively, that is, ∂i = ∂i1 ∪∂i2. Define the following functions for k = 1, 2, . . . , K: fk(z1, z2) = z1 1 −z2 1 −z2 2  −z2 1 + z6 1 + z6 2 , (18) (18) 123 123 emical Systems with Limit Cycles… Page 9 of 29 76 Chemical Systems with Limit Cycles… Page 9 of 29 76 Chemical Systems with Limit Cycles… Page 9 of 29 76 gk(z1, z2) = z2 1 −z2 1 −z2 2  + z1 1 + z6 1 + z6 2 . (19) gk(z1, z2) = z2 1 −z2 1 −z2 2  + z1 1 + z6 1 + z6 2 . Lemma 2 Let us assume that (19) (19) Then, the ODE system (10–11) can be rewritten as Then, the ODE system (10–11) can be rewritten as Then, the ODE system (10–11) can be rewritten as dx dt = f (x, y), (20) dy dt = g(x, y), (21) dx dt = f (x, y), (20) dy dt = g(x, y), (21) (20) (21) where where f (x, y) = K  k=1 fk(x −ak, y −bk) and g(x, y) = K  k=1 gk(x −ak, y −bk). (22) First, we will show that i for i = 1, 2, . . . , K does not contain any equilibrium points. Let us consider any point (x∗, y∗) ∈i. Substituting First, we will show that i for i = 1, 2, . . . , K does not contain any equilibrium points. Let us consider any point (x∗, y∗) ∈i. Substituting x∗= ai + r cos θ, y∗= bi + r sin θ, (23) (23) in the terms for k = i in (22), we obtain in the terms for k = i in (22), we obtain n the terms for k = i in (22), we obtain f (x∗, y∗)= r cos θ {1 −r2} −r sin θ 1 + r6 cos6 θ + r6 sin6 θ + K  k=1,k̸=i fk(x∗−ak, y∗−bk), (24) g(x∗, y∗)= r sin θ {1 −r2} + r cos θ 1 + r6 cos6 θ + r6 sin6 θ + K  k=1,k̸=i gk(x∗−ak, y∗−bk). (25) (24) (25) he first terms in (24) and (25) can be rewritten as 4r  (r2 −1)2 + 1 sin(θ + ˜θ) 4 + r6 4 −3 sin2 2θ , (26) (26) where ˜θ = α with tan α = r2 −1 and π/2 < α < 3π/2 in the case of (24) and ˜θ = α −π/2 in the case of (25). Since we have where ˜θ = α with tan α = r2 −1 and π/2 < α < 3π/2 in the case of (24) and ˜θ = α −π/2 in the case of (25). Lemma 2 Let us assume that Since we have max(| sin(θ + α)|, | sin(θ + α −π/2)|) > 1/ √ 2 max(| sin(θ + α)|, | sin(θ + α −π/2)|) > 1/ √ 2 for any θ and α, at least one of the two terms expressed in the form (26) is greater than for any θ and α, at least one of the two terms expressed in the form (26) is greater than 1 √ 2 r  (r2 −1)2 + 1 1 + r6 , 1 √ 2 r  (r2 −1)2 + 1 1 + r6 , 123 123 Page 10 of 29 R. Erban, H.-W. Kang 76 which has a minimum √ 2/9 when 1/2 < r2 < 2. Therefore, at least one of the absolute values of the i-th components, fi(x∗−ai, y∗−bi) and gi(x∗−ai, y∗−bi), in (24) and (25) at any point (x∗, y∗) ∈i is greater than or equal to √ 2/9. Without loss of generality, we assume | fi(x∗−ai, y∗−bi)| ≥|gi(x∗−ai, y∗−bi)|. Then we have | fi(x∗−ai, y∗−bi)| ≥ √ 2/9. We want to show that the second term in (24) (i.e., the sum) has a smaller magnitude than the first term fi(x∗−ai, y∗−bi) so that we could conclude that f (x∗, y∗) ̸= 0. The k-th component in the second term in (24) is bounded by | fk(z1, z2)| ≤|z1| |1 −z2 1 −z2 2| + |z2| |1 + z6 1 + z6 2| (27) (27) where (z1, z2) = (x∗−ak, y∗−bk). Denoting c2 = z2 1 + z2 2, we have where (z1, z2) = (x∗−ak, y∗−bk). Denoting c2 = z2 1 + z2 2, we have 1 + c6 4 ≤1 + z6 1 + z6 2 ≤1 + c6. (28) (28) Using |zi| ≤c and (28), we estimate (27) as Using |zi| ≤c and (28), we estimate (27) as | fk(z1, z2)| ≤c |1 −c2| + 1 1 + c6/4 . (29) (29) Since (x∗, y∗) ∈i and (ak, bk) ∈k where k ̸= i, our assumption (14) implies that c2 ≥2. Thus, (29) becomes | fk(z1, z2)| ≤ c3 1 + c6/4 ≤4 c3 . (30) (30) Therefore, the magnitude of the second term in (24) is bounded by 4(K −1)/c3. Since | fi(x∗−ai, y∗−bi)| ≥ √ 2/9, a sufficient condition for f (x∗, y∗) ̸= 0 is Therefore, the magnitude of the second term in (24) is bounded by 4(K −1)/c3. Lemma 2 Let us assume that Since | fi(x∗−ai, y∗−bi)| ≥ √ 2/9, a sufficient condition for f (x∗, y∗) ̸= 0 is 4(K −1) c3 < √ 2 9 . (31) (31) Using the assumption (14), the distance c =  (x∗−ak)2 + (y∗−bk)2 is bounded by Using the assumption (14), the distance c =  (x∗−ak)2 + (y∗−bk)2 is bounded by c >  (ai −ak)2 + (bi −bk)2 − √ 2 >  15 K 2/3 + 2 − √ 2 , (32) (32) which implies the sufficient condition (31). Therefore, (x∗, y∗) is not an equilibrium point. which implies the sufficient condition (31). Therefore, (x∗, y∗) is not an equilibrium point. 123 123 emical Systems with Limit Cycles… Page 11 of 29 76 Chemical Systems with Limit Cycles… Page 11 of 29 76 76 Next, consider an arbitrary point (xb, yb) ∈∂i1. Let us calculate the scalar product of vectors (xb −ai, yb −bi) and f (xb, yb), g(xb, yb) . (33) (33) Using (22), we obtain this scalar product as Using (22), we obtain this scalar product as (xb −ai) fi(xb −ai, yb −bi) + (yb −bi)gi(xb −ai, yb −bi) +(xb −ai) K  k=1,k̸=i fk(xb −ak, yb −bk) + (yb −bi) K  k=1,k̸=i gk(xb −ak, yb −bk). (34) The first two terms in (34) become first two terms in (34) become The first two terms in (34) become The first two terms in (34) become The first two terms in (34) become −2 1 + (xb −ai)6 + (yb −ai)6 , −2 1 + (xb −ai)6 + (yb −ai)6 , which has a magnitude greater than 2/9 by using (28) with c2 = (xb −ai)2 + (yb − bi)2 = 2. Using (30), |xb −ai| ≤ √ 2 and |yb −bi| ≤ √ 2, we can estimate the third and fourth terms in (34), namely, we have |(xb −ai) fk(z1, z2)| ≤4 √ 2 d3 and |(yb −bi) gk(z1, z2)| ≤4 √ 2 d3 , (35) (35) where d2 = (xb −ak)2 + (yb −bk)2. Then the sum of the third and fourth terms in (34) is bounded by 8 √ 2(K −1)/d3. Therefore, the sufficient condition that the scalar product in (34) is negative is where d2 = (xb −ak)2 + (yb −bk)2. Then the sum of the third and fourth terms in (34) is bounded by 8 √ 2(K −1)/d3. Lemma 2 Let us assume that Therefore, the sufficient condition that the scalar product in (34) is negative is 8 √ 2(K −1) d3 < 2 9. (36) (36) Using the assumption (14), the distance d =  (xb −ak)2 + (yb −bk)2 is bounded by d >  (ai −ak)2 + (bi −bk)2 − √ 2 >  15 K 2/3 + 2 − √ 2 , (37) (37) which implies the sufficient condition (36). Therefore, the vector f (xb, yb), g(xb, yb) f (xb, yb), g(xb, yb) always points inside the domain i for each boundary point (xb, yb) ∈∂i1. Similarly, for an arbitrary point (xb, yb) ∈∂i2, we can show that the scalar product of vectors in (33) is always positive due to that the sum of the first two terms in (34) 123 R. Erban, H.-W. Kang Page 12 of 29 76 is equal to 1/4 1 + (xb −ai)6 + (yb −bi)6 , 1 + (xb −ai)6 + (yb −bi)6 , which is greater than 2/9 by using (28) with c2 = 1/2, and the sum of the third and fourth terms in (34) is bounded by 8(K −1)/(d3√ 2). Therefore, the sufficient condition that the scalar product in (34) is positive is 8(K −1) d3√ 2 < 2 9 , 8(K −1) d3√ 2 < 2 9 , 8(K −1) d3√ 2 < 2 9 , which is a weaker condition than the condition (36), i.e., it is again satisfied because of our assumption (14). This implies that the scalar product in (34) is positive. Thus, the directional vector always points inside the domain i on all parts of the boundary ∂i. which is a weaker condition than the condition (36), i.e., it is again satisfied because of our assumption (14). This implies that the scalar product in (34) is positive. Thus, the directional vector always points inside the domain i on all parts of the boundary ∂i. Therefore, applying Poincaré-Bendixson theorem (Strogatz 2015), we conclude that each i contains at least one stable limit cycle. Since i, i = 1, 2, . . . , K, are pairwise disjoint, this implies that the ODE system (10–11) has at least K stable limit cycles. ⊓⊔ 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles The dynamics of the original ODE system (10–11) with the initial condition (x(0), y(0)) = (x0, y0) is the same as the dynamics of the extended ODE system (39–41), when we initialize the additional variables by for i = 1, 2, . . . , K. The dynamics of the original ODE system (10–11) with the initial condition (x(0), y(0)) = (x0, y0) is the same as the dynamics of the extended ODE system (39–41), when we initialize the additional variables by ui(0) = 1 1 + (x0 −ai)6 + (y0 −bi)6 , for i = 1, 2, . . . , K. (42) (42) However, when we use a general initial condition, However, when we use a general initial condition, (x(0), y(0), u1(0), u2(0), . . . , uK (0)) ∈RK+2, the trajectory of the extended ODE system (39–41) may become unbounded and it may not converge to a limit cycle. To illustrate this behavior, let us consider the initial condition the trajectory of the extended ODE system (39–41) may become unbounded and it may not converge to a limit cycle. To illustrate this behavior, let us consider the initial condition ui(0) = c 1 + (x0 −ai)6 + (y0 −bi)6 , for i = 1, 2, . . . , K, (43) (43) where c > 0 is a constant. If c = 1, then the initial condition (43) reduces to (42). In particular, Fig.1a shows an illustrative behavior of both the extended ODE system (39– 41) for c = 1 and the planar ODE system (10–11), assuming that we use a sufficiently accurate numerical method to solve ODEs (39–41) and plot the projection of the calculated trajectory to the (x, y)-plane. Changing c = 1 to c = 0.5, we plot the dynamics of the extended ODE system in Fig.2a, where the black dots denote the end points of the calculated trajectories at the final time (t = 100). We observe that only the trajectories which started ‘inside a limit cycle’ (i.e., their projections to the (x, y)-plane are initially inside a black dashed circle) seem to converge to it, while the other trajectories do not seem to approach the ‘limit cycles’. This is indeed the case even if we continue these trajectories for times t > 100. 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles Considering an auxiliary variable ui = 1 1 + (x −ai)6 + (y −bi)6 , for i = 1, 2, . . . , K, (38) (38) we can formally convert the non-polynomial ODE system (10–11) to a system of (K + 2) ODEs with polynomial right-hand sides (Kerner 1981). We obtain we can formally convert the non-polynomial ODE system (10–11) to a system of (K + 2) ODEs with polynomial right-hand sides (Kerner 1981). We obtain dx dt = K  k=1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  , (39) dy dt = K  k=1 uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (40) dui dt = −6u2 i (x −ai)5 K  k=1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  −6u2 i (y −bi)5 K  k=1 uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (41) dx dt = K  k=1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  , (39) dy dt = K  k 1 uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (40)  1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  , (39) (39) (40) dui dt = −6u2 i (x −ai)5 K  k=1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  −6u2 i (y −bi)5 K  uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , dui dt = −6u2 i (x −ai)5  k=1 uk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  K −6u2 i (y −bi)5 K  k=1 uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , −6u2 i (y −bi)5 K  k=1 uk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (41) 123 emical Systems with Limit Cycles… Page 13 of 29 76 Chemical Systems with Limit Cycles… Page 13 of 29 76 76 for i = 1, 2, . . . , K. 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles In fact, depending on the accuracy of the numerical method used, all trajectories eventually stop somewhere in the phase plane, because ui(t) →0 as t →∞. p p On the other hand, considering the extended ODE system (39–41) with the initial condition (43) for c > 1, some additional variables ui(t) tend to infinity as t →∞, and we again do not observe sustained oscillations in our numerical experiments (results not shown). In particular, the formal conversion of the non-polynomial ODE system (10–11) into the polynomial system (39–41) does not preserve the dynamics well. Therefore, we augment our polynomial ODE system (10–11) in a different way. We introduce K new variables vi, i = 1, 2, . . . , K, and formulate the extended ODE system as the following (K + 2) equations: dx dt = K  k=1 vk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  , (44) dy dt = K  k=1 vk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (45) (44) (45) 123 76 Page 14 of 29 R. Erban, H.-W. Kang 76 (b) (a) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Fig. 2 a Twenty illustrative trajectories of the ODE system (39–41) for K = 4, the parameter choices a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6 and initial condition (43) with c = 1/2. The black dots denote the final position of each calculated trajectory at time t = 100. The black dashed lines are limit cycles shown in Fig.1a. b Twenty illustrative trajectories of the ODE system (44–46) for K = 4, the parameter choices ε = 1, a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6 and the initial condition (43) with c = 1/2. As t →∞, all trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. The black dots denote the final position of each calculated trajectory at time t = 100 (Color figure online) (a) 0 2 4 6 8 0 2 4 6 8 (b) 0 2 4 6 8 0 2 4 6 8 (a) (b) Fig. 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles 2 a Twenty illustrative trajectories of the ODE system (39–41) for K = 4, the parameter choices a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6 and initial condition (43) with c = 1/2. The black dots denote the final position of each calculated trajectory at time t = 100. The black dashed lines are limit cycles shown in Fig.1a. b Twenty illustrative trajectories of the ODE system (44–46) for K = 4, the parameter choices ε = 1, a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6 and the initial condition (43) with c = 1/2. As t →∞, all trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. The black dots denote the final position of each calculated trajectory at time t = 100 (Color figure online) ε dvi dt =1 −vi  1 + (x −ai)6 + (y −bi)6 , for i = 1, 2, . . . , K, (46) (46) where ε > 0 is a constant. The first two ODEs (44–45) are the same as ODEs (39–40) with vk taking place of uk. The difference is in the dynamics of the additional vari- ables, i.e., in Eq. (46) which removes the non-polynomial factor (38) in a different way. Rather than defining new variable ui in the form (38) and deriving ODEs which have equivalent dynamics to the ODE system (10–11) for very special initial condi- tion (42), we have written the ODE (46) in such a way that it formally recovers the non-polynomial factor (38) in the limit ε →0, which will be used in our proof of Lemma 3, where we consider small values of ε. However, even for larger values of ε, the ODE system (44–46) has multiple limit cycles for general initial conditions, as it is illustrated for ε = 1 and K = 4 in Fig.2b, where all plotted trajectories finish on a limit cycle (see the final calculated positions, at time t = 100, plotted as black dots). y p p Next, we prove that the extended ODE system (44–46) has K limit cycles in the sense of Definition 3 for general values of K. 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles Since (44–46) is a system of (K + 2) ODEs, we cannot directly apply the Poincaré-Bendixson theorem as we did for the planar system in the proof of Lemma 2. While one possible approach to proving the existence of limit cycles is to work with generalizations of the Poincaré-Bendixson the- orem to higher-dimensional ODEs (Hirsch 1982; Li and Muldowney 1996; Sanchez 2010), we will base our proof of Lemma 3 on the application of Tikhonov’s theo- rem (Tikhonov 1952; Klonowski 1983) and the result of Lemma 2. In particular, we show that the extended system (44–46) is a polynomial system which has K limit cycles for sufficiently small values of ε provided that the points (ai, bi) are suffi- 123 Page 15 of 29 76 Chemical Systems with Limit Cycles… ciently separated, as we have previously established in Lemma 2 for the planar ODE system (10–11). ciently separated, as we have previously established in Lemma 2 for the planar ODE system (10–11). Lemma 3 Let us assume that parameters ai > 0 and bi > 0, i = 1, 2, . . . , K, satisfy the inequality (14). Then there exists ε0 > 0 such that the ODE system (44–46) has at least K stable limit cycles for all ε ∈(0, ε0). Proof Let us consider ε = 0. Then the right-hand side of the ODE (46) is equal to zero. This equation can be solved for vi, i = 1, 2, . . . , K, to obtain vi = qi(x, y), where we define qi(x, y) = 1 1 + (x −ai)6 + (y −bi)6 . (47) (47) Substituting vi = qi(x, y) into (44–45), we obtain that the reduced problem in the sense of Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983) is equal to Substituting vi = qi(x, y) into (44–45), we obtain that the reduced problem in the sense of Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983) is equal to dx dt = f (x, y), (48) dy dt = g(x, y), (49) (48) (49) where functions f (·, ·) and g(·, ·) are defined in (10) and (11). This means that the reduced system (48–49) corresponding to the fast–slow extended ODE system (44– 46) is the same as our original non-polynomial ODE system (10–11). 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles Therefore, using Lemma 2, we know that the reduced system (48–49) has (at least) K stable limit cycles in the sense of Definition 3, i.e., there exist K solutions (xc,i(t), yc,i(t)) for t ∈[0, ∞), i = 1, 2, . . . , K, (50) (50) of the reduced system (48–49) which are periodic with period Ti > 0 for i = 1, 2, . . . , K. Moreover, there exist εi > 0, i = 1, 2, . . . , K, such that any solution (x(t), y(t)) of the reduced systems (48–49) approaches the limit cycle (xc,i(t), yc,i(t)) as t →∞provided that the initial condition (x(0), y(0)) satisfies min t∈[0,Ti] x(0) −xc,i(t) 2 + y(0) −yc,i(t) 2 < εi. (51) (51) Next, we define pairwise disjoint sets i ⊂RK+2 for i = 1, 2, . . . , K by Next, we define pairwise disjoint sets i ⊂RK+2 for i = 1, 2, . . . , K by i =  (x, y, v1, v2, . . . , vK ) ∈RK+2 such that min t∈[0,Ti] x −xc,i(t) 2 + y −yc,i(t) 2 + K  j=1 v j −q j(xc,i(t), yc,i(t)) 2 < εi  , (52) i =  (x, y, v1, v2, . . . , vK ) ∈RK+2 such that  min t∈[0,Ti] x −xc,i(t) 2 + y −yc,i(t) 2 + K  j=1 v j −q j(xc,i(t), yc,i(t)) 2 < εi  , (52) 123 123 6 Page 16 of 29 R. Erban, H.-W. Kang 76 where functions q j(·, ·) are defined by (47). Let us define where functions q j(·, ·) are defined by (47). Let us define ε0 = min i∈{1,2,...,K} εi. ε0 = min i∈{1,2,...,K} εi. Let ε ∈(0, ε0) be chosen arbitrarily. To show that the extended fast–slow polynomial ODE system (44–46) has (at least) K stable limit cycles, it is sufficient to show that each set i contains one stable limit cycle. We will do this by applying Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983). Considering the ODEs (46) for i = 1, 2, . . . , K, where x > 0 and y > 0 are taken as parameters, we obtain the adjoined system as a K-dimensional system of ODEs with an isolated stable equilibrium point [q1(x, y), q2(x, y), . . . , qK (x, y)], where qi(·, ·) is defined in (47). 4 ODE Systems with Polynomial Right-Hand Sides and Arbitrary Number of Limit Cycles This equilibrium point attracts the solutions of the adjoined system for any initial condition. Therefore, the ODE system (44–46) has a limit cycle in i. Moreover, this limit cycle attracts any solution x(t), y(t), v1(t), v2(t), . . . , vK (t) of the ODE system (44–46) with the initial condition satisfying x(0), y(0), v1(0), v2(0), . . . , vK (0) ∈i. ⊓⊔ ⊓⊔ 5 Chemical Systems with Arbitrary Many Limit Cycles To construct a CRN with K limit cycles, we first construct a system of ODEs with polynomial right-hand sides which satisfy the condition (9) in Lemma 1, i.e., it will be a system of reaction rate equations, which correspond to a CRN. Once we have such reaction rate equations, there are infinitely many CRNs described by them, so we conclude this section by specifying some illustrative CRNs corresponding to the derived reaction rate equations. Our starting point is the polynomial ODE system (44–46), which has K limit cycles provided that the conditions of Lemma 3 are satisfied. The reaction rate equations are constructed by applying the so-called x-factorable transformation (Plesa et al. 2016) to the right-hand sides of equations (44) and (45). We do not modify the right-hand sides of ODEs (46), because they already satisfy the conditions of Definition 2. We obtain the ODE system: dx dt = K  k=1 x vk  (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk)  , (53) dy dt = K  k=1 y vk  (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak)  , (54) ε dvi dt =1 −vi  1 + (x −ai)6 + (y −bi)6 , for i = 1, 2, . . . , K. (55) (53) (55) The illustrative dynamics of the ODE system (53–55) is presented in Fig.3a, where we use the same parameters as we use in Fig.2b for the ODE system (44–46). We observe that the presented trajectories converge to one of the four limit cycles as in Fig.2b. The shape of the limit cycles is slightly modified by using the x-factorable transformation, but the limit cycles are still there as we formally prove in Sect.6. 123 Page 17 of 29 76 Chemical Systems with Limit Cycles… Page 17 of 29 76 76 (b) (a) 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 2 4 6 8 10 2 4 6 8 10 Fig. 3 a Twenty illustrative trajectories of the ODE system (53–55) for K = 4, the parameter choices a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6, ε = 1 and the initial condition (43) with c = 1/2. 5 Chemical Systems with Arbitrary Many Limit Cycles As t →∞, all trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. As in Fig.2, the black dots denote the final position of each calculated trajectory at time t = 100. b Twenty illustrative trajectories of the ODE system (53–55) for K = 9, the parameter choices a1 = b1 = a2 = b3 = a6 = b7 = 2, a3 = b2 = a4 = b4 = a5 = b8 = 6, a7 = a8 = a9 = b5 = b6 = b9 = 10, ε = 1 and the initial condition (56). As t →∞, all trajectories approach one of the nine limit cycles, which are plotted as the black dashed lines. The black dots denote the final position of each calculated trajectory at time t = 100 (Color figure online) (a) 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 (b) 2 4 6 8 10 2 4 6 8 10 (b) (a) Fig. 3 a Twenty illustrative trajectories of the ODE system (53–55) for K = 4, the parameter choices a1 = b1 = a2 = b3 = 2, a3 = b2 = a4 = b4 = 6, ε = 1 and the initial condition (43) with c = 1/2. As t →∞, all trajectories approach one of the four limit cycles, which are plotted as the black dashed lines. As in Fig.2, the black dots denote the final position of each calculated trajectory at time t = 100. b Twenty illustrative trajectories of the ODE system (53–55) for K = 9, the parameter choices a1 = b1 = a2 = b3 = a6 = b7 = 2, a3 = b2 = a4 = b4 = a5 = b8 = 6, a7 = a8 = a9 = b5 = b6 = b9 = 10, ε = 1 and the initial condition (56). As t →∞, all trajectories approach one of the nine limit cycles, which are plotted as the black dashed lines. The black dots denote the final position of each calculated trajectory at time t = 100 (Color figure online) The x-factorable transformations modify the dynamics on the x-axis and y-axis. 5 Chemical Systems with Arbitrary Many Limit Cycles In Fig.3a, we present illustrative trajectories which all start with the positive values of x(0) and y(0), while in Fig.2b, some of the illustrative trajectories have zero initial values of x(0) and y(0). To get a comparable plot, we use the same initial conditions in both Fig.2b and Fig.3a, with the only exception that all initial conditions with x(0) = 0 (resp. y(0) = 0) in Fig.2(b) are replaced by x(0) = 1/2 (resp. y(0) = 1/2) in Fig.3a. We note that if we start a trajectory of the ODE system (53–55) on the x-axis or the y-axis, then it stays on the axis. In Fig.3b, we present illustrative dynamics of the ODE system (53–55) for K = 9, showing that each computed trajectory converges to one of the 9 limit cycles denoted by black dashed lines. To illustrate that this behavior does not require special choices of initial conditions, we used different initial conditions for x(0) and y(0) together with the initial conditions for variables vi satisfying vi(0) = 1, for i = 1, 2, . . . , K. (56) (56) However, a similar figure can be obtained if we replace (56) with the initial condi However, a similar figure can be obtained if we replace (56) with the initial condition (43) or if we initialize all values of vi i = 1 2 K as zero (results not shown) However, a similar figure can be obtained if we replace (56) with the initial condition (43), or if we initialize all values of vi, i = 1, 2, . . . , K as zero (results not shown). A CRN corresponding to reaction rate equations (53–55) can be obtained (by apply- ing the construction in the proof of Lemma 1) as the CRN with K +2 chemical species, i i h i i D fii i 1 h (43), or if we initialize all values of vi, i = 1, 2, . . . , K as zero (results not shown). A CRN corresponding to reaction rate equations (53–55) can be obtained (by apply- ing the construction in the proof of Lemma 1) as the CRN with K +2 chemical species, i.e., using the notation in Definition 1, we have S = {X, Y, V1, V2, . . . , VK } . (57) (57) 76 Page 18 of 29 R. Erban, H.-W. 5 Chemical Systems with Arbitrary Many Limit Cycles Kang To specify the reaction complexes and chemical reactions, we expand the right-hand side of reaction rate equations (53–55). First, we rewrite ODEs (55) as dvi dt = −ki,1 vi + ki,2 vi x + ki,3 vi y −ki,4 vi x2 −ki,5 vi y2 + ki,6 vi x3 + ki,7 vi y3 −ki,8 vi x4 −ki,9 vi y4 + ki,10 vi x5 + ki,11 vi y5 −vi x6/ε −vi y6/ε + 1/ε, (58) (58) (58) where ki, j, i = 1, 2, . . . , K, j = 1, 2, . . . , 11, are positive constants given by where ki, j, i = 1, 2, . . . , K, j = 1, 2, . . . , 11, are positive constants given by ki,1 = (1 + a6 i + b6 i )/ε, ki,2 = 6a5 i /ε, ki,3 = 6b5 i /ε, ki,4 = 15a4 i /ε, ki,5 = 15b4 i /ε, ki,6 = 20a3 i /ε, ki,7 = 20b3 i /ε, ki,8 = 15a2 i /ε, ki,9 = 15b2 i /ε, ki,10 = 6ai/ε and ki,11 = 6bi/ε. (59) (59) Consequently, the right-hand side of Eq. (55) can be interpreted as the set of 14 chemical reactions for each i = 1, 2, . . . , K. We define it as Consequently, the right-hand side of Eq. (55) can be interpreted as the set of 14 chemical reactions for each i = 1, 2, . . . , K. We define it as Ri =  Vi ki,1 −→∅, Vi + X ki,2 −→2Vi + X, Vi + Y ki,3 −→2Vi + Y, Vi + 2X ki,4 −→2X, Vi + 2Y ki,5 −→2Y, Vi + 3X ki,6 −→2Vi + 3X, Vi + 3Y ki,7 −→2Vi + 3Y, Vi + 4X ki,8 −→4X, Vi + 4Y ki,9 −→4Y, Vi + 5X ki,10 −→2Vi + 5X, Vi + 5Y ki,11 −→2Vi + 5Y, Vi + 6X 1/ε −→6X, Vi + 6Y 1/ε −→6Y, ∅ 1/ε −→Vi  . (60) (60) Consequently, reaction rate equation (55) corresponds to 14 K chemical reactions in sets Ri, i = 1, 2, . . . , K. Similarly, we rewrite ODEs (53–54) as Consequently, reaction rate equation (55) corresponds to 14 K chemical reactions in sets Ri, i = 1, 2, . . . , K. 5 Chemical Systems with Arbitrary Many Limit Cycles Similarly, we rewrite ODEs (53–54) as dx dt = K  i=1  −vi x4 + ki,12 vi x3 −ki,13 vi x2 + ki,14 vi x + ai vi xy2 dx dt = K  i=1  −vi x4 + ki,12 vi x3 −ki,13 vi x2 + ki,14 vi x + ai vi xy2 +ki,15 vi x2y −ki,16 vi xy −vi x2y2  , (61) i 1 +ki,15 vi x2y −ki,16 vi xy −vi x2y2  , (61) K (61) dy dt = K  k=1  −vi y4 + ki,17 vi y3 −ki,18 vi y2 + ki,19 vi y + bi vi x2y +ki,20 vi xy2 −ki,21 vi xy −vi x2y2  , (62) (62) where ki, j, i = 1, 2, . . . , K, j = 12, 13, . . . , 21, are constants given by where ki, j, i = 1, 2, . . . , K, j = 12, 13, . . . , 21, are constants given by where ki, j, i = 1, 2, . . . , K, j = 12, 13, . . . , 21, are constants given by ki,12 = 3ai, ki,13 = 3a2 i + b2 i −1, ki,14 = a3 i + aib2 i + bi −ai, ki,15 = 2bi, ki,16 = 1 + 2aibi, ki,17 = 3bi, ki,18 = a2 i + 3b2 i −1, 123 Page 19 of 29 76 Chemical Systems with Limit Cycles… 76 ki,19 = b3 i + a2 i bi −ai −bi, ki,20 = 2ai, ki,21 = 2aibi −1. (63) Considering sufficiently large ai and bi (say, for ai > 1 and bi > 1), the constants (63) are positive. Moreover, since the term −vi x2y2 appears in both Eqs. (61) and (62), the right-hand sides of Eqs. (53–54) can be interpreted as the set of 15 K chemical reactions. 5 Chemical Systems with Arbitrary Many Limit Cycles We define R∗ i =  Vi + 4X 1 −→Vi + 3X, Vi + 3X ki,12 −→Vi + 4X, Vi + 2X ki,13 −→Vi + X Vi + X ki,14 −→Vi + 2X, Vi + X + 2Y ai −→Vi + 2X + 2Y, Vi + 2X + Y ki,15 −→Vi + 3X + Y, Vi + X + Y ki,16 −→Vi + Y, Vi + 2X + 2Y 1 −→Vi + X + Y, Vi + 4Y 1 −→Vi + 3Y, Vi + 3Y ki,17 −→Vi + 4Y, Vi + 2Y ki,18 −→Vi + Y, Vi + Y ki,19 −→Vi + 2Y, Vi + 2X + Y bi −→Vi + 2X + 2Y, Vi + X + 2Y ki,20 −→Vi + X + 3Y, Vi + X + Y ki,21 −→Vi + X  , for i = 1, 2, . . . , K. ( Vi + X + Y ki,21 −→Vi + X  , for i = 1, 2, . . . , K. (64) (64) Then, we conclude that the reaction rate Eqs. (53–55) correspond to the CRN with N = K + 2 chemical species and 29 K chemical reactions of at most seventh order given by Then, we conclude that the reaction rate Eqs. (53–55) correspond to the CRN with N = K + 2 chemical species and 29 K chemical reactions of at most seventh order given by R = K  i=1 Ri ∪R∗ i . (65) (65) The CRN (S, C, R) consisting of chemical species S given by (57) and chemical reactions R given by (65) is the CRN which we will use to prove Theorem 1 in Sect.6. The corresponding set of reaction complexes C can be inferred from the provided lists of reactions Ri and R∗ i , i = 1, 2, . . . , K, given by (60) and (64). 6 Proof of Theorem 1 Theorem 1 Let K be an arbitrary positive integer. Then there exists a CRN with N(K) chemical species which are subject to M(K) chemical reactions of at most seventh order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤K + 2 and M(K) ≤29 K. (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤K + 2 and M(K) ≤29 K. The idea of the proof of Theorem 1 is similar to the one chosen in Sects.3 and 4, where we have first proven Lemma 2 about the existence of K limit cycles in the planar ODE system (10–11) and then we have used it to prove the existence of K limit cycles in the (K + 2)-dimensional ODE system in Lemma 3. In this section, we will again start 123 76 Page 20 of 29 R. Erban, H.-W. Kang 76 by formulating Lemma 4 for a planar ODE system, establishing that it has K stable limit cycles. This is again proven by using K disjoint sets (15). The result of Lemma 4 is then used in Lemma 5 to prove Theorem 1 by considering the (K + 2)-dimensional ODE system (53–55). by formulating Lemma 4 for a planar ODE system, establishing that it has K stable limit cycles. This is again proven by using K disjoint sets (15). The result of Lemma 4 is then used in Lemma 5 to prove Theorem 1 by considering the (K + 2)-dimensional ODE system (53–55). The planar ODE system in Lemma 4 is derived by applying the x-factorable trans- formation to the planar ODE system (10–11). We obtain dx dt = K  k=1 x fk(x −ak, y −bk)=x f (x, y), (66) dy dt = K  k=1 y gk(x −ak, y −bk)= y g(x, y), (67) (66) (67) where we have used the notation fk(·, ·) and gk(·, ·) introduced in Eqs. (18), (19) and (22). The dynamics of the ODE system (66–67) is similar to the dynamics of the original planar ODE system (10–11) in the same way as the dynamics of the (K + 2)-dimensional extended ODE system (53–55) is similar to the dynamics of the (K + 2)-dimensional extended ODE system (44–46). 6 Proof of Theorem 1 We have already observed in Fig.3(a) that the limit cycle around the point (ai, bi) = (6, 6) of the ODE sys- tem (44–46) is relatively circular. On the other hand, the shape of the limit cycles can more significantly differ between Figs.2b and 3a if the corresponding parameters ai and bi are not equal to each other. Motivated by this observation, we will study the case ai = bi in Lemma 4 and prove that it is possible to choose these parameters in a way that the planar ODE system (66–67) have (at least) K stable limit cycles. This result is sufficient for the proof of Theorem 1. However, we also note that the existence of limit cycles of the ODE system (66–67) is not restricted to the case ai = bi and a more general lemma could be stated and proven, as we did in Lemma 2 where the existence of K limit cycles has been proven under a relatively general condition (14). The advantage of the case ai = bi is that it simplifies the proof of Lemma 4, because we can use the approach and notations introduced in the proof of Lemma 2. Lemma 4 Let us assume that ai = bi = 8i K (68) (68) for i = 1, 2, . . . , K. Then the ODE system (66–67) has at least K stable limit cycles. for i = 1, 2, . . . , K. Then the ODE system (66–67) has at least K stable limit cycles. Proof Let us define regions i ⊂R2, i = 1, 2, . . . , K, together with their boundary parts ∂i1 and ∂i2 by (15), (16) and (17). Our choice of values of ai and bi in (68) satisfies the assumption (14) in Lemma 2. Therefore, the ODE system (10–11) has with parameters given by (68) at least K stable limit cycles. Moreover, in the proof of Lemma 2, we have shown that each region i does not include any equilibrium of the planar ODE system (10–11). Any equilibrium of the ODE system (66–67) is either located on the x-axis or y-axis, or it is also an equilibrium of the ODE system (10–11). However, our assumption (68) implies that no region i, i = 1, 2, . . . 6 Proof of Theorem 1 , K, intersects 123 Page 21 of 29 76 Chemical Systems with Limit Cycles… 76 with the x-axis or y-axis. Therefore, we conclude that each i, for i = 1, 2, . . . , K, does not contain any equilibrium of the ODE system (66–67). Next, consider any point (xb, yb) ∈∂i. We will compute the scalar product of vectors with the x-axis or y-axis. Therefore, we conclude that each i, for i = 1, 2, . . . , K, does not contain any equilibrium of the ODE system (66–67). Next, consider any point (xb, yb) ∈∂i. We will compute the scalar product of vectors (xb −ai, yb −bi) and xb f (xb, yb), yb g(xb, yb) (69) (69) by rewriting the second vector as a sum of two vectors xb f (xb, yb), yb g(xb, yb) = xb f (xb, yb), g(xb, yb) + 0, (yb −xb) g(xb, yb) . (70) (70) (70) The scalar product of vectors The scalar product of vectors Since i, i = 1, 2, . . . , K, are pairwise disjoint, this implies that the ODE system (66–67) has at least K stable limit cycles. ⊓⊔ ⊓⊔ The following lemma shows that the extended ODE system (53–55) has at least K stable limit cycles when ε is small enough. The detailed proof is omitted since one can use similar steps as in the proof of Lemma 3. Lemma 5 Let us assume that constants ai, bi, i = 1, 2, . . . , K are given by (68). Then there exists ε0 > 0 such that the reaction rate equations (53)–(55) have at least K stable limit cycles for all ε ∈(0, ε0). Lemma 5 Let us assume that constants ai, bi, i = 1, 2, . . . , K are given by (68). Then there exists ε0 > 0 such that the reaction rate equations (53)–(55) have at least K stable limit cycles for all ε ∈(0, ε0). Proof This follows directly from Lemma 4 and Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983). ⊓⊔ Proof This follows directly from Lemma 4 and Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983). ⊓⊔ The existence of K limit cycles in the CRN (65) follows by application of Lemma 5. The Chemical system (65) has (K +2) chemical species X, Y, V1, V2, …, VK , which are subject to 29K chemical reactions, so, by construction, we also establish bounds in part (ii) of Theorem 1 on N(K) and M(K). This concludes the proof of Theorem 1. The scalar product of vectors (xb −ai, yb −bi) and xb f (xb, yb), g(xb, yb) (71) (71) has already been calculated in the proof of Lemma 2 starting with Eq. (33). We obtained that it is negative for (xb, yb) ∈∂i1 and positive for (xb, yb) ∈∂i2. Therefore, the vector xb f (xb, yb), g(xb, yb) always points inside the domain i on all parts of the boundary ∂i. Next, we want to show that this conclusion also holds if vec- tor xb f (xb, yb), g(xb, yb) is modified by adding the vector 0, (yb −xb) g(xb, yb) as it is done in Eq. (70). To do this, we note that our choice of parameters (68) implies that (ai −a j)2 + (bi −b j)2 = 128(i −j)2K 2 for all i, j = 1, 2, . . . , K, which not only satisfies the assumption (14), but it can be used in Eq. (37) to make a stronger conclusion that the scalar product of vectors (71) is at most −1.45 for (xb, yb) ∈∂i1 and at least 1.45 for (xb, yb) ∈∂i2. Thus, we only need to show that the scalar product of vectors (xb −ai, yb −bi) and 0, (yb −xb) g(xb, yb) (72) (72) is in absolute value less than 1.45 to conclude that the original scalar product (69) is negative for (xb, yb) ∈∂i1 and positive for (xb, yb) ∈∂i2. Using the definition of g(·, ·) in (22) and the notation z1 = xb −ai, z2 = yb −bi introduced in the proof of Lemma 2, we have yb −xb = z2 −z1 and the scalar product (72) can be written as (z2 −z1) z2 gi(z1, z2) + (z2 −z1) z2 K  k=1,k̸=i gk(xb −ak, yb −ak). (73) (73) Since we have max (xb,yb)∈∂i (z2 −z1) z2 gi(z1, z2)  = max z2 1+z2 2=2 (or 1/2) (z2 −z1) z2 gi(z1, z2)  ≤1.4 123 Page 22 of 29 R. Erban, H.-W. Kang 76 and the second term in (73) is also less than 0.05, we conclude that the scalar prod- uct (69) is negative for (xb, yb) ∈∂i1 and positive for (xb, yb) ∈∂i2. Therefore, the vector xb f (xb, yb), yb g(xb, yb) always points inside the domain i on all parts of the boundary ∂i. In particular, applying Poincaré-Bendixson theorem (Strogatz 2015), we conclude that each i contains at least one stable limit cycle. 7 Proof of Theorem 2 , 6 evolve according to reaction rate equations dx dt = K  i=1  −xzi,3 + ki,12 viw2 −ki,13 xzi,1 + ki,14 vi x + ai viw11 +ki,15 viw12 −ki,16 xzi,2 −xzi,5  , (75) dy dt = K  k=1  −yzi,4 + ki,17 viw7 −ki,18 yzi,2 + ki,19 vi y + bi viw12  dx dt = K  i=1  −xzi,3 + ki,12 viw2 −ki,13 xzi,1 + ki,14 vi x + ai viw11 +ki,15 viw12 −ki,16 xzi,2 −xzi,5  , (7 K  dx dt = K  i=1  −xzi,3 + ki,12 viw2 −ki,13 xzi,1 + ki,14 vi x + ai viw11 +ki,15 viw12 −ki,16 xzi,2 −xzi,5  , (75) K (75) dy dt = K  k=1  −yzi,4 + ki,17 viw7 −ki,18 yzi,2 + ki,19 vi y + bi viw12 +ki,20 viw11 −ki,21 yzi,1 −yzi,6  , (76) (76) dvi dt =−ki,1 vi + ki,2 vi x + ki,3 vi y −ki,4 viw1 −ki,5 viw6 + ki,6 viw2 + ki,7 viw7 −ki,8 viw3 −ki,9 viw8 (77) + ki,10 viw4 + ki,11 viw9 −viw5/ε −viw10/ε + 1/ε, dvi dt =−ki,1 vi + ki,2 vi x + ki,3 vi y −ki,4 viw1 −ki,5 viw6 + ki,6 viw2 + ki,7 viw7 −ki,8 viw3 −ki,9 viw8 (77) + ki,10 viw4 + ki,11 viw9 −viw5/ε −viw10/ε + 1/ε, dvi dt =−ki,1 vi + ki,2 vi x + ki,3 vi y −ki,4 viw1 −ki,5 viw6 + ki,6 viw2 + ki,7 viw7 −ki,8 viw3 −ki,9 viw8 (77) + ki,10 viw4 + ki,11 viw9 −viw5/ε −viw10/ε + 1/ε, δ dw1 dt = x2 −w1, δ dw2 dt = xw1 −w2, δ dw3 dt = xw2 −w3, (78) δ dw4 dt = xw3 −w4, δ dw5 dt = xw4 −w5, δ dw6 dt = y2 −w6, (79) δ dw7 dt = yw6 −w7, δ dw8 dt = yw7 −w8, δ dw9 dt = yw8 −w9, (80) δ dw10 dt = yw9 −w10, δ dw11 dt = xw6 −w11, δ dw12 dt = yw1 −w12, (81) δ dzi,1 dt =vi x −zi,1, δ dzi,2 dt = vi y −zi,2, δ dzi,3 dt = viw2 −zi,3, (82) δ dzi,4 dt =viw7 −zi,4, δ dzi,5 dt = viw11 −zi,5, δ dzi,6 dt = viw12 −zi,6, (83) (77) where δ > 0, ε > 0 and ki, j, i = 1, 2, . . . , K, j = 1, 2, . . . 7 Proof of Theorem 2 Theorem 2 Let K be an arbitrary positive integer. Then there exists a CRN with N(K) chemical species which are subject to M(K) chemical reactions of at most second order such that (i) Reaction rate equations (3) have at least K stable limit cycles, (ii) We have N(K) ≤7K + 14 and M(K) ≤42 K + 24. In Theorem 1, we have established that the reaction rate equations (53–55) describ- ing the CRN (65) have at least K stable limit cycles. Since the right-hand sides of ODEs (53–55) include polynomials up to the order 7, the resulting chemical reac- tions (65) are reactions of the order at most 7. However, in practice, every higher-order reactions can be subdivided into elementary steps, which are at most bimolecular (sec- ond order). Therefore, we focus here on the proof of Theorem 2 which restricts our considerations to at most second-order kinetics. We prove it by further extending the number of variables in the reaction rate equations (53–55), i.e., by adding intermediary chemical species and elementary reactions into the CRN (65). The resulting CRN has N = 7K + 14 chemical species denoted by S = X, Y, W1, W2, . . . , W12  ∪ K  i=1 Vi, Zi,1, Zi,2, Zi,3, Zi,4, Zi,5, Zi,6  , (74) 123 Chemical Systems with Limit Cycles… Page 23 of 29 76 76 where we use the notation introduced in Definition 1 of CRNs. The concentrations x, y, vi, w1, w2, . . . , w12, zi, j for i = 1, 2, . . . , K and j = 1, 2, . . . , 6 evolve according to reaction rate equations where we use the notation introduced in Definition 1 of CRNs. The concentrations x, y, vi, w1, w2, . . . , w12, zi, j for i = 1, 2, . . . , K and j = 1, 2, . . . 7 Proof of Theorem 2 , 21, are positive constants given by (59) and (63). Considering the limit δ →0 in Eqs. (78–83), we obtain where δ > 0, ε > 0 and ki, j, i = 1, 2, . . . , K, j = 1, 2, . . . , 21, are positive constants given by (59) and (63). Considering the limit δ →0 in Eqs. (78–83), we obtain w1 = x2, w2 = x3, w3 = x4, w4 = x5, w5 = x6, w6 = y2, w7 = y3, w8 = y4, w9 = y5, w10 = y6, w11 = xy2, w12 = x2y, zi,1 = vi x, zi,2 = vi y, zi,3 = vi x3, zi,4 = vi y3, zi,5 = vi xy2, zi,6 = vi x2y. (84) (84) Substituting the limiting values (84) for wℓand zi, j, ℓ= 1, 2, . . . , 12, i = 1, 2 . . . , K, j = 1, 2, . . . , 6, into Eqs. (75–77), we obtain Eqs. (61), (62) and (58), which are equal to the reaction rate equations (53–55). In particular, we deduce the following lemma, establishing that the extended ODE system (75–83) with N = 7K + 14 variables has at least K stable limit cycles when δ and ε are small enough. 123 76 Page 24 of 29 R. Erban, H.-W. Kang 76 Lemma 6 Let us assume that constants ai, bi, i = 1, 2, . . . , K are given by (68). Then there exist δ0 > 0 and ε0 > 0 such that reaction rate equations (75–83) have at least K stable limit cycles for all δ ∈(0, δ0) and ε ∈(0, ε0). Proof This follows directly from Lemma 5 and Tikhonov’s theorem (Tikhonov 1952; Klonowski 1983). ⊓⊔ ⊓⊔ The right-hand sides of reaction rate equations (75–83) only include quadratic terms. Therefore, there exists a CRN corresponding to the reaction rate Eqs. (75–83) which includes(atmost)second-orderreactions.Wecanobtainitbyapplyingtheconstruction in the proof of Lemma 1. The right-hand sides of Eqs. 7 Proof of Theorem 2 (75) and (76) can be interpreted as the set of 16 K chemical reactions (compare with (64) for ODEs (53–54)) Rs,∗ i =  X + Zi,3 1 −→Zi,3, Vi + W2 ki,12 −→Vi + W2 + X, X + Zi,1 ki,13 −→Zi,1, Vi + X ki,14 −→Vi + 2X, Vi + W11 ai −→Vi + W11 + X, Vi + W12 ki,15 −→Vi + W12 + X, X + Zi,2 ki,16 −→Zi,2, X + Zi,5 1 −→Zi,5, Y + Zi,6 1 −→Zi,6, Y + Zi,4 1 −→Zi,4, Vi + W7 ki,17 −→Vi + W7 + Y, Y + Zi,2 ki,18 −→Zi,2, Vi + Y ki,19 −→Vi + 2Y, Vi + W12 bi −→Vi + W12 + Y, Vi + W11 ki,20 −→Vi + W11 + Y, Y + Zi,1 ki,21 −→Zi,1  , for i = 1, 2, . . . , K. (85) Y + Zi,1 ki,21 −→Zi,1  , for i = 1, 2, . . . , K. (85) (85) The right-hand side of equations (77) can be interpreted as the set of 14 chemical reactions for each i = 1, 2, . . . , K (compare with (60) for the right-hand side of ODE (55)) The right-hand side of equations (77) can be interpreted as the set of 14 chemical reactions for each i = 1, 2, . . . , K (compare with (60) for the right-hand side of ODE (55)) Rs i =  Vi ki,1 −→∅, Vi + X ki,2 −→2Vi + X, Vi + Y ki,3 −→2Vi + Y, Vi + W1 ki,4 −→W1, Vi + W6 ki,5 −→W6, Vi + W2 ki,6 −→2Vi + W2, Vi + W7 ki,7 −→2Vi + W7, Vi + W3 ki,8 −→W3, Vi + W8 ki,9 −→W8, Vi + W4 ki,10 −→2Vi + W4, Vi + W9 ki,11 −→2Vi + W9, Vi + W5 1/ε −→W5, Vi + W10 1/ε −→W10, ∅ 1/ε −→Vi  . ( (86) Consequently, reaction rate equations (75–77) correspond to 30 K chemical reactions in sets Rs,∗ i and Rs i , i = 1, 2, . . . , K. This is already more than 29 K chemical reactions used in Theorem 1, because we did not combine two terms on the right-hand sides into one reaction as we did in the set R∗ i (this is further discussed in Eq. (92) in Sect.9). 7 Proof of Theorem 2 Moreover, there are additional chemical reactions corresponding to the 123 Chemical Systems with Limit Cycles… Page 25 of 29 76 76 dynamics of additional chemical species in Eqs. (78–83). The right-hand sides of equations (78–81) can be interpreted as the set of 24 chemical reactions given as dynamics of additional chemical species in Eqs. (78–83). The right-hand sides of equations (78–81) can be interpreted as the set of 24 chemical reactions given as Rw =  2X 1/δ −→2X + W1, 2Y 1/δ −→2Y + W6, X + W j 1/δ −→X + W j + W j+1, for j = 1, 2, 3, 4, Y + W j 1/δ −→Y + W j + W j+1, for j = 6, 7, 8, 9, X + W6 1/δ −→X + W6 + W11, Y + W1 1/δ −→Y + W1 + W12, Wℓ 1/δ −→∅, for ℓ= 1, 2, . . . , 12  . (87) Wℓ 1/δ −→∅, for ℓ= 1, 2, . . . , 12  . (87) (87) Finally, the right-hand sides of Eqs. (82–83) can be interpreted as the set of 12 chemical reactions for each i = 1, 2, . . . , K given by Finally, the right-hand sides of Eqs. (82–83) can be interpreted as the set of 12 chemical reactions for each i = 1, 2, . . . , K given by Rz i =  X + Vi 1/δ −→X + Vi + Zi,1, Y + Vi 1/δ −→Y + Vi + Zi,2, Vi + W2 1/δ −→Vi + W2 + Zi,3, Vi + W7 1/δ −→Vi + W7 + Zi,4, Vi + W11 1/δ −→Vi + W11 + Zi,5, Vi + W12 1/δ −→Vi + W12 + Zi,6, Zi, j 1/δ −→∅, for j = 1, 2, . . . , 6  . (88 (88) In summary, we conclude that the reaction rate equations (75–83) correspond to the CRN with N = 7K + 14 chemical species and 42 K + 24 chemical reactions given by In summary, we conclude that the reaction rate equations (75–83) correspond to the CRN with N = 7K + 14 chemical species and 42 K + 24 chemical reactions given by R = Rw ∪ K  i=1 Rs i ∪Rs,∗ i ∪Rz i . 7 Proof of Theorem 2 (89) (89) Using Lemma 6, we deduce that the CRN (S, C, R) consisting of chemical species S given by (74) and chemical reactions R given by (89) is an example of a CRN which satisfies Theorem 2. The corresponding set of reaction complexes C can be inferred from the provided lists of reactions Rs,∗ i , Rs i , Rw and Rz i , for i = 1, 2, . . . , K, given by (85), (86), (87) and (88). 8 Proof of Theorem 3 Theorem 3 Let C(n) be the maximum number of stable limit cycles of reaction rate equations (1–2) corresponding to CRNs with two chemical species undergoing chem- 123 Page 26 of 29 R. Erban, H.-W. Kang 76 ical reactions of at most n-th order. Then we have ical reactions of at most n-th order. Then we have ical reactions of at most n-th order. Then we have C(n) ≥ n + 2 6  , (4) (4) where the floor function ⌊.⌋denotes the integer part of a positive real number. Given an arbitrarily large integer K ∈N, we will show that there exists a CRN with two chemical species such that its reaction rate equations have at least K stable limit cycles and the order of the chemical reactions is at most n(K) = 6K −2. To do that, we start with the planar ODE system (10–11) and renormalize time t to get a planar system with polynomial ODEs. Using an auxiliary function h(x, y) = K  k=1  1 + (x −ak)6 + (y −bk)6 , we define our new time variable τ by we define our new time variable τ by τ =  t 0 1 h(x(s), y(s)) ds. τ =  t 0 1 h(x(s), y(s)) ds. Then we obtain Then we obtain dx dτ = dx dt dt dτ = h(x, y) K  k=1 (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk) 1 + (x −ak)6 + (y −bk)6 , (90) dy dτ = dy dt dt dτ = h(x, y) K  k=1 (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak) 1 + (x −ak)6 + (y −bk)6 , (90) (91) k 1 (91) which is a planar ODE system with its right-hand side given as polynomials of degree n(K)−1 = 6K −3. Since we only rescaled the time, Fig.1(a) provides an illustrative dynamics of the ODE system (90–91) for K = 4. The illustrative trajectories are calculated in Fig.1a by solving ODEs (10–11) in time interval t ∈[0, 100], and we can obtain the same result by solving ODEs (90–91) numerically in time interval τ ∈[0, 10−9]. 9 Discussion The main results of this paper have been formulated as Theorems 1, 2 and 3, which show that there exist CRNs with K stable limit cycles for any integer K ∈N. The CRN presented in our proof of Theorem 1 consisted of N(K) = K + 2 chemical species S given by (57) and M(K) = 29 K chemical reactions R (of at most seventh order) given by (65). The number of species and chemical reactions further increases in our proof of Theorem 2, where we restrict our investigation to CRNs with (at most) second-order kinetics. On the other hand, if we restrict to CRNs with only N = 2 chemical species, then the order of the chemical reactions increases with K as n(K) = 6K −2 in our proof of Theorem 3. An important question is whether we can further decrease N(K) (the number of chemical species) and M(K) (the number of chemical reactions) in Theorems 1 and 2 and still obtain a CRN with K stable limit cycles. One possibility to decrease M(K) is to use one chemical reaction to interpret multiple terms on the right-hand sides of ODEs (53–55). We have already done this in the reaction set R∗ i given by (64) with the reaction Vi + 2X + 2Y 1 −→Vi + X + Y, (92) (92) which corresponds to terms of the form −vi x2y2 appearing in both equations (53) and (54). Another way to construct a CRN with reactions modeling the two terms, −vi x2y2, in the reaction rate equations (53–54), is to use one chemical reaction per one term on the right-hand side. That is, the chemical reaction (92) could be replaced by two chemical reactions Vi + 2X + 2Y 1 −→Vi + X + 2Y, and Vi + 2X + 2Y 1 −→Vi + 2X + Y without modifying the form of the reaction rate equations (53–54). In particular, if our aim is to decrease the number M(K) of chemical reactions, we could consider to ‘merge’ some other reactions, which have the same reactants. For example, reaction lists (60) and (64) contain chemical reactions Vi + 3Y ki,7 −→2Vi + 3Y, Vi + 3Y ki,17 −→Vi + 4Y. 8 Proof of Theorem 3 Applying x-factorable transformation to ODEs (90–91), we obtain dx dτ = x h(x, y) K  k=1 (x −ak) 1 −(x −ak)2 −(y −bk)2 −(y −bk) 1 + (x −ak)6 + (y −bk)6 , dy dτ = y h(x, y) K  k=1 (y −bk) 1 −(x −ak)2 −(y −bk)2 + (x −ak) 1 + (x −ak)6 + (y −bk)6 , 3 Page 27 of 29 76 Chemical Systems with Limit Cycles… which is a kinetic system of ODEs with polynomials of degree n(K) = 6K −2 and which has K stable limit cycles. Solving for K, we obtain K = (n(K) + 2)/6, which establishes the lower bound (4) in Theorem 3. which is a kinetic system of ODEs with polynomials of degree n(K) = 6K −2 and which has K stable limit cycles. Solving for K, we obtain K = (n(K) + 2)/6, which establishes the lower bound (4) in Theorem 3. 9 Discussion If these chemical reactions had the same reaction rate constants ki,7 and ki,17, then we could replace them by one chemical reaction given by If these chemical reactions had the same reaction rate constants ki,7 and ki,17, then we could replace them by one chemical reaction given by Vi + 3Y ki,7 −→2Vi + 4Y 123 Page 28 of 29 76 Page R. Erban, H.-W. Kang 76 and we would obtain a CRN which has 28 K chemical reactions rather than 29 K, which we use in Theorem 1. Consequently, there is potential to decrease the size of the constructed CRN by a careful choice of our parameters or by modifying the right- hand sides of reaction rate equations (53–55). However, the focus of our paper was on the existence proofs and we leave the improvement of bounds on N(K) and M(K) to future work. Another possible direction to investigate is to consider more detailed stochastic description of CRNs, written as continuous-time discrete space Markov chains and simulated by the Gillespie algorithm (Erban and Chapman 2020). Such simulations would help us to investigate how our parameters ai, bi, i = 1, 2, . . . , K, need to be chosen that the system not only has the limit cycles of comparable size (as we visualized in Fig.3 in the ODE setting), but it also follows each of these limit cycles with a similar probability (comparable to 1/K). This could also be achieved by using the noise-control algorithm (Plesa et al. 2018) for designing CRNs. This algorithm structurally modifies a given CRN under mass-action kinetics, in such a way that (i) controllable state-dependent noise is introduced into the stochastic dynamics, while (ii) the reaction rate equations are preserved. In particular, it could be used to introduce additional chemical reactions (which do not change the ODE dynamics), but lead to controllable noise-induced switching between different limit cycles. Author Contributions Both authors have contributed equally to this work. Funding This work was supported by the Engineering and Physical Sciences Research Council, grant number EP/V047469/1, awarded to Radek Erban. This work was also supported by the National Sci- ence Foundation, grant number DMS-1620403, and a Visiting Research Fellowship from Merton College, Oxford, awarded to Hye-Won Kang. Data availability Not applicable. No data are associated with this article. Conflict of interest Authors have no conflict of interest. Conflict of interest Authors have no conflict of interest. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,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/. Author Contributions Both authors have contributed equally to this work. Declarations Ethical approval Not applicable. 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A New Family of Macrocyclic Polyamino Biphenolic Ligands: Acid-Base Study, Zn(II) Coordination and Glyphosate/AMPA Binding
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Citation: Giacomazzo, G.E.; Paderni, D.; Giorgi, L.; Formica, M.; Mari, L.; Montis, R.; Conti, L.; Macedi, E.; Valtancoli, B.; Giorgi, C.; et al. A New Family of Macrocyclic Polyamino Biphenolic Ligands: Acid-Base Study, Zn(II) Coordination and Glyphosate/AMPA Binding. Molecules 2023, 28, 2031. https:// doi.org/10.3390/molecules28052031 Academic Editor: Antonella Dalla Cort Received: 30 January 2023 Revised: 16 February 2023 Accepted: 17 February 2023 Published: 21 February 2023 Citation: Giacomazzo, G.E.; Paderni, D.; Giorgi, L.; Formica, M.; Mari, L.; Montis, R.; Conti, L.; Macedi, E.; Valtancoli, B.; Giorgi, C.; et al. A New Family of Macrocyclic Polyamino Biphenolic Ligands: Acid-Base Study, Zn(II) Coordination and Glyphosate/AMPA Binding. Molecules 2023, 28, 2031. https:// doi.org/10.3390/molecules28052031 Academic Editor: Antonella Dalla Cort Received: 30 January 2023 Revised: 16 February 2023 Accepted: 17 February 2023 Published: 21 February 2023 Citation: Giacomazzo, G.E.; Paderni, D.; Giorgi, L.; Formica, M.; Mari, L.; Montis, R.; Conti, L.; Macedi, E.; Valtancoli, B.; Giorgi, C.; et al. A New Family of Macrocyclic Polyamino Biphenolic Ligands: Acid-Base Study, Zn(II) Coordination and Glyphosate/AMPA Binding. Molecules 2023, 28, 2031. https:// doi.org/10.3390/molecules28052031 Keywords: glyphosate; AMPA; Zn(II)-complexes; polyamines; macrocycles; anion recognition Academic Editor: Antonella Dalla Cort Article A New Family of Macrocyclic Polyamino Biphenolic Ligands: Acid-Base Study, Zn(II) Coordination and Glyphosate/AMPA Binding Gina Elena Giacomazzo 1,† , Daniele Paderni 2,† , Luca Giorgi 2, Mauro Formica 2 , Lorenzo Mari 1 , Riccardo Montis 2, Luca Conti 1,* , Eleonora Macedi 2,* , Barbara Valtancoli 1, Claudia Giorgi 1,* and Vieri Fusi 2 Gina Elena Giacomazzo 1,† , Daniele Paderni 2,† , Luca Giorgi 2, Mauro Formica 2 , Lorenzo Mari 1 , Riccardo Montis 2, Luca Conti 1,* , Eleonora Macedi 2,* , Barbara Valtancoli 1, Claudia Giorgi 1,* and Vieri Fusi 2 1 Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy y 2 Department of Pure and Applied Sciences, University of Urbino “Carlo Bo”, Via della Stazione 4, 61029 Urbino, Italy * Correspondence: luca.conti@unifi.it (L.C.); eleonora.macedi@uniurb.it (E.M.); claudia.giorgi@unifi.it (C.G.) † These authors contributed equally to this work. * Correspondence: luca.conti@unifi.it (L.C.); eleonora.macedi@uniurb.it (E.M.); claudia.giorgi@unifi.it (C.G.) † These authors contributed equally to this work. Abstract: In this study, the ligands 23,24-dihydroxy-3,6,9,12- tetraazatricyclo[17.3.1.1(14,18)] eicosatetra-1(23),14,16,18(24),19,21-hexaene, L1, and 26,27-dihidroxy-3,6,9,12,15-pentaazatricyclo [20.3.1.1(17,21)] eicosaepta-1(26),17,19,21(27),22,24-hexaene, L2, were synthesized: they represent a new class of molecules containing a biphenol unit inserted into a macrocyclic polyamine fragment. The previously synthesized L2 is obtained herein with a more advantageous procedure. The acid-base and Zn(II)-binding properties of L1 and L2 were investigated through potentiometric, UV-Vis, and fluorescence studies, revealing their possible use as chemosensors of H+ and Zn(II). The new peculiar design of L1 and L2 afforded the formation in an aqueous solution of stable Zn(II) mono (LogK 12.14 and 12.98 for L1 and L2, respectively) and dinuclear (LogK 10.16 for L2) complexes, which can be in turn exploited as metallo-receptors for the binding of external guests, such as the popular herbicide glyphosate (N-(phosphonomethyl)glycine, PMG) and its primary metabolite, the aminomethylphos- phonic acid (AMPA). Potentiometric studies revealed that PMG forms more stable complexes than AMPA with both L1- and L2-Zn(II) complexes, moreover PMG showed higher affinity for L2 than for L1. Fluorescence studies showed instead that the L1-Zn(II) complex could signal the presence of AMPA through a partial quenching of the fluorescence emission. These studies unveiled therefore the utility of polyamino-phenolic ligands in the design of promising metallo-receptors for elusive environmental targets. molecules molecules 1. Introduction N-(phosphonomethyl)glycine (glyphosate or PMG) is one of the most frequently used broad-spectrum organophosphorus herbicides worldwide [1,2]. Its popularity mainly derives from its effectiveness in removing unwanted weeds in pre-harvest treatments and in non-crop areas, which can be attributed to its capacity to inhibit the activity of the 5-enol- pyruvyl-shikimate-3-phosphate synthase (EPSPS) enzymes, impairing the biosynthesis of essential amino acids for the plant growth [3]. Several additional features, such as low perceived toxicity, rapid absorption by plants, and slow evolution of PMG-resistant weeds have then contributed to further extending its use. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). g Since its first introduction into the market in 1974, as an active ingredient of Monsanto’s herbicide Roundup® [4], the global sales volume of glyphosate has increased dramatically, with over 1.6 billion kilograms of PMG that have been used only in the US in 2016 [5]. This https://www.mdpi.com/journal/molecules Molecules 2023, 28, 2031. https://doi.org/10.3390/molecules28052031 Molecules 2023, 28, 2031 2 of 20 has led to the accumulation of PMG, along with its primary metabolite aminomethylphos- phonic acid (AMPA), in different environmental matrices, such as top-soil layers [6] and surface water [7], as well as in various harvest/food products [8], raising serious concerns about its use (abuse) and regulation. Actually, there is an ongoing scientific and social controversy regarding the potential risks for the ecosystems and in particular for human health [9,10]. Indeed, contrary to the US Environmental Protection Agency (EPA), which stated that “there are no risks to public health when glyphosate is used in accordance with its current label”, other countries and international agencies reached diametrically opposed conclusions. The International Agency for Research on Cancer (IARC) classified PMG as probably carcinogenic to humans (Group 2A) [11], and several studies reported on the possible acute and chronic biological effects associable with PMG, such as cytotoxicity, carcinogenicity, teratogenicity, endocrine disruption and metabolic alterations [12–15]. In 2017, the European Union (EU) contro- versially voted to re-license glyphosate use for a limited period of five years and a recent extension, until December 2023, was granted to allow the European Food Safety Authority (EFSA) to conclude its examination on this timely and debated issue. Analogously, raising concerns about the chronic toxicity associated with AMPA has led to its inclusion with PMG in pollution monitoring programs [16]. It is therefore of utmost importance to develop effective methods to monitor and detect PMG and AMPA in environmental samples. Apart from some recent examples of biosensors [17,18] and SERS-based sensors [19,20], the majority of the techniques currently employed still rely on liquid-gas chromatography, capillary electrophoresis, and mass spectrometry analysis [21–23]. However, the zwitterionic nature, high polarity, and low molecular weights of the targeted analytes make difficult both their extraction from samples and their retention on chromatographic phases. As a consequence, preliminary time- consuming derivatization procedures and the optimization of a high number of parameters (temperature, reaction time, laboratory handling time, etc.) are usually required [24–26], making important the research of suitable alternatives for PMG and AMPA recognition [27]. In this respect, fluorescent chemosensors, e.g., molecules composed of a binding unit linked through a spacer to a fluorogenic unit, hold great promise in the design of novel and effective systems for anion recognition and sensing [28–30]. Indeed, the structural features of the spacer, the binding, and the signaling unit can be finely tuned to realize low-cost and versatile systems with increased sensitivity and selectivity. Concerning the choice of binding units, polyamine scaffolds represent optimal solutions. Indeed, besides their typically high water solubility, a fundamental pre-requisite for the application in real matrices, the high number of positively charged polyammonium groups formed in solution allows for the efficient interaction with anionic guests, stabilized via hydrogen bonding and charge-charge interactions [31,32]. Moreover, these frameworks can bind metal ions [33–37], affording promising metallo-receptors for anionic targets [38,39]. More in particular, a number of Zn(II) coordination compounds have been shown to possess promising features as receptors for anions of environmental and biological importance, mainly thanks to the presence of coordinatively unsaturated metal centers which can in turn act as suitable anchoring sites for the coordination of anionic guests [40–42]. With this regard, the use of macrocyclic ligands could ensure both selectivity towards a specific metal cation and the formation of stable complexes, featuring high binding constants [35,36,43]. p g g g Of particular interest is the coupling of polyamine-based receptor units with phe- nolic groups as signaling moieties [37,43,44], in the realization of versatile and effective polyamino-phenolic receptors for anionic targets. Indeed, the phenolic functions confer to these systems peculiar photochemical behaviors, making it possible to detect a selected substrate via an optical response, namely by fluorescence signaling [44,45], but they can be also involved, as phenolates, in metal-coordination, affording efficient metallo-receptors for anionic guests [38,46,47]. This scenario prompted us to recently consider the class of polyamino-phenolic ligands, in their Zn(II)-receptor forms, for the recognition and sensing of PMG and AMPA [48]. Molecules 2023, 28, 2031 3 of 20 ns, imp 3 of 20 ns, imp We showed that the coordination of these analytes by open chain diethylentriamine (dien) frameworks spaced by a phenol or biphenol moiety was remarkably strengthened when Zn(II) ions are present in the binding pockets of ligands. Metal ions not only had a great impact on the anion binding properties of receptors but also affected their behavior as fluorescent chemosensors. Importantly, if compared to the metal-free ligands, the metallo- receptors exhibited stricter binding selectivity patterns, imparted by the combination between the structural features of the metallo-receptors and the coordination requirements of their Zn(II) centers. quirements of their Zn(II) centers. In an effort to further explore and optimize the ability of this cl possible receptors for PMG and AMPA, we inserted herein the fluoro nol) group (BPH) into two differently sized macrocyclic frameworks ands 23,24-dihydroxy-3,6,9,12-tetraazatricyclo[17.3.1 1(23),14,16,18(24),19,21-hexaene, L1, and 26,27-dihidroxy-3,6,9 In an effort to further explore and optimize the ability of this class of compounds as possible receptors for PMG and AMPA, we inserted herein the fluorogenic 1,1′-bis(2-phenol) group (BPH) into two differently sized macrocyclic frameworks, to give the two ligands 23,24-dihydroxy-3,6,9,12-tetraazatricyclo[17.3.1.1(14,18)]eicosatetra-1(23),14,16,18(24),19,21- hexaene, L1, and 26,27-dihidroxy-3,6,9,12,15-pentaazatricyclo[20.3.1.1(17,21)]eicosaepta- 1(26),17,19,21(27),22,24-hexaene, L2 (Figure 1). The two macrocyclic structures bear four (L1) or five (L2) nitrogen donor atoms spaced by ethylene linkers. Following the synthesis of the compounds, which, in case of L2, was accomplished by adopting a new procedure, the acid-base and Zn(II)-binding properties of the ligands were investigated by means of potentiometric, UV-Vis and fluorescence measurements. The same techniques were then used to inspect the ability of the Zn(II) complexes of L1 and L2 to bind and sense the presence of elusive anions such as PMG and AMPA in aqueous solution. clo[20.3.1.1(17,21)]eicosaepta-1(26),17,19,21(27),22,24-hexaene, L2 ( macrocyclic structures bear four (L1) or five (L2) nitrogen donor atom inkers. Following the synthesis of the compounds, which, in case of L by adopting a new procedure, the acid-base and Zn(II)-binding prop were investigated by means of potentiometric, UV-Vis and fluoresc The same techniques were then used to inspect the ability of the Zn and L2 to bind and sense the presence of elusive anions such as PMG ous solution. Figure 1. Polyamino-biphenolic ligands of this work and anionic substrate Figure 1. Polyamino-biphenolic ligands of this work and anionic substrates herein investigated. Figure 1 Polyamino biphenolic ligands of this work and anionic substrat Figure 1. Polyamino-biphenolic ligands of this work and anionic substrates herein investigated. 2 Results a 2.1. Synthesis The protonation constants of L1 and L2 were potentiometrically determined e Cl aqueous solution at 298 1 K and are reported in Table 1 as LogK values 2.2. Protonation and Zn(II) Binding by L1 and L2 2.2.1. Basicity of L1 and L2 absorption and fluorescence emission measurements. The protonation constants of L1 and L2 were potentiometrically determined e Cl aqueous solution at 298 1 K and are reported in Table 1 as LogK values 2.2. Protonation and Zn(II) Binding by L1 and L2 2.2.1. Basicity of L1 and L2 e4Cl aqueous solution at 298.1 K and are reported in Table 1 as LogK values. ution diagrams of the species for L1 and L2 are reported in Figures 2 and 3 The acid-base properties of L1 and L2 were investigated by potentiometric and UV-Vis absorption and fluorescence emission measurements. ly. e 1. Protonation constants (LogK) of L1 and L2 determined in NMe4Cl 0.1 M at 298 ± The protonation constants of L1 and L2 were potentiometrically determined in 0.1 M NMe4Cl aqueous solution at 298.1 K and are reported in Table 1 as LogK values. The distri- bution diagrams of the species for L1 and L2 are reported in Figures 2 and 3, respectively. LogK Table 1. Protonation constants (LogK) of L1 and L2 determined in NMe4Cl 0.1 M at 298 ± 0.1 K. ction LogK L1 L2 L− + H+ = L 10.34 (9) 1 10.22 (8) H+ = HL+ 10.14 (6) 9.99 (8) + H+ = H2L2+ 6.89 (9) 8.70 (9) 2+ + H+ = H3L3+ 3.60 (9) 4.32 (8) 3+ + H+ = H4L4+ 2.28 (9) 3.47 (8) lues in parentheses are standard deviations on the last significant figures Table 1. Protonation constants (LogK) of L1 and L2 determined in NMe4Cl 0.1 M at 298 ± 0.1 K. Reaction LogK L1 L2 H−1L−+ H+ = L 10.34 (9) 1 10.22 (8) L + H+ = HL+ 10.14 (6) 9.99 (8) HL+ + H+ = H2L2+ 6.89 (9) 8.70 (9) H2L2+ + H+ = H3L3+ 3.60 (9) 4.32 (8) H3L3+ + H+ = H4L4+ 2.28 (9) 3.47 (8) 1 Values in parentheses are standard deviations on the last significant figures. ues i pa e t eses a e sta da d de iatio s o t e ast sig i ica t igu es Both neutral L species behave as tetraprotic bases and as monoprotic acids erimental conditions. 2 Results a 2.1. Synthesis 2. Results and Discussion 2.1. Synthesis The synthetic pathway used to obtain the ligands L1 and L2 is r The tosylated, phenol methyl-protected macrocycles 4 and 5 were o cation of the Richman-Atkins method, involving the cyclization of th yamines 2 and 3, respectively, with one equivalent of the 3,3′-bis(b methoxybiphenyl (1), in the presence of the alkaline carbonate base K pounds were obtained by deprotection of the macrocycles 4 and 5 Th The synthetic pathway used to obtain the ligands L1 and L2 is reported in Scheme 1. The tosylated, phenol methyl-protected macrocycles 4 and 5 were obtained by a modi- fication of the Richman-Atkins method, involving the cyclization of the poly-tosylated polyamines 2 and 3, respectively, with one equivalent of the 3,3′-bis(bromomethyl)-2,2′- dimethoxybiphenyl (1), in the presence of the alkaline carbonate base K2CO3. The final compounds were obtained by deprotection of the macrocycles 4 and 5. The cleavage reac- tions of the tosyl groups were carried out with lithium in liquid ammonia. The reducing conditions of the treatment also led to the demethylation reaction of the two ethereal methyl groups on the biphenol moiety, obtaining, after the described work-up, the ligands L1 and L2. Both compounds were further purified as hydrochloride salts. 4 of 20 vious o d Molecules 2023, 28, 2031 Scheme 1. Synthetic pathway of ligands L1 and L2. (n refers to the number of -CH2-N(Ts units in the macrocycle). Scheme 1. Synthetic pathway of ligands L1 and L2. (n refers to the number of -CH2-N(Ts)-CH2- units in the macrocycle). me 1. Synthetic pathway of ligands L1 and L2. (n refers to the number of -CH2-N(Ts in the macrocycle). Scheme 1. Synthetic pathway of ligands L1 and L2. (n refers to the number of -CH2-N(Ts)-CH2- units in the macrocycle). Protonation and Zn(II) Binding by L1 and L2 1. Basicity of L1 and L2 The acid-base properties of L1 and L2 were investigated by potentiometric In the present paper, an alternative synthetic pathway to obtain L2 was employed compared to the previously reported procedure [49]. This consisted of a template synthesis involving the use of toxic cadmium(II) ion as templating agent. Although the present synthesis returned a little bit lower overall yield with respect to the previous one, it allowed for avoiding the use of a toxic metal ion and was therefore preferred. absorption and fluorescence emission measurements. 2 Results a 2.1. Synthesis Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). Figure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). igure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent em tration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). gure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in Me4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emi ration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). Figure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). The high value of the first protonation constant for both ligands suggests tha H−1L− and L species behave as strong bases in the addition of the first two protons (L anging between 10.34 and 9.99); a drop in the LogK values is observed thereafter. 2 Results a 2.1. Synthesis rend could be rationalized in terms of minimization of the electrostatic repulsions, esting that the first two protons locate on sites placed at a certain distance in the ma ycles then the following protons additions occur on positions close to already proton The high value of the first protonation constant for both ligands suggests that −1L− and L species behave as strong bases in the addition of the first two protons (L anging between 10.34 and 9.99); a drop in the LogK values is observed thereafter. end could be rationalized in terms of minimization of the electrostatic repulsions, esting that the first two protons locate on sites placed at a certain distance in the ma ycles, then the following protons additions occur on positions close to already proton Spectrophotometric UV-Vis absorption and fluorescence spectra of L1 and L2 were recorded in aqueous solution as a function of pH (Figures S1 and 4) to get insights about the role of BPH in the fluorescence behavior and its dependence on pH. The BPH group shows indeed fluorescence properties depending on its protonation degree, moving from the least emitting neutral species to the most emitting monodeprotonated species. Usually, the fully deprotonated BPH could not be obtained unless at very high pH values [51–55]. ycles, then the following protons additions occur on positions close to already proton ites [50]. In the case of L2, a greater grouping of the LogK values for the addition reactio h fi h i b d l i h L K l f hi h h f L tes [50]. In the case of L2, a greater grouping of the LogK values for the addition reaction he first three protons is observed along with LogK values often higher than for L The trend of the absorbance and fluorescence emission intensity at selected wave- lengths (■) vs. pH, together with the distribution curves of the species for the two ligands, are reported in Figures 2 and 3 for L1 and L2, respectively. he first three protons is observed along with LogK values often higher than for L ccordance with the greater macrocyclic dimensions and number of protonatable sit 2, which allow a better minimization of the electrostatic repulsion. 2 Results a 2.1. Synthesis Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). olecules 2023, 28, x FOR PEER REVIEW 5 o Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). Figure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). accordance with the greater macrocyclic dimensions and number of protonatable sites of L2, which allow a better minimization of the electrostatic repulsion. accordance with the greater m L2, which allow a better minim p Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 2. 2 Results a 2.1. Synthesis The monoanionic H−1L− species are forming at alkaline (Figures 2 and 3), suggesting that only one acidic hydrogen atom of the BPH ost under these conditions, as previously reported for similar systems [47]. M Both neutral L species behave as tetraprotic bases and as monoprotic acids in these experimental conditions. The monoanionic H−1L−species are forming at alkaline pH values (Figures 2 and 3), suggesting that only one acidic hydrogen atom of the BPH unit can be lost under these conditions, as previously reported for similar systems [47]. Moreover, only L1, contrarily to L2, can be fully protonated in the analyzed pH range, as observed for analogous compounds bearing a phenol instead of a BPH group [43]. p y p y y L1, contrarily to L2, can be fully protonated in the analyzed pH range, as o analogous compounds bearing a phenol instead of a BPH group [43]. The high value of the first protonation constant for both ligands suggests that the H−1L−and L species behave as strong bases in the addition of the first two protons (LogK ranging between 10.34 and 9.99); a drop in the LogK values is observed thereafter. This trend could be rationalized in terms of minimization of the electrostatic repulsions, suggesting that the first two protons locate on sites placed at a certain distance in the macrocycles, then the following protons additions occur on positions close to already protonated sites [50]. In the case of L2, a greater grouping of the LogK values for the addition reactions of the first three protons is observed along with LogK values often higher than for L1, in Molecules 2023, 28, 2031 5 of 20 accordance with the greater macrocyclic dimensions and number of protonatable sites of L2, which allow a better minimization of the electrostatic repulsion. olecules 2023, 28, x FOR PEER REVIEW 5 Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 3. 2 Results a 2.1. Synthesis Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emis titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emi itration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0.1 M NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emission titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). igure 2. Distribution diagrams of the species of L1 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L1] = 1 × 10−3 M) and (a) absorption titration at λ = 308 nm and (b) fluorescent emis tration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). ( ) ( ) p ( ) titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). Figure 3. Distribution diagrams of the species of L2 (—) in aqueous solution at 298 ± 0.1 K in 0 NMe4Cl ([L2] = 1 × 10−3 M) and (a) absorption titration at λ = 313 nm and (b) fluorescent emi titration (■) (λex = 288, λem = 403 nm) ([L2] = 1 × 10−5 M). titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10−5 M). titration (■) (λex = 288, λem = 402 nm) ([L1] = 1 × 10 5 M). Figure 3. 2 Results a 2.1. Synthesis p; this can be attributed to the deprotonation of one phenolic function of the BPH gr he suggested BPH deprotonation is confirmed by the absorption spectra, where the b t 281 nm, by moving towards higher pH values, is gradually replaced by a band max at 308 or 313 nm for L1 and L2, respectively (Figures S1 and 4), ascribable to p p In the case of L1 a similar behavior can be described, the emission intensity increasing with pH from 2 to 7.5 (H3L13+ →HL1+), then decreasing up to pH 10 (neutral L1 species), rising again to reach the maximum emission at pH 12, together with the formation of the H−1L1−species. up; this can be attributed to the deprotonation of one phenolic function of the BPH gr The suggested BPH deprotonation is confirmed by the absorption spectra, where the b t 281 nm, by moving towards higher pH values, is gradually replaced by a band λmax at 308 or 313 nm for L1 and L2, respectively (Figures S1 and 4), ascribable to In the case of L1 a similar behavior can be described, the emission intensity increasing with pH from 2 to 7.5 (H3L13+ →HL1+), then decreasing up to pH 10 (neutral L1 species), rising again to reach the maximum emission at pH 12, together with the formation of the H−1L1−species. , p y ( g ), eprotonated BPH group. Therefore, as in similar systems, the fluorescence emissio oth L1 and L2 depends on the protonation degree of the BPH fluorophore. Howe ontrarily to similar but open chain systems [45,47], where the emission stays low onstant up to pH 5, in the present cases the emission intensity starts increasing at p More in details, in the case of L2, the emission increase at pH > 2 goes along with ppearance of the H3L23+ species and reaches a relative maximum at pH 5, where H prevalent in solution. The emission remains constant as long as H2L22+ is prevale olution (5 ≤ pH ≤ 8), then a moderate drop is observed with the appearance of the H pecies. 2 Results a 2.1. Synthesis Spectrophotometric UV-Vis absorption and fluorescence spectra of L1 and L2 w ecorded in aqueous solution as a function of pH (Figures S1 and 4) to get insights a he role of BPH in the fluorescence behavior and its dependence on pH The BPH gr he first three protons is observed along with LogK values often higher than for L1 ccordance with the greater macrocyclic dimensions and number of protonatable site 2, which allow a better minimization of the electrostatic repulsion. Spectrophotometric UV-Vis absorption and fluorescence spectra of L1 and L2 w ecorded in aqueous solution as a function of pH (Figures S1 and 4) to get insights ab he role of BPH in the fluorescence behavior and its dependence on pH. The BPH gr At acidic pH values (pH = 2), where the protonated H4L4+ species are mainly present in solution, both ligands are very low emissive: the observed low intensity band is attributed to the BPH fluorophore in its neutral form, as can be inferred from the absorption spectrum (band with λmax at 281 nm, see Figures S1 and 4). By increasing the pH, whereas the emission wavelength remains substantially constant, the emission intensity also grows up; this can be attributed to the deprotonation of one phenolic function of the BPH group. The Molecules 2023, 28, 2031 6 of 20 suggested BPH deprotonation is confirmed by the absorption spectra, where the band at 281 nm, by moving towards higher pH values, is gradually replaced by a band with λmax at 308 or 313 nm for L1 and L2, respectively (Figures S1 and 4), ascribable to the deprotonated BPH group. Therefore, as in similar systems, the fluorescence emission of both L1 and L2 depends on the protonation degree of the BPH fluorophore. However, contrarily to similar but open chain systems [45,47], where the emission stays low and constant up to pH 5, in the present cases the emission intensity starts increasing at pH > 2. W 6 suggested BPH deprotonation is confirmed by the absorption spectra, where the band at 281 nm, by moving towards higher pH values, is gradually replaced by a band with λmax at 308 or 313 nm for L1 and L2, respectively (Figures S1 and 4), ascribable to the deprotonated BPH group. Therefore, as in similar systems, the fluorescence emission of both L1 and L2 depends on the protonation degree of the BPH fluorophore. 2 Results a 2.1. Synthesis However, contrarily to similar but open chain systems [45,47], where the emission stays low and constant up to pH 5, in the present cases the emission intensity starts increasing at pH > 2. W 6 Figure 4. (a) Absorption and (b) fluorescence spectra of L2 at different pH values. [L2] = 1 × 10 λex = 288 λem = 403 nm Figure 4. (a) Absorption and (b) fluorescence spectra of L2 at different pH values. [L2] = 1 × 10−5 M, λex = 288, λem = 403 nm. gure 4. (a) Absorption and (b) fluorescence spectra of L2 at different pH values. [L2] = 1 × 10 = 288 λ = 403 nm Figure 4. (a) Absorption and (b) fluorescence spectra of L2 at different pH values. [L2] = 1 × 10−5 M, λex = 288, λem = 403 nm. , At acidic pH values (pH = 2), where the protonated H4L4+ species are mainly pre n solution, both ligands are very low emissive: the observed low intensity band i ributed to the BPH fluorophore in its neutral form, as can be inferred from the absorp pectrum (band with λmax at 281 nm, see Figures S1 and 4). By increasing the pH, whe he emission wavelength remains substantially constant, the emission intensity also gr thi b tt ib t d t th d t ti f h li f ti f th BPH More in details, in the case of L2, the emission increase at pH > 2 goes along with the appearance of the H3L23+ species and reaches a relative maximum at pH 5, where H2L22+ is prevalent in solution. The emission remains constant as long as H2L22+ is prevalent in solution (5 ≤pH ≤8), then a moderate drop is observed with the appearance of the HL2+ species. A relative minimum in the emission intensity is observed when the neutral L2 species is completely formed, then the intensity steeply increases again at pH > 10, reaching the maximum at pH 12, ascribable to the formation of the anionic H−1L2−species. Reaction Both ligands are able to form mononuclear species with Zn(II) ions, while only L2 can form dinuclear complexes with the metal ion. When the L2/Zn(II) molar ratio is 1:1, the mononuclear species prevail in solution in the whole tested pH range; on the contrary, when the L2/Zn(II) molar ratio is 1:2, the dinuclear species are mainly present in solution at pH > 6. The distribution diagrams of the species for the L1/Zn(II) (1:1 molar ratio) and L2/Zn(II) (1:1 and 1:2 molar ratios) systems as a function of pH are reported in Figures 5 and 6. By comparing the mononuclear complexes of the two ligands having the same sto- ichiometry, L2 forms more stable species than L1 (Table 2); this can be explained by the higher number of donor atoms in L2 than in L1. For both ligands, the values for the addition constants (LogK) of Zn(II) to the H−1L− species are not so different from those for the addition to the L species, suggesting a similar coordination environment for Zn(II) in the two species and the involvement in the first protonation step of an amine function non-coordinated to the metal center. Moreover, while the [Zn(H−1L1)]+ mononuclear complex can only add one hydrogen ion, the [Zn(H−1L2)]+ mononuclear complex can add up to three hydrogen ions; the LogK values for the addition of the first two protons to [Zn(H−1L2)]+ are similar to each other and to the third proto- nation step of the free ligand (LogK = 8.70), suggesting that such protonation processes involve nitrogen atoms not engaged in the coordination. This means that the Zn(II) ion in the mononuclear [Zn(H−1L)]+ complexes is probably coordinated by three amine functions of the macrocycles, besides probably only one oxygen atom of the monoanionic BPH. Finally, both [Zn(H−1L)]+ complexes seem to be able to bind hydroxide species, sug- gesting that they are prone to add external groups. However, an alternative process such as a further deprotonation of the BPH moiety could not be ruled out for both ligands. L2 also forms dinuclear Zn(II) species; the LogK value for the addition of the second Zn(II) ion to [Zn(H−1L2)]+ is much lower than that for the first one (5.49 vs. 12.98), which can be rationalized both in terms of addition of a cation to a positively charged species and of a lower number of binding sites available in [Zn(H−1L2)]+ with respect to H−1L2−species. 2 Results a 2.1. Synthesis A relative minimum in the emission intensity is observed when the neutra pecies is completely formed, then the intensity steeply increases again at pH > 10, re th i t H 12 ib bl t th f ti f th i i H L2 i The observed fluorescence trend for L2 could be rationalized in terms of deprotonation of the BPH fluorophore, to give the H3L23+ species, along with the formation of O . . . HN and/or O . . . HO hydrogen bonds, that could either negatively or positively impact the fluorescence emission. Indeed, the formation of an intramolecular H-bond between the two oxygen functions of BPH, that stabilizes the hydrogen atom in the monoanionic BPH, is known to rise the fluorescence emission, due to the increase of the co-planarity and the rigidity of the biphenyl system; on the contrary, the H-bond formation between the BPH oxygen and close amine functions could decrease the emission through a nonradiative relaxation process of the excited species, due to the loss of co-planarity between the two aromatic rings [51–55]. Therefore, it can be suggested that in both H2L22+ and H-1L2− species, that feature a maximum of emission, the hydrogen atom of the monoanionic BPH group is only stabilized by an H-bond between the two oxygen functions and no other H-bonds are present. ng the maximum at pH 12, ascribable to the formation of the anionic H−1L2− species. In the case of L1 a similar behavior can be described, the emission intensity increa with pH from 2 to 7.5 (H3L13+ → HL1+), then decreasing up to pH 10 (neutral L1 spec i i h h i i i H 12 h i h h f i p Finally, for both ligands, the absence of fluorescence changes at the highest pH value tested suggest that even in these systems, as in previous ones, the full deprotonation of the BPH group could not be achieved under the present experimental conditions. Molecules 2023, 28, 2031 7 of 20 7 of 20 2.2.2. Coordination of Zn(II) by L1 and L2 2.2.2. Coordination of Zn(II) by L1 and L2 2.2.2. Coordination of Zn(II) by L1 and L2 The coordination behavior of L1 and L2 towards Zn(II) was investigated by potentio- metric, UV-Vis absorption and fluorescence emission measurements. The stability constants for the equilibrium reactions were potentiometrically deter- mined in 0.1 M NMe4Cl aqueous solution at 298.1 K and are reported in Table 2. Table 2. Stability constants of the Zn(II) complexes with L1 and L2 determined in NMe4Cl 0.1 M at 298 ± 0.1 K. Table 2. Stability constants of the Zn(II) complexes with L1 and L2 determined in NMe4Cl 0.1 M at 298 ± 0.1 K. Reaction LogK L1 L2 H−1L−+ Zn2+ = [Zn(H−1L)]+ 12.14 (9) 1 12.98 (9) [Zn(H−1L)]+ + H+ = [ZnL]2+ 7.54 (8) 7.89 (9) L + Zn2+ = [ZnL]2+ 9.54 (7) 10.88 (8) [ZnL]2+ + H+ = [Zn(HL)]3+ - 7.39 (8) [Zn(HL)]3+ + H+ = [Zn(H2L)]4+ - 5.21 (7) [Zn(H−1L)]+ + OH−= [Zn(H−1L)(OH)] - 2.44 (8) [Zn(H−1L)]+ + 2OH−= [Zn(H−1L)(OH)2]− 7.32 (8) - H−1L−+ 2Zn2+ = [Zn2(H−1L)]3+ - 18.47 (8) H−1L−+ 2Zn2+ = [Zn2(H−2L)2+ + H+ - 10.16 (8) [Zn(H−1L)]+ + Zn2+ = [Zn2(H−1L)]3+ - 5.49 (8) [Zn2(H−2L)]2+ + 2OH−= [Zn2(H−2L)(OH)2] - 9.16 (7) 1 Values in parentheses are standard deviations on the last significant figures. Reaction Above pH 6 only dinuclear species are present in solution, each one existing in a narrow range of pH. No further protons can be added to the [Zn2(H−1L2)]3+ species, suggesting the involvement of all available donor atoms in the coordination of the two metal centers. The species is however able to bind further hydroxide groups, that Molecules 2023, 28, 2031 8 of 20 probably contribute to saturate the coordination requirement of the two Zn(II) ions, making this species a suitable metallo-receptor for external ligands. However, even in this case, an alternative full deprotonation of BPH cannot be totally ruled out. EW Figure 5. Fluorescence emission titration (λex = 288 nm) (■), absorption wa sion wavelength trend (---) (a); absorption titration at λ = 299 nm (■), dis species (−) for the Zn(II)/L1 system as a function of pH in aqueous solutio Figure 5. Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend ( . . . ), emission wavelength trend (—) (a); absorption titration at λ = 299 nm (■), distribution diagrams of the species (−) for the Zn(II)/L1 system as a function of pH in aqueous solution (b): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M. igure 5. Fluorescence emission titration (λex = 288 nm) (■), absorption wa on wavelength trend (---) (a); absorption titration at λ = 299 nm (■), dis pecies (−) for the Zn(II)/L1 system as a function of pH in aqueous solution Figure 5. Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend ( . . . ), emission wavelength trend (—) (a); absorption titration at λ = 299 nm (■), distribution diagrams of the species (−) for the Zn(II)/L1 system as a function of pH in aqueous solution (b): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M. 98.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M. In the case of L2 in the presence of an equimolar amount of Z ormation of the [Zn(H2L2)]4+ species comes with the increase of flu 402 nm, that is due to both Zn(II) coordination and deprotonation highlighted by the increase of absorbance and a change of the absor 281 to 313 nm. Reaction The deprotonation of [Zn(H2L2)]4+ to give [Zn(H ZnL2]2+ induces a drop of both the fluorescence emission intensity To try to understand the degree of protonation of the BPH group in the complexes and its role in the emission properties, UV-Vis and fluorescence experiments at different pH values were performed. The fluorescence spectra of L1 + 1 equiv. of Zn(II), L2 + 1 equiv. of Zn(II) and L2 + 2 equiv. of Zn(II) (λex 288 nm) recorded in aqueous solution in the pH range 2–12 are reported in Figures S2–S4. The trend of the fluorescence emission intensity (■) as a function of pH, together with the maximum absorption ( . . . ) and emission (—) wavelength is reported in Figure 5a for L1 and Figure 6a,c for L2. In Figures 5b and 6b,d is depicted the trend of the absorption titration at λ = 299 nm (L1) and λ = 303 nm (L2: Zn(II) 1:1) and 300 nm (L2: Zn(II) 1:2) (■) along with the distribution curves for the species of the two ligands. ZnL2]2+, induces a drop of both the fluorescence emission intensity 02 to 391 nm; also, the absorption wavelength blue shifts from 31 ncrease of both the fluorescence emission intensity and wavelen b d t H 8 5 h th [Z (H L2)] i il i g In the case of L1 the formation of the [ZnL1]2+ species is accompanied by a fluorescence emission increase, due to both the coordination of Zn(II) (chelation enhanced fluorescence (CHEF) effect) and the deprotonation of BPH, as confirmed by the absorption increase of the phenolate band (Figure 5b). observed at pH > 8.5, where the [Zn(H−1L2)]+ species prevails in so onuclear species the only significantly present in solution, the obser The deprotonation of [ZnL1]2+ to give [Zn(H−1L1)]+ goes along with an emission decrease and a blue shift of the emission wavelength from 400 to 395 nm. A rearrangement Molecules 2023, 28, 2031 9 of 20 of the species leading to a lesser conjugation of the BPH group could explain this behavior, in agreement with the small variation of the absorption wavelength from 307 to 298 nm. of the species leading to a lesser conjugation of the BPH group could explain this behavior, in agreement with the small variation of the absorption wavelength from 307 to 298 nm. Figure 6. Reaction Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend (…), em sion wavelength trend (---) (a); absorption titration at λ = 303 nm (L2: Zn(II) 1:1, (b) and 300 nm Zn(II) 1:2, (d) (■), distribution diagrams of the species (−) for the Zn(II)/L2 system as a function pH in aqueous solution (c): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10− (left), [Zn2+] = 2 × 10−3 M (right). Figure 6. Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend ( . . . ), emission wavelength trend (—) (a); absorption titration at λ = 303 nm (L2: Zn(II) 1:1, (b) and 300 nm (L2: Zn(II) 1:2, (d) (■), distribution diagrams of the species (−) for the Zn(II)/L2 system as a function of pH in aqueous solution (c): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (left), [Zn2+] = 2 × 10−3 M (right). gure 6. Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend (…), e on wavelength trend (---) (a); absorption titration at λ = 303 nm (L2: Zn(II) 1:1, (b) and 300 nm n(II) 1:2, (d) (■), distribution diagrams of the species (−) for the Zn(II)/L2 system as a functio H in aqueous solution (c): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10 eft), [Zn2+] = 2 × 10−3 M (right). Figure 6. Fluorescence emission titration (λex = 288 nm) (■), absorption wavelength trend ( . . . ), emission wavelength trend (—) (a); absorption titration at λ = 303 nm (L2: Zn(II) 1:1, (b) and 300 nm (L2: Zn(II) 1:2, (d) (■), distribution diagrams of the species (−) for the Zn(II)/L2 system as a function of pH in aqueous solution (c): I = 0.1 M NMe4Cl at 298.1 ± 0.1 K, [L1] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (left), [Zn2+] = 2 × 10−3 M (right). 3. Reaction PMG and AMPA Binding by Zn(II) Complexes of L1 and L2 In the previous section we showed that L1 and L2 could be considered as us hemosensors of H+ and Zn(II), since they are able to change their optical absorption mission properties as a function of pH and in the presence of the metal cation. As for Finally, the addition of two hydroxide groups to give the [Zn(H−1L1)(OH)2]−species probably increases the rigidity of the BPH-Zn(II) system: this could be due to the removal of the possible coordinated water molecules that would favor the vibrational dissipation of energy, thus inducing a fluorescence emission increase. However, a full deprotonation of BPH cannot be totally ruled out, as already suggested above. p p p p n(II) complexes, most of them showed to be prone to add external anionic guests, s s OH−. Moreover, in the case of dinuclear complexes of L2, two Zn(II) centers are pla short distance between each other, thus providing distinct anchoring sites for the ltaneous binding of multiple negatively charged residues of a given substrate. A ether, these considerations make the metal complex species of L1 and L2, hereina eferred to as R systems (R1 = Zn(II) mononuclear complexes of L1 and R2 = Zn(II) m nd dinuclear complexes of L2), particularly attractive as metallo-receptors for anions his reason, and in view of the growing need for suitable tools to recognize the envi mentally relevant glyphosate and AMPA, herein we decided to investigate the abilit he R metallo-receptors to bind such important analytes in aqueous solution, as we y y gg In the case of L2 in the presence of an equimolar amount of Zn(II) (Figure 6a,b), the formation of the [Zn(H2L2)]4+ species comes with the increase of fluorescence emission at 402 nm, that is due to both Zn(II) coordination and deprotonation of the BPH group, as highlighted by the increase of absorbance and a change of the absorption wavelength from 281 to 313 nm. The deprotonation of [Zn(H2L2)]4+ to give [Zn(HL2)]3+, first, and then [ZnL2]2+, induces a drop of both the fluorescence emission intensity and wavelength, from 402 to 391 nm; also, the absorption wavelength blue shifts from 313 to 305 nm. A further increase of both the fluorescence emission intensity and wavelength (391 to 397 nm) is observed at pH > 8.5, where the [Zn(H−1L2)]+ species prevails in solution. 2.3. PMG and AMPA Binding by Zn(II) Complexes of L1 and L2 In the previous section we showed that L1 and L2 could be considered as useful chemosensors of H+ and Zn(II), since they are able to change their optical absorption and emission properties as a function of pH and in the presence of the metal cation. As for the Zn(II) complexes, most of them showed to be prone to add external anionic guests, such as OH−. Moreover, in the case of dinuclear complexes of L2, two Zn(II) centers are placed at short distance between each other, thus providing distinct anchoring sites for the simultaneous binding of multiple negatively charged residues of a given substrate. Altogether, these considerations make the metal complex species of L1 and L2, hereinafter referred to as R systems (R1 = Zn(II) mononuclear complexes of L1 and R2 = Zn(II) mono- and dinuclear complexes of L2), particularly attractive as metallo-receptors for anions. For this reason, and in view of the growing need for suitable tools to recognize the environmen- tally relevant glyphosate and AMPA, herein we decided to investigate the ability of the R metallo-receptors to bind such important analytes in aqueous solution, as well as their capability to detect their presence via fluorescence signaling. Reaction Being the mononuclear species the only significantly present in solution, the observed behavior could be attributed to a rearrangement of the species that affect the conjugation of the BPH moiety. he R metallo receptors to bind such important analytes in aqueous solution, as we heir capability to detect their presence via fluorescence signaling. 3.1. Potentiometric Measurements The capacity of R metallo-receptors to bind PMG and AMPA was investig hrough potentiometric measurements in NMe4Cl 0.1 M solution at 298.1 ± 0.1 K. R:A In the case of L2 in the presence of a double amount of Zn(II) compared to the ligand (Figure 6c,d), the mononuclear species are prevalent in solution up to pH 7. Up to this pH value, a similar discussion as for L2: Zn(II) 1:1 molar ratio can be made also for the L2: Zn(II) 1:2 molar ratio. Starting from pH 7 the dinuclear species prevail in solution: the formation of [Zn2(H−1L2)]3+ induces a small increase of the fluorescence emission and a blue-shift of the emission wavelength from 390 to 386 nm. In this range of pH (7–9) a change Molecules 2023, 28, 2031 10 of 20 10 of 20 of the absorption wavelength from 305 to 299 nm is also observed. All these phenomena could be attributed to the formation of the [Zn2(H−1L2)]3+ dinuclear species. The formation of the next [Zn2(H−2L2)]2+ species in the pH range 9–10 is accompanied by a small drop of both absorption and fluorescence emission, with a red-shift of the emission wavelength from 386 to 392 nm. The absorption wavelength keeps on blue shifting, reaching 297 nm. All these observations are related to the change of the protonation degree of BPH, that becomes fully deprotonated, along with the simultaneous coordination of each Zn(II) ion by one phenolate function of BPH, as already reported [45]. Finally, the formation of the neutral hydroxylated species [Zn2(H−2L2)(OH)2] at pH > 10 makes the fluorescence emission increasing again, suggesting again an increase in the BPH conjugation upon binding of OH−. 2.3.1. Potentiometric Measurements The capacity of R metallo-receptors to bind PMG and AMPA was investigated through potentiometric measurements in NMe4Cl 0.1 M solution at 298.1 ± 0.1 K. R:A (A = PMG or AMPA) molar rations varying from 0.2:1 to 2:1 was employed, to ascertain the stoi- chiometries of the ternary adducts formed in solution. The resulting stability constants are summarized in Table 3, whereas the corresponding distribution diagrams of the species present in solutions are respectively shown in Figure 7. Table 3. Logarithms of the equilibrium constants (LogK) for the coordination of Zn(II) complexes of L1 and L2 with glyphosate and AMPA determined by means of potentiometric measurements in 0.1 M NMe4Cl at 298 ± 0.1 K. Table 3. Logarithms of the equilibrium constants (LogK) for the coordination of Zn(II) complexes of L1 and L2 with glyphosate and AMPA determined by means of potentiometric measurements in 0.1 M NMe4Cl at 298 ± 0.1 K. Reaction L1 LogK [Zn(H−1L1)]+ + HPMG2−= [Zn(H−1L1)(HPMG)]− 3.54 (9) 1 [ZnL1]2+ + HPMG2−= [ZnL1(HPMG)] 4.46 (8) [Zn(H−1L1)]+ + HAMPA−= [Zn(H−1L1)(HAMPA)] 3.47 (9) [ZnL1]2+ + HAMPA−= [ZnL1(HAMPA)]+ 2.74 (8) L2 LogK [Zn(H−1L2)]+ + HPMG2−= [Zn(H−1L2)(HPMG)]− 6.97 (8) [ZnL2]2+ + HPMG2−= [ZnL2(HPMG)] 8.08 (9) [Zn2(H−2L2)]2+ + HPMG2−= [Zn2(H−2L2)(HPMG)] 5.97 (8) [Zn2(H−1L2)]3+ + HAMPA−= [Zn2(H−1L2)(HAMPA)]2+ 4.25 (8) [Zn2(H−2L2)]2+ + HAMPA−= [Zn2(H−2L2)(HAMPA)]+ 4.74 (7) 1 Values in parentheses are standard deviations on the last significant figures. 11 of 20 Molecules 2023, 28, 2031 3, 8, O EE E Figure 7. Distribution diagrams of the species formed in solution for the systems Zn(I Zn(II)/L1/AMPA (b), Zn(II)/L2/PMG (c), 2Zn(II)/L2/PMG (d) and 2Zn(II)/L2/AMPA = [PMG] = [AMPA] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (a–c) and 2 × 10−3 M (d,e), NMe4C 0.1 K. The superior binding properties of R2 towards PMG as compared to R1 Figure 7. Distribution diagrams of the species formed in solution for the systems Zn(II)/L1/PMG (a), Zn(II)/L1/AMPA (b), Zn(II)/L2/PMG (c), 2Zn(II)/L2/PMG (d) and 2Zn(II)/L2/AMPA (e [L1] = [L2] = [PMG] = [AMPA] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (a–c) and 2 × 10−3 M (d,e NMe4Cl 0.1 M, 298.1 ± 0.1 K. A first analysis of data reveals that PMG and AMPA form stable adducts with both th mono- and dinuclear complexes of L1 and L2, with the only exception of the mononuclea e 7. 2.3.1. Potentiometric Measurements Overall, the LogK values relative to the PMG coordination emerge to be comparable or up to 2 log units higher relative to the ones reported in literature for the binding of this substrate by analogous Zn(II)-polyamine receptors [56–58]. As shown in the distribution diagrams reported in Figure 7, the ternary adducts are formed at intermediate pH values, within 4 and 11, as expected due to the presence in this interval of pH of highly charged forms of both the metallo-receptors and the anionic substrates. In particular, the most relevant species formed by PMG with the two recep- tor systems emerged to be [ZnL1(HPMG)], [Zn(H−1L1)(HPMG)]−, [ZnL2(HPMG)] and [Zn2(H−2L2)(HPMG)] (Figure 7a,c,d), which are present in solution in a wide range of pH, from 4 to 11. On the other side, AMPA is mainly present in the adducts [ZnL1(HAMPA)]+, [Zn(H−1L1)(HAMPA)], [Zn2(H−1L2)(HAMPA)]2+ and [Zn2(H−2L2)(HAMPA)]+ (Figure 7b,e), which occur in a slightly narrower range of pH, from 5 to 11. For more alkaline pH values, the hydroxylated species of the metallo-receptors become to be predominant in solution. y y p p p The superior binding properties of R2 towards PMG as compared to R1 can be better evidenced by the selectivity diagrams calculated for a competitive system containing PMG, R1 and R2 in equimolar amounts (R1 = ∑Zn(II)-mononuclear species of L1 bound and R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound). Indeed, as shown in Figure 8, where are reported the overall percentages of the adducts formed by the different metallo- receptors with PMG (in its monoprotonated form HPMG2−) as a function of pH, this substrate is preferentially bound to R2 over a wide range of pH, from 4 to 11. In strict analogy with our previous study [48], this finding can be possibly rationalized by consid- ering that, in the dinuclear complexes of L2, two coordinatively unsaturated Zn(II) ions can cooperate in the binding of PMG, with the latter being likely coordinated through its phosphate and carboxylate groups in a bridge disposition, with a Zn-O-C-C-N-C-P-O-Zn arrangement (Figure 8b). The simultaneous involvement of two distinct Zn(II) ions in R2 would be therefore clearly advantageous if compared to the anion coordination by R1, where only one metal-based anchoring site is available. 2.3.1. Potentiometric Measurements Distribution diagrams of the species formed in solution for the systems Zn(II)/ /L1/AMPA (b), Zn(II)/L2/PMG (c), 2Zn(II)/L2/PMG (d) and 2Zn(II)/L2/AMPA (e MG] = [AMPA] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (a–c) and 2 × 10−3 M (d,e), NMe4Cl 0 Figure 7. Distribution diagrams of the species formed in solution for the systems Zn(II)/L1/PMG (a), Zn(II)/L1/AMPA (b), Zn(II)/L2/PMG (c), 2Zn(II)/L2/PMG (d) and 2Zn(II)/L2/AMPA (e). [L1] = [L2] = [PMG] = [AMPA] = 1 × 10−3 M, [Zn2+] = 1 × 10−3 M (a–c) and 2 × 10−3 M (d,e), NMe4Cl 0.1 M, 298.1 ± 0.1 K. . The superior binding properties of R2 towards PMG as compared to R1 ca enced by the selectivity diagrams calculated for a competitive system G, R1 and R2 in equimolar amounts (R1 = ∑Zn(II)-mononuclear species o R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound). Indeed, as show here are reported the overall percentages of the adducts formed by th A first analysis of data reveals that PMG and AMPA form stable adducts with both the mono- and dinuclear complexes of L1 and L2, with the only exception of the mononuclear complexes of L2, for which no evidence of AMPA binding was found. Only AR adducts with 1:1 stoichiometry was observed under our experimental conditions, thus ruling out the coordination of multiple substrates to the same receptor unit but rather hinting at the simultaneous binding of a single guest to distinct anchoring sites of the host, likely through a bridge disposition (vide infra). Molecules 2023, 28, 2031 12 of 20 12 of 20 As it can be easily appreciated from Table 3, among the two substrates PMG led to the formation of the most stable adducts with the different forms of the two metallo-receptors. For example, the addition of HPMG2−to [Zn2(H−2L2)]2+ occurs with a LogK value of 5.97, whereas the addition of HAMPA−to the same species takes place with a LogK of 4.74; similar considerations can be made for L1. The comparison between the two metallo-receptors unveils instead a higher affinity of PMG for L2, in both its mono- and dinuclear Zn(II) complex forms. This can be highlighted, for example, by LogK values of 6.97 and 3.54, respectively found for the addition of HPMG2−to [Zn(H−1L2)]+ and [Zn(H−1L1)]+. re omitted for clarity (b). 2.3.2. Fluorescence Measurements 3.2. Fluorescence Measurements Besides the analysis of the binding properties of the Zn(II) complexed form nd L2 towards PMG and AMPA, the ability of these metallo-receptors to underg tion of their fluorescence emission as a result of the coordination of the same ani so evaluated. To this aim, aqueous solutions of R systems were added with inc mounts of a selected anionic substrate and the resulting spectra were collected. F ystem, measurements were performed at a fixed pH value, which was selected he formation of a solution of the most stable adducts between the metallo-recep he anionic substrates. The coordinative selectivity highlighted by potentiometr which indicated the R2 systems as the most effective in the coordination of PMG, aralleled by the same optical trend. In fact, in general, the luminescent propert ystems were only poorly affected by the coordination of the tested anions, with xception being represented by the adducts formed by R1 with AMPA. In this hown in Figure 9a, the presence of increasing concentrations of AMPA in a sol H 9 of R1 determined a progressive quenching of the fluorescence emission of t em, resulting in ca. 35% decrease induced by a relatively small amount of an quivalents). In all the other cases, the same amount of PMG/AMPA led to sma ons of fluorescence emission that did not exceed 15% of the signal of the unb metallo-receptors (Figure 9b). Since a PET mechanism could not be usually invoke Besides the analysis of the binding properties of the Zn(II) complexed forms of L1 and L2 towards PMG and AMPA, the ability of these metallo-receptors to undergo a variation of their fluorescence emission as a result of the coordination of the same anions was also evaluated. To this aim, aqueous solutions of R systems were added with increasing amounts of a selected anionic substrate and the resulting spectra were collected. For each system, measurements were performed at a fixed pH value, which was selected to allow the formation of a solution of the most stable adducts between the metallo-receptors and the anionic substrates. The coordinative selectivity highlighted by potentiometric data, which indicated the R2 systems as the most effective in the coordination of PMG, was not paralleled by the same optical trend. 2.3.1. Potentiometric Measurements Interestingly enough, the different binding abilities observed between the mononuclear complexes of the two ligands (Figure S5) could be tentatively explained by assuming that these metallo-receptors still display two potential anchoring sites, being represented by a coordinated Zn(II) ion on one side and by protonated nitrogen atom/s (-NH2+-) on the other side. In this view, the prolonged O-C-C-N-C-P-O scaffold of the guest would better match the larger Zn(II)-NH2+ distance in the 5-membered macrocycle of L2, compared to the smaller cavity (4-membered macrocycle) of L1, thus justifying the higher affinity of PMG towards the mononuclear complex species of L2. Accordingly, the lower affinity of both R1 and R2 towards AMPA would be the result of the worse fit between the smaller anionic bite of this guest (the O-donors are gathered on the same phosphate group) and the above proposed ditopic motif of the metallo-receptors. 13 of 20 ed ditop 13 of 20 ed ditop Molecules 2023, 28, 2031 Figure 8. Selectivity diagrams showing the affinity of glyphosate (HPMG2−) with the metal species of L1 and L2 (R1 and R2, respectively) as a function of pH. Percentages were calcula respect to ligand concentrations ([R1] = [R2] = [HPMG] = 1 × 10−3 M, R1 = ∑Zn(II)-mononuc cies of L1 bound and R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound) (a). Models p for the coordination of HPMG2− by the metallo-receptors [Zn(H−1L1)]+ and [Zn2(H−2L2)]2+ are omitted for clarity (b). Figure 8. Selectivity diagrams showing the affinity of glyphosate (HPMG2−) with the metal complex species of L1 and L2 (R1 and R2, respectively) as a function of pH. Percentages were calculated with respect to ligand concentrations ([R1] = [R2] = [HPMG] = 1 × 10−3 M, R1 = ∑Zn(II)-mononuclear species of L1 bound and R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound) (a). Models proposed for the coordination of HPMG2−by the metallo-receptors [Zn(H−1L1)]+ and [Zn2(H−2L2)]2+, charges are omitted for clarity (b). igure 8. Selectivity diagrams showing the affinity of glyphosate (HPMG2−) with the metal pecies of L1 and L2 (R1 and R2, respectively) as a function of pH. Percentages were calcula espect to ligand concentrations ([R1] = [R2] = [HPMG] = 1 × 10−3 M, R1 = ∑Zn(II)-mononuc es of L1 bound and R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound) (a). 2.3.1. Potentiometric Measurements Models p or the coordination of HPMG2− by the metallo-receptors [Zn(H−1L1)]+ and [Zn2(H−2L2)]2+ re omitted for clarity (b) Figure 8. Selectivity diagrams showing the affinity of glyphosate (HPMG2−) with the metal complex species of L1 and L2 (R1 and R2, respectively) as a function of pH. Percentages were calculated with respect to ligand concentrations ([R1] = [R2] = [HPMG] = 1 × 10−3 M, R1 = ∑Zn(II)-mononuclear species of L1 bound and R2 = ∑Zn(II)-mono- and dinuclear species of L2 bound) (a). Models proposed for the coordination of HPMG2−by the metallo-receptors [Zn(H−1L1)]+ and [Zn2(H−2L2)]2+, charges are omitted for clarity (b). re omitted for clarity (b). 2.3.2. Fluorescence Measurements In fact, in general, the luminescent properties of R systems were only poorly affected by the coordination of the tested anions, with the only exception being represented by the adducts formed by R1 with AMPA. In this case, as shown in Figure 9a, the presence of increasing concentrations of AMPA in a solution at pH 9 of R1 determined a progressive quenching of the fluorescence emission of this system, resulting in ca. 35% decrease induced by a relatively small amount of analyte (5 equivalents). In all the other cases, the same amount of PMG/AMPA led to small variations of fluorescence emission that did not exceed 15% of the signal of the unbounded metallo-receptors (Figure 9b). Since a PET mechanism could not be usually invoked in the case of BPH-containing ligands, the observed quenching upon addition of AMPA to R1 could be rationalized in terms of coplanarity of the BPH group: a distortion of this moiety could be hypothesized in the binding of a non-well-fitting substrate such as AMPA, as discussed above. The lost in coplanarity would diminish the conjugation of BPH, and, finally, the fluorescence emission. 14 of 20 14 of 20 Molecules 2023, 28, 2031 Figure 9. Fluorescence spectra of aqueous solutions containing L1 and Zn(II) in 1:1 mol pH 9 and collected by adding increasing amounts of AMPA; in the inset is reported the va the fluorescence emission at 395 nm as a function of the concentration of the anion (a). H showing the variations at the maximum fluorescence emission of metal complex species L2 (R systems) in the presence of 5 equivs. of PMG (gray bars) and AMPA (light grey b blue color highlights the starting point of the fluorescence titration whereas the red line h the last measurement of the titration, registered in the presence of an excess of anionic sp ([R] = 5 µM, λex = 270 nm). (The grey scales further highlight the data collected in the prese different anionic guests tested, whereas the dark grey bar evidences the reponse of the Figure 9. Fluorescence spectra of aqueous solutions containing L1 and Zn(II) in 1:1 molar ratio at pH 9 and collected by adding increasing amounts of AMPA; in the inset is reported the variation of the fluorescence emission at 395 nm as a function of the concentration of the anion (a). plexed species o 3. Conclusions onclusions In this study, we explored the potential as metallo-receptors for glyphosat AMPA of the Zn(II) complexes of two polyamino phenolic ligands characte In this study, we explored the potential as metallo-receptors for glyphosate (PMG) and AMPA of the Zn(II) complexes of two polyamino-phenolic ligands, characterized by a fluorogenic biphenolic unit nicely placed into macrocyclic polyamine fragments of four (L1) and five (L2) nitrogen atom members. AMPA of the Zn(II) complexes of two polyamino-phenolic ligands, characte uorogenic biphenolic unit nicely placed into macrocyclic polyamine fragmen and five (L2) nitrogen atom members. Among the two ligands, L2 was prepared by employing a novel synthetic a g Among the two ligands, L2 was prepared by employing a novel synthetic approach, which consisted of a modification of the Richman-Atkins method. This allowed us to avoid the use of the toxic cadmium(II) ion, which was instead necessary for the previous template synthesis. Among the two ligands, 2 was prepared by employing a novel synthetic a ch consisted of a modification of the Richman-Atkins method. This allow id the use of the toxic cadmium(II) ion, which was instead necessary for the plate synthesis. Following the study of the acid-base properties of L1 and L2, their ability Following the study of the acid-base properties of L1 and L2, their ability to bind Zn(II) in an aqueous solution was investigated by means of potentiometric, UV-Vis, and fluorescence measurements. Thanks to their peculiar macrocyclic design, these ligands allowed the formation of stable mono (L1) and both mono and dinuclear complexes (L2) with Zn(II), resulting in promising metallo-receptors for anionic targets. g y p p , y II) in an aqueous solution was investigated by means of potentiometric, UV orescence measurements. Thanks to their peculiar macrocyclic design, these wed the formation of stable mono (L1) and both mono and dinuclear compl The ability of the Zn(II) complexes of L1 and L2 (R1 and R2) to act as metallo-receptors for glyphosate and AMPA was inspected through potentiometric measurements. These studies highlighted that R1 and R2 strongly interact with the anionic substrates, with both receptors preferentially binding PMG over AMPA. h Zn(II), resulting in promising metallo-receptors for anionic targets. re omitted for clarity (b). 2.3.2. Fluorescence Measurements (The grey scales further highlight the data collected in the presence of the different anionic guests tested, whereas the dark grey bar evidences the reponse of the of metal complexed species of L1 and L2 (R systems) for comparison.) re omitted for clarity (b). 2.3.2. Fluorescence Measurements Histogram showing the variations at the maximum fluorescence emission of metal complex species of L1 and L2 (R systems) in the presence of 5 equivs. of PMG (gray bars) and AMPA (light grey bars) (The blue color highlights the starting point of the fluorescence titration whereas the red line highlights the last measurement of the titration, registered in the presence of an excess of anionic species.) (b) ([R] = 5 µM, λex = 270 nm). (The grey scales further highlight the data collected in the presence of the different anionic guests tested, whereas the dark grey bar evidences the reponse of the of metal complexed species of L1 and L2 (R systems) for comparison.) ure 9. Fluorescence spectra of aqueous solutions containing L1 and Zn(II) in 1:1 mol 9 and collected by adding increasing amounts of AMPA; in the inset is reported the v fluorescence emission at 395 nm as a function of the concentration of the anion (a). H wing the variations at the maximum fluorescence emission of metal complex species R systems) in the presence of 5 equivs. of PMG (gray bars) and AMPA (light grey b color highlights the starting point of the fluorescence titration whereas the red line last measurement of the titration, registered in the presence of an excess of anionic sp = 5 µM, λex = 270 nm). (The grey scales further highlight the data collected in the prese erent anionic guests tested, whereas the dark grey bar evidences the reponse of th Figure 9. Fluorescence spectra of aqueous solutions containing L1 and Zn(II) in 1:1 molar ratio at pH 9 and collected by adding increasing amounts of AMPA; in the inset is reported the variation of the fluorescence emission at 395 nm as a function of the concentration of the anion (a). Histogram showing the variations at the maximum fluorescence emission of metal complex species of L1 and L2 (R systems) in the presence of 5 equivs. of PMG (gray bars) and AMPA (light grey bars) (The blue color highlights the starting point of the fluorescence titration whereas the red line highlights the last measurement of the titration, registered in the presence of an excess of anionic species.) (b) ([R] = 5 µM, λex = 270 nm). 4.1. Synthesis Ligands L1 and L2 were obtained following the synthetic procedure reported in Scheme 1. 3,3′-Bis(bromomethyl)-2,2′-dimethoxybiphenyl (1) [59–61], 1,4,7,10-tetrakis (p-tolylsulphonyl)-1,4,7,10-tetrazadecane (2) [62] and 1,4,7,10,13-pentakis(p-tolylsulphonyl)- 1,4,7,10,13-pentaazatridecane (3) [63] were prepared as previously described. All other chemicals were purchased, using the highest quality commercially available. The solvents were RP grade, unless otherwise indicated. 4.1.1. 23,24-Dimethoxy-3,6,9,12-tetrakis(4-methylbenzenesufonyl)-3,6,9,12- tetraazatricyclo[17.3.1.1(14,18)]eicosatetra-1(23),14,16,18(24),19,21-hexaene (4) Over a period of 4 h, a solution of 1 (2.0 g, 5.0 mmol) in 100 mL of anhydrous acetonitrile was added to a refluxing suspension of 2 (3.8 g, 5.0 mmol) and K2CO3 (6.9 g, 50 mmol) in 250 mL of anhydrous acetonitrile, under nitrogen. The reaction mixture was refluxed for further 24 h. Subsequently, the mixture was cooled down to room temperature (R.T.) and the resulting suspension was concentrated under reduced pressure to one third of the initial volume, then poured into stirred cold water (1 dm3). The resulting white precipitate was filtered off, washed with cold water, dried under vacuum and purified by flash chromatography (hydrated alumina, dichloromethane/chloroform 70/30 v/v) obtaining 4 as a white solid (1.8 g, 36%). 1H NMR (CDCl3, 25 ◦C): δ = 2.41 (s, 6H), 2.48 (s, 6H), 2.92 (s, 6H), 2.97–3.52 (m, 12H), 3.94 (d, J = 14.3 Hz, 2H), 4.65 (d, J = 14.3 Hz, 2H), 7.15–7.23 (m, 4H), 7.30 (d, J = 8.1 Hz, 4H), 7.37 (d, J = 8.1 Hz, 4H), 7.53–7.62 (m, 2H), 7.70 (d, J = 8.3 Hz, 4H), 7.79 (d, J = 8.3 Hz, 4H) ppm. 13C NMR: δ = 21.5, 21.6, 47.3, 47.8, 48.9, 49.0, 61.2, 125.1, 127.3, 127.5, 129.8, 129.9, 130.5, 131.3, 132.0, 132.1, 135.2, 136.7, 143.5, 143.6, 155.7 ppm. 4.1.2. 23,24-Dihydroxy-3,6,9,12-tetraazatricyclo[17.3.1.1(14,18)]eicosatetra- 1(23),14,16,18(24),19,21-hexaene Tetrahydrochloride (L1·4HCl) plexed species o 3. Conclusions The ability of the Zn(II) complexes of L1 and L2 (R1 and R2) to act as metal for glyphosate and AMPA was inspected through potentiometric measu t di hi hli ht d th t R1 d R2 t l i t t ith th i i Comparative studies especially proved that PMG was preferentially coordinated by R2, also in competition with R1. This was assumed to be due to a ditopic motif for PMG coordination by R2, where the carboxylate and phosphate residues of the anionic guest are simultaneously coordinated to metal centers, in a bridge disposition among them. se studies highlighted that R1 and R2 strongly interact with the anionic su h both receptors preferentially binding PMG over AMPA. Comparative studies especially proved that PMG was preferentially coordi also in competition with R1 This was assumed to be due to a ditopic motif Interestingly, R2 systems displayed a higher affinity for PMG over R1, even in their Zn(II) mononuclear forms. This led us to speculate that the ditopic coordination motif could be preserved in the mononuclear forms of L2, through the possible involvement, besides the metal, of protonate nitrogen atom/s (-NH2+-) of the polyamine framework. Molecules 2023, 28, 2031 15 of 20 15 of 20 Lastly, the ability of R systems to detect the presence of the selected analytes through fluorescence signaling in an aqueous solution was evaluated. In general, the luminescent properties of R1 and R2 were slightly affected by the coordination of the tested anions, with the only exception being represented by the adducts formed by R1 with AMPA. In conclusion, this work further highlights the intriguing perspectives arising from the use of polyamino-phenolic ligands in the design of promising metallo-receptors for the recognition and sensing of elusive anions of great environmental relevance. It es- pecially shows that slight differences in the structural architectures of this class of com- pounds may strongly affect not only their binding properties but even their behavior as fluorimetric chemosensors. 4.1.4. 26,27-Dihidroxy-3,6,9,12,15-pentaazatricyclo[20.3.1.1(17,21)]eicosaepta- 1(26),17,19,21(27),22,24-hexaenepentahidrochloride (L2·5HCl) 4.1.4. 26,27-Dihidroxy-3,6,9,12,15-pentaazatricyclo[20.3.1.1(17,21)]eicosaepta- 1(26),17,19,21(27),22,24-hexaenepentahidrochloride (L2·5HCl) L2·5HCl was obtained following the same procedure used for L1·4HCl (5 (1.5 g, 1.2 mmol); L2·5HCl (490 mg white solid, 68%)). Anal. Calcd. for C22H38N5O2Cl5: C 45.41; H 6.58; N 12.04. Found: C 45.4; H 6.5; N 12.1. MS m/z (ESI): 400.5 (M + H+). 1H NMR (D2O, pH 2, 25 ◦C): δ = 3.33–3.42 (m, 8H), 3.45–3.53 (m, 8H), 4.37 (s, 4H), 7.11 (t, J = 7.7 Hz, 2H), 7.34 (dd, J = 1.7, 7.6 Hz, 2H), 7.41 (dd, J = 1.5, 7.6 Hz, 2H) ppm. 13C NMR: δ = 42.4, 43.7, 44.2, 44.6, 47.3, 118.6, 122.0, 125.1, 132.4, 133.7, 152.4 ppm. 4.1.2. 23,24-Dihydroxy-3,6,9,12-tetraazatricyclo[17.3.1.1(14,18)]eicosatetra- 1(23),14,16,18(24),19,21-hexaene Tetrahydrochloride (L1·4HCl) Ammonia (300 mL) was condensed in a suspension of 4 (1.5 g, 1.5 mmol) in diethyl ether (30 mL) and methanol (1 mL) and cooled down to −70 ◦C. Small pieces of lithium were carefully added to the mixture until the suspension turned blue. After thirty minutes, NH4Cl (12 g, 0.2 mol) was added. The white solid obtained after the evaporation of the solvent was treated with 3 mol dm−3 HCl (3 × 100 mL). The acidic solution was filtered and then evaporated to dryness, then the resulting solid was dissolved in the minimum amount of water and the solution made alkaline with concentrated NaOH. The liquid was extracted with CHCl3 (6 × 50 mL). The organic phase was dried over Na2SO4 and vacuum-evaporated to obtain a solid that was dissolved in ethanol and treated with 37% HCl/ethanol 1:1 v/v until complete precipitation of a white solid, which was filtered off to obtain L1 as tetrahydrochloride salt (490 mg, 68%). Anal. Calcd. for C20H32N4O2Cl4: C 47.82; H 6.42; N 11.15. Found: C 47.7; H 6.5; N 11.1. MS m/z (ESI): 357.5 (M + H+). 1H NMR (D2O, pH 2, 25 ◦C): δ = 3.18–3.57 (m, 12H), 4.39 (s, 4H), 7.14 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H) ppm. 13C NMR: δ = 41.4, 43.0, 43.8, 46.0, 118.6, 122.4, 126.1, 132.5, 133.6, 152.5 ppm. Molecules 2023, 28, 2031 16 of 20 4.1.3. 26,27-Dimethoxy-3,6,9,12,15-pentakis(4-methylbenzenesufonyl)-3,6,9,12,15- pentaazatricyclo[20.3.1.1(17,21)]eicosaepta-1(26),17,19,21(27),22,24-hexaene (5) 4.1.3. 26,27-Dimethoxy-3,6,9,12,15-pentakis(4-methylbenzenesufonyl)-3,6,9,12,15- pentaazatricyclo[20.3.1.1(17,21)]eicosaepta-1(26),17,19,21(27),22,24-hexaene (5) 5 was obtained following the same procedure used for 4 (1 (1.9 g, 4.7 mmol); 3 (4.5 g, 4.7 mmol) and K2CO3 (6.5 g,47 mmol); 5 (1.2 g white solid, 21%)). g g 1H NMR (CDCl3, 25 ◦C): δ = 2.40 (s, 6H), 2.41 (s, 6H), 2.47 (s, 3H), 2.89 (s, 6H), 2.93–3.21 (m, 6H), 3.23–3.61 (m, 10H), 4.24 (d, J = 14.2 Hz, 2H), 4.57 (d, J = 14.4 Hz, 2H), 7.06–7.15 (m, 4H), 7.24–7.38 (m, 10H), 7.49–7.58 (m, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 8.3 Hz, 4H), 7.75 (d, J = 8.3 Hz, 4H) ppm. 13C NMR: δ = 21.4, 21.5, 21.6, 47.2, 48.2, 49.3, 49.5, 51.0, 60.8, 124.3, 127.4, 127.5, 127.6, 129.6, 129.7, 129.8, 129.9, 131.7, 131.8, 132.2, 135.1, 136.8, 143.2, 143.6, 143.9, 156.1 ppm. 4.2. Potentiometric Measurements The equilibrium constants for protonation, Zn(II)-complexation, glyphosate and AMPA binding by the Zn(II)-complexes of L1 and L2, were determined by means of poten- tiometric (pH-metric) titrations in degassed 0.10 M NMe4Cl at 298.1 ± 0.1 K, employing equipment and procedures which have been already described [64–66]. Briefly, a combined glass electrode was calibrated as a hydrogen-ion concentration probe by titrating known amounts of HCl with CO2-free NaOH solutions, employing an Ag/AgCl electrode in saturated KCl as reference electrode. The equivalent point was determined by the Gran’s method [67], which afforded to obtain the standard potential Eo and the ionic product of water (pKw = 13.83 ± 0.01 in our experimental conditions). All the employed solutions were prepared by using freshly boiled, doubly deionized water, satu- rated with anhydrous nitrogen prior to uses; NaOH solutions were standardized against carbonate free potassium hydrogen phthalate and stored under nitrogen atmosphere. p y g p g p Measurements were performed by using a total ligand concentration of 1 × 10−3 M, in a range of pH within 2–11. For each system were performed at least three titration experiments, consisting of ca. 100 data points each. The relative equilibrium constants were determined from EMF data by using the program HYPERQUAD [68] while the distribution diagrams of the species present in the solution were obtained by the Hyss program [69]. 1. Maggi, F.; la Cecilia, D.; Tang, F.H.M.; McBratney, A. The global environmental hazard of glyphosate use. Sci. Total Environ. 2020, 717, 137167. [CrossRef] [PubMed] 1. Maggi, F.; la Cecilia, D.; Tang, F.H.M.; McBratney, A. The global environmental hazard of glyphosate use. Sci. Total Environ. 2020, 717, 137167. [CrossRef] [PubMed] 2. Richmond, M.E. Glyphosate: A review of its global use, environmental impact, and potential health effects on humans and other species. J. Environ. Stud. Sci. 2018, 8, 416–434. [CrossRef] 3. Gravena, R.; Filho, R.V.; Alves, P.L.C.A.; Mazzafera, P.; Gravena, A.R. Glyphosate has low toxicity to citrus plants growing in the field. Can. J. Plant Sci. 2012, 92, 119–127. [CrossRef] 4. Duke, S.O.; Powles, S.B. Glyphosate: A once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319– S.O.; Powles, S.B. Glyphosate: A once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319–325. [CrossRef ook, C.M. Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. Eur. 2016, 28, 3 les, S.B. Glyphosate: A once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319–325. [CrossRef] Duke, S.O.; Powles, S.B. Glyphosate: A once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319–325. [C Benbrook, C.M. Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. Eur. 201 , yp y g , , [ ] Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. Eur. 2016, 28, 3. [CrossRe , ; , yp y g , , 5. Benbrook, C.M. Trends in glyphosate herbicide use in the United States and globally. Environ. Sci. 6. Kanissery, R.; Gairhe, B.; Kadyampakeni, D.; Batuman, O.; Alferez, F. Glyphosate: Its Environmental Persistence and Impact on Crop Health and Nutrition. Plants 2019, 8, 499. [CrossRef] [PubMed] p 7. Hendlin, Y.H.; Arcuri, A.; Lepenies, R.; Hüesker, F. Like Oil and Water: The Politics of (Not) Assessing Glyphosate Concentrations in Aquatic Ecosystems. Eur. J. Risk Regul. 2020, 11, 539–564. [CrossRef] 8. Soares, D.; Silva, L.; Duarte, S.; Pena, A.; Pereira, A. Glyphosate Use, Toxicity and Occurrence in Food. Foods 2021, 10, 2785. [CrossRef] [PubMed] 9. Benbrook, C.M. How did the US EPA and IARC reach diametrically opposed conclusions on the genotoxicity of glyphosate-based herbicides? Environ. Sci. Eur. 2019, 31, 2. [CrossRef] 10. Meftaul, I.M.; Venkateswarlu, K.; Dharmarajan, R.; Annamalai, P.; Asaduzzaman, M.; Parven, A.; Megharaj, M. Controversies over human health and ecological impacts of glyphosate: Is it to be banned in modern agriculture? Environ. Pollut. 2020, 263, 114372. [CrossRef] 11. IARC, W. Evaluation of Five Organophosphate Insecticides and Herbicides; IARC: Lyon, France, 2015. 12. 4.3. Spectrophotometric and Fluorescence Measurements The authors would also like to thank G.E.A., Green Economy and Agriculture Centro per la Ricerca s.r.l., and MIUR for “Progetto Diparti- menti di Eccellenza 2023–2027” allocated to the Department of Chemistry “Ugo Schiff”, for financial support. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The publication was made by a researcher with a research contract co-funded by the European Union—PON Research and Innovation 2014–2020 in accordance with Article 24, paragraph (3a), of Law No. 240 of 30 December 2010, as amended, and Ministerial Decree No. 1062 of 10 August 2021. Ms Anna Rita Pierleoni is acknowledged for her help with NMR measurements. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 4.3. Spectrophotometric and Fluorescence Measurements 4.3. Spectrophotometric and Fluorescence Measurements Electronic UV-Vis absorption spectra were collected on a Perkin-Elmer Lambda 6 spec- trophotometer and on a Varian Cary-100 spectrophotometer equipped with a temperature control unit, whereas fluorescence emission spectra were registered on a spectrofluorometer Horiba FluoroMax Plus and a Varian Cary-Eclipse spectrofluorimeter (spectra are uncor- rected) by using an excitation wavelength of 288 nm. All measurements were performed at 298 ± 0.1 K. Molecules 2023, 28, 2031 17 of 20 17 of 20 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28052031/s1, Figure S1: (a) Absorption and (b) flu- orescence spectra of L1 at different pH values. [L1] = 1 × 10−5 M, λex = 288, λem = 403 nm; Figure S2: Fluorescence spectra of L1 + 1 equiv. of Zn(II) at different pH values (λex 288 nm); Figure S3: Fluorescence spectra of L2 + 1 equiv. of Zn(II) at different pH values (λex 288 nm); Figure S4: Fluorescence spectra of L2 + 2 equiv. of Zn(II) at different pH values (λex 288 nm); Figure S5: Selectivity diagrams showing the affinity of glyphosate (HPMG2−) with the mononu- clear Zn(II) complex species of L1 and L2 (R1 and R2, respectively) as a function of pH. Percent- ages were calculated with respect to ligand concentrations ([R1] = [R2] = [HPMG] = 1 × 10−3 M, R1 = ΣZn(II)-mononuclear species of L1 bound and R2 = ΣZn(II)-mononuclear species of L2 bound). Author Contributions: Conceptualization, V.F., L.G., L.C. and C.G.; investigation, D.P., G.E.G., M.F., L.M. and L.G.; writing—original draft preparation, L.C. and E.M.; writing—review and editing, V.F., C.G., B.V., L.C., G.E.G., E.M., L.G., M.F. and R.M.; supervision, V.F. and C.G.; funding acquisition, V.F., E.M., L.C., C.G., M.F. and B.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Italian Ministero dell’Istruzione dell’Università e della Ricerca (MIUR, project 2017EKCS35) and University of Urbino (Grant DISPEA_ASSEGNAZIONE_ ATENEO_SICUREZZA_ALIMENTARE, DISPEA_MACEDI_PROG21). The authors would also like to thank G.E.A., Green Economy and Agriculture Centro per la Ricerca s.r.l., and MIUR for “Progetto Diparti- menti di Eccellenza 2023–2027” allocated to the Department of Chemistry “Ugo Schiff”, for financial support. 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Goldene Konfirmation des Jahrgangs 1940/41 Von Ilse Fleischmann Am Sonntag, den 10. April 2005 trafen sich 87 ehemalige Konfirmandinnen und Konfirman­ den des Jahrgangs 1940/41 in Backnang, um das Jubiläum ihrer 50-jährigen Konfirmation miteinander zu feiern. Ein erster Treff - vor dem Festgottesdienst in der Stiftskirche - war auf dem Freithof, und es gab mit viel Hallo man­ ches Wiedersehen mit alten Freunden, die Jahr­ zehnte lang verschollen waren. Unter festlichen Trompeten- und Orgelklängen fand dann der gemeinsame Einzug in die Stiftskirche statt. Die Predigt in dem von einigen Jubilaren mitgestal­ teten Gottesdienst hielt Dekan i. R. Dieter Eisenhardt. Das Predigtthema „Wanderschaft und Heimkehr" brachte manchen zum Nach­ denken über seine zurückliegende, über 60-jährige Lebenswanderschaft mit Nachkriegszeit, Schulzeit, Berufs- und Arbeitswelt, und in der Regel mit nunmehrigem Ruhestand. Anschlie­ ßend begab man sich ins Bürgerhaus zu einem Sektempfang und Mittagessen. Der Nachmittag stand dann unter dem Moto: weißt du noch? Eine kleine Diaschau mit alten Aufnahmen rief längst vergessene Erinnerungen wach an viele gemeinsame Erlebnisse in einer doch schönen Jugendzeit, ohne Fernsehen und Handy, bei einem erst langsam sich abzeichnenden Wohl­ stand in unserem Lande. Nach dem obligatori­ schen Gruppenfoto klang das Fest bei Kaffee und Kuchen langsam aus, und wohl alle - und nicht nur der Festausschuss - konnten sich über eine harmonische und gelungene Veranstaltung freuen, mit neu geknüpften Kontakten die nun sicher intensiver gepflegt werden. Die Namen der Goldenen Konfirmanden/Innen sind (in alphabetischer Reihenfolge): Horst Adam, Walter Bareither, Elfriede Bauer geb. Roos, Dieter Bäuml, Harald Behnert, Irma Binder-Schaal geb. Schaal, Renate Bock geb. Wurst, Doris Bohn geb. Schmidgall, Siegrun Braun geb. Entenmann, Rolf Burkhardtsmeier, 282 Heide Bürkle, Irene Eckstein geb. Schenk, Kurt Eisenmann, Brigitte Erkert geb. Anger, Ursel Esenwein geb. Jungmann, Otto Feiger, Lore Fichtner geb. Klotz, Ilse Fleischmann geb. Pfuderer, Helga Gehring geb. Dais, Ursula Goltz geb. Leicht, Doris Gräter geb. Rothfuß, Hans Guth, Elke Haas geb. Rais, Erwin Här, Margarete Henn geb. Hoffmann, Bärbel Hermann geb. Schad, Ilse Hessler geb. Vogel, Maria Hinderer geb. Bischof, Rose Hinderer geb. Franke, Sieghart Hummel, Günther Ibler, Heiderose Jäger geb. Elser, Walter Jauch, Heiderose Kemmler geb. Wurst, Hans Knörzer, Heide Körner geb. Endres, Gerhard Körner, Ernst Kreß, Ursel Greß geb. Scholpp, Helga Krull geb. Schiefer, Rolf Kübler, Herbert Kühner, Renate Kühnle geb. Noller, Heinz Kühnle, Sigmar Langbein, Uta Langhoff geb. lunger, Margarete Lauterwasser geb. Benz, Dieter Lenz, Bärbel Marbaz geb. Lange, Helga Mayer geb. Gier, Heide Möchel geb. Bauer, Brigitte Müller geb. Klett, Werner Müller, Volker Müller, Manfred Packmor, Ilse Petershans geb. Benignus, Doris Pfeil geb. Bäuerle, Klaus Platzek, Herbert Rieger, Kurt Rieger, Inge Riester geb. Reusch, Adolf Schaal, Helga Schäfer geb. Seitel, Sieglinde Schatz geb. Krauter, Ulrike Schintz geb. Kapphan, Herbert Schmidgall, Margarete Schmitt geb. Tritt, Helga Schreiber, Willi Schwab, Emil Schwarz, Werner Schwarz, Manfred Seitel, Edith Silbermann geb. Oppenländer, Irmgard Stängle geb. Stätter, Waltraud Stitz geb. Morgenthaler, Gerturd Ten Brink geb. Schneck, Heiderose Traub geb. Waibel, Günter Trefz, Erika Vermehren geb. Tschache, Inge Wahl geb. Zügel, Adelheid Warkentin geb. Schad, Margret Wehle geb. Scheib, Ilse Wendt geb. Conradt, Rosemarie Widmer geb. Krauter, Rudolf Wiesenauer, Dieter Wildermuth, Rudolf Wurst und Traude Zehender geb. Bürkler. 283 Gruppenfoto im Bürgerhaus.
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https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/97/e3sconf_bft2023_06024.pdf
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Calculation of the speed of movement of the car along the entire length of the path profile with different inclines of the sorting slide
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Calculation of the speed of movement of the car along the entire length of the path profile with different inclines of the sorting slide Nassim Rustamov1, Asal Kasimova2, Rashida Tursunkhodjaeva2, and Mukaddas 1 Khoja Akhmet Yassawi International Kazakh-Turkish University, 161200 Turkestan, Kazakhistan 2 Tashkent State Transport University, 100069 Tashkent, Uzbekistan Abstract. In the article, a formula derived on the basis of the kinetic energy change theorem for a non-free material point in a finite form is used to calculate the speed of movement of the car on various sections of the sorting slide. Examples of calculations show that in the intermediate section of the sorting slide to the dividing switch, the relative calculation error is 10.3%, in the section of the switching zone of the sorting slide of the second dividing switch  δv6s2 ≈ 9.0 %, in the section of the first sorting path  δv7 ≈ 16.1%. 1 Introduction As it is known [1 – 10], the existing theoretical provisions of the design and technological calculations of the designed sections of the sorting slide [11 – 18] are not devoid of inaccuracies. In [4], in order to really take into account the operational conditions of the sorting slides, it is recommended to use the parameters of the specific resistance to movement w, which reflect the generalized characteristics of the modern wagon fleet and sorting paths. Taking into account this factor, the formula (2) in [4] is given, supposedly having an expanded universal form: 𝑣𝑒𝑣𝑖𝑔𝑖𝑤𝑙𝑔𝑏 𝑣𝑣𝑒𝑒 2 = 𝑣𝑣𝑖𝑖 2 + 2𝑔𝑔′(𝑖𝑖−𝑤𝑤)10-3 ⋅𝑙𝑙−2𝑔𝑔′ℎ𝑏𝑏 (1) (1) However, formula (1) contains a number of inaccuracies and gross errors in its components, some of which are noted in [3, 9]. At the same time, for the convenience of analysis, we present separately the reduced and subtracted in (1) in the form: 𝑣𝑣𝑔𝑖𝑤𝑙 𝑣𝑣𝑒𝑒𝑒𝑒 2 = 𝑣𝑣𝑖𝑖𝑖𝑖 2 + 2𝑔𝑔′(𝑖𝑖𝑖𝑖−𝑤𝑤𝑖𝑖)10-3𝑙𝑙𝑖𝑖ሺʹሻ  𝑣𝑣eb𝑖𝑖 2 = 𝑣𝑣𝑖𝑖𝑖𝑖 2 −2𝑔𝑔′ℎ𝑏𝑏ሺ͵ሻ ሺʹሻ ሺ͵ሻ Where vibi = [veni] = [vcdi] – the maximum permissible speed of the entrance of the car to the car decelerators [23]; * Corresponding author: toshmatova.2021@mail.ru https://doi.org/10.1051/e3sconf/202346006024 https://doi.org/10.1051/e3sconf/202346006024 E3S Web of Conferences 460, 06024 (2023) BFT-2023 1.3 Research method The practical problem of determining the speed of the carriage along the incline of the sorting slide is solved on the basis of the theorem on the change of kinetic energy for a non-free material point in a finite form [21, 22]. * Corresponding author: toshmatova.2021@mail.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/e3sconf/202346006024 E3S Web of Conferences 460, 06024 (2023) BFT-2023 hbi – the power of the braking positions (according to Table. 2 in [20] the power of Мbm braking means) from the hump of the slide to the park braking position, depending on the type and number of retarders. Note [9] that formula (2) is necessary to determine the speed of the car on the high-speed sections of the slide profile, and (3) – for the sections of the braking positions (BP). Although, it is known that the derivation of formula (2) and/or formula (2) in [4] based on the kinetic energy change theorem is well known (see formula (30) on page 142 in [12]). However, in the reduced formula (2) and/or (2) in [4], the unit of measurement of the incline of the profile of the path i in ‰, is equated to the unit of measurement of the resistivity of the movement w of the off-system unit of measurement in kgfpt (i.e., ‰ = kgfpt) (see page 141 in [11], p. 9 in [12]), which is unacceptable in theoretical and engineering mechanics [21, 22]. 1.2 Problem statement With the calculated data, check the applicability of formula (2), as part of the universal form formula in [4] on various sections of the sorting slide (except for brake positions). 1.1 The purpose of this article The results of calculations of the rolling speed of the car on the intermediate section, the switch zone after the second switch, the first sorting path of the slide prove that the formula (2) describing the movement of the car on the high-speed sections of the slide, presented in a universal form in [4], is not applicable for sections of the slide with a small incline. 2 Example of performing hill calculations To determine the limit and/or the limit of applicability of formula (2) (without incorrectly included deductible) in [4] or formula (2) for the entire length of the path profile with different inclines, we present below the results of studies to determine the kinematic parameters of the car on various sections of the slide. So, for example, we determine the speed of the car on the first intermediate section of the slide (IN) to the dividing switch (S), the switch zone (SZ) of the second switch (S2), as well as on the first sorting path (ST1) by formula (2), as a subtractible formula (2) in [4]. Calculation example 1. For example, we examine the intermediate section (IN) to the dividing switch (S) of the slide. The initial data for the section of the IN hill are as follows: vi4 = 1.519 – the accepted value of the speed of entry of the car on the IN section after the car leaves the zone of braking of the car retarder of the second braking position (2BP), mps; g Calculation example 1. For example, we examine the intermediate section (IN) to the dividing switch (S) of the slide. 2 Example of performing hill calculations The initial data for the section of the IN hill are as follows: vi4 = 1.519 – the accepted value of the speed of entry of the car on the IN section after the car leaves the zone of braking of the car retarder of the second braking position (2BP), mps; g = 9.635 – acceleration of the free fall of the body taking into account the mass of rotating parts, mps2; l4 = 20,001 – the length of the intermediate section, m; i4Сk = 11 – the incline of the main section, ‰; Fo4 = ko4G = 0,001G = 0,908 – the force of the main resistance to the movement of the car to the dividing switch (S) of the section IN hill (where ko4 = 0.001 g = 9.635 – acceleration of the free fall of the body taking into account the mass of rotating parts, mps2; l4 = 20,001 – the length of the intermediate section, m; i4Сk = 11 – the incline of the main section, ‰; Fo4 = ko4G = 0,001G = 0,908 – the force of the main resistance to the movement of the car to the dividing switch (S) of the section IN hill (where ko4 = 0.001 2 E3S Web of Conferences 460, 06024 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346006024 – a coefficient that takes into account the resistance from the rolling friction force with sliding of the wheelset, taking into account the tailwind, while ko4 = ωо4 = 0,5 – the main resistivity the movement of a very good runner (VG), kgfpts (see Table 4.2 in [15]), kN; Fra4 = kra4G = 0,0005G = 0,454 – force resistance from the air and wind (where kra4 = 0.0005 – resistance from the environment and wind). ) Calculation results [23]. We will perform the calculations in the following sequence. Calculate the total specific resistance to the movement of the car, taken into account as a dimensionless value: |w4| = |ko4 + kra4| = – 0.0015 Note that the rolling speed of the wagon v4, calculated by the formula of elementary physics with the acceleration value a4 = 0,128 mps2, is equal to v4 = 2.723 mps. 2 Example of performing hill calculations Let's calculate the rolling speed of the car to the dividing switch (S) of the intermediate section (IN) of the slide v4 without taking into account the projection of the force of the tailwind Fwх according to the formula (2), mps.      4 -3 4 4 2 i4 4 )10 ( g 2 l w i v v 2 -3 1.519 2 9.635(11.0 1.5)10 20.0 2.443.      The relative error of calculations in comparison with the data of the formula of elementary physics is δv4S = 10.3 %, which is almost 2 times higher than the accuracy of engineering calculations (5%). Let's calculate the rolling speed of the car to the dividing switch (S) of the intermediate section (IN) of the vx4 slide according to the formula (2) taking into account the projection of the force of the tailwind Fwх, mps:      4 -3 4 4 2 i4 4 )10 ( g 2 l w i v v x x 2 -3 1.519 2 9.635(14.515 1.5)10 20.0 2.706.      The relative calculation error performed according to the formulas of elementary physics is equal to δvх4s ≈ 0.6 %, which is negligible. 4) It is interesting to note that if we vary the value of the initial speed of the carriage vi4 in the range from 1 to 2.0 mps with a step of ∆vi4 = 0.125 at w4 = 1.5 (dimensionless value) and i4 = 11, then the value of the rolling speed of the carriage in the calculated section v4Sk increases from 2.159 to 2.768 mps. p The graphical change v4 = f(vi4) is shown in Fig. 1. The graphical change v4 = f(vi4) is shown in Fig. 1. Fig. 1. Graphical change v4S = f(vi4S) 3 Fig. 1. Graphical change v4S = f(vi4S) Fig. 1. Graphical change v4S = f(vi4S) 3 E3S Web of Conferences 460, 06024 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346006024 Note that even if we vary the value of the specific resistance to the movement of the wagon w4 in the range from 0.5 to 3.0 (dimensionless value) with a step of ∆w4 = 0.25 at vi4 = 1.529 m/s and i4S = 11, the value of the rolling speed of the wagon in the calculated section v4Сk decreases from 2.521 to 2.332 mps. 2 Example of performing hill calculations p The graphical change of v41 = f(w4S) is shown in Fig. 2. Fig. 2. Graphical change v41 = f(w4) Fig. 2. Graphical change v41 = f(w4) If we vary the value of the incline of the path i4 in the range from 10.0 to 12.0 with a step of ∆i4 = 0.25 ‰ at vi4 = 1,529 mps and w4 = 1.5 (dimensionless value), then the value of the rolling speed of the car on the calculated section v4 increases from 2.363 to 2.521 mps. The graphical change v4i = f(i4) is shown in Fig. 3. Fig. 3. Graphical change v4i = f(i4) Fig. 3. Graphical change v4i = f(i4) Example of calculation 2. For an example of calculation, we examine the section of the switch zone (SZ) after the second switch (S2). 2 Example of performing hill calculations ( ) Calculation results [23]. 1) Calculate the total resistivity of the movement of the car: ts [23]. 1) Calculate the total resistivity of the movement of the car: |w6S2| = |km6S2 + ksw + kcur6S2 + kra + ksn| = = – (0.001 + 0.00025 + 0.0002463 + 0.0005+ 0.00025) = – 0.002246. |w6S2| = |km6S2 + ksw + kcur6S2 + kra + ksn| = We present the results of calculating the speed of movement of the car according to the formulas of elementary physics, the possibility of using which is analytically proved in [24] (see formulas (16), (19) – (20)). Let's calculate the time of movement of the car at the initial speed and/or the speed of the entrance of the car to the investigated section of the slide SZ vi6s2 = 2.654 mps and acceleration a6s2 = 0.032 mps2 with equidistant motion, taking into account the projection of the force of the tailwind Fwх: t6s2 = 7.567 s. Calculate the rolling speed of the car taking into account the projection of the tailwind force Fwх at vi6s2 = 2.654 mps, a6s2 = 0.032 mps2 and t6s2 = 7.567 s: p , p v6s2 = 2.897 mps and/or v6s2 ≈ 10.43 kmph. p , p v6s2 = 2.897 mps and/or v6s2 ≈ 10.43 kmph. p , p v6s2 = 2.897 mps and/or v6s2 ≈ 10.43 kmph. 2) Let's calculate the rolling speed of the car after the dividing switch (S2) of the section of the slide SZ according to the formula (2) without taking into account the projection of the force of the tailwind FWх, mps:      2 s 6 -3 2 s 6 2 s 6 2 i6s2 6s2 )10 ( g 2 l w i v v 2 -3 2.654 2 9.635(2 2.2246)10 21.0 2.635      Here, the resulting calculation result v6s2 = 2.635 mps is less than the initial velocity vi6s2 = 2.654 mps, since |w6s2| > i6s2. Here, the resulting calculation result v6s2 = 2.635 mps is less than the initial velocity vi6s2 = 2.654 mps, since |w6s2| > i6s2. The relative calculation error performed by formulas (2) and by formulas of elementary physics is equal to δv6s2 ≈ 9.0%, which is not small, i.e. almost 2 times exceeds the limits of accuracy of engineering calculations (5%). 2 Example of performing hill calculations The initial data of the section of the SZ are as follows: G = 650 – the gravity of the load on the car, kN; G0 = 908 – the gravity of the car with the load, kN; sinψ6s2 = 0.002 and cosψ6s2 = 1.0 or i6с2 = 2 ‰ – the incline of the  profile of the path of the slide SZ, rad.; g = 9.635 – acceleration of the free fall of the body with taking into account the mass of the rotating parts, calculated with a relative calculation error δg ≈ 0.184% at g = 9.81 mps2, n = 4 pcs., Q = G0 = 92.56 ts and/or G = 908 kN (according to Table 4.2 in [15] – this is a very good runner (VG)), γ = 0.00185 (see page 183 in [11]), m/s2; l6s2 = 21.0 – the length of the section of the SZ after the second switch (S2), m; vi6s2 = 2.654 – the accepted value of the speed of the entrance of the car to the section of the SZ after the second switch (S2) of the slide after the exit of the car from the first dividing switch (S1) of this zone, mps; Fm6s2 = km6s2G = 0.001 G = 0.908 – the force of the main resistance to the movement of the car on the section of the second switch (S2) of the slide SZ (where km6s2 = 0.001 is a coefficient that takes into account the resistance from the rolling friction force with sliding of the wheelset, taking into account the tailwind of a small value profile of the path of the slide SZ, rad.; g = 9.635 – acceleration of the free fall of the body with taking into account the mass of the rotating parts, calculated with a relative calculation error δg ≈ 0.184% at g = 9.81 mps2, n = 4 pcs., Q = G0 = 92.56 ts and/or G = 908 kN (according to Table 4.2 in [15] – this is a very good runner (VG)), γ = 0.00185 (see page 183 in [11]), m/s2; l6s2 = 21.0 – the length of the section of the SZ after the second switch (S2), m; vi6s2 = 2.654 – the accepted value of the speed of the entrance of the car to the section of the SZ after the second switch (S2) of the slide after the exit of the car from the first dividing switch (S1) of this zone, mps; Fm6s2 = km6s2G = 0.001 G = 0.908 – the force of the main resistance to the movement of the car on the section of the second switch (S2) of the slide SZ (where km6s2 = 0.001 is a coefficient that takes into account the resistance from the rolling friction force with sliding of the wheelset, taking into account the tailwind of a small value 4 E3S Web of Conferences 460, 06024 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346006024 Fwx ≈ 3.2 kN), kN; Fsw = kswG = 0.00025G = 0.227 – force resistance at the transition of the curves of the sections of the path (where ksw = 0.00025– resistance from the switchs), kN; Fcur6S2 = kcur6S2G = 0.0002463G = 0.224 – force resistance at the transition of the curves of the sections of the path (where kcur6S2 = 0.0002463 – resistance from the curves), kN; Fra = kraG = 0.0005G = 0.454 – force resistance from the air and wind (where kra = 0.0005– resistance from the medium), kN; Fsn = ksnG = 0.00025G = 0.227 – force resistance from the air and wind (where ksn = 0.00025– resistance from the medium), kN. 2 Example of performing hill calculations If for the main specific resistance to movement, according to Table 4.2 in [15], take ωо1 = 0.5 kgfpts for a very good runner (VG), then the rolling speed of the wagon v6s2о after the dividing switch (S2) on the section of the SZ, mps:      2 s 6 -3 о 2 s 6 2 s 6 2 i6s2 6s2о )10 ( g 2 l w i v v 2 -3 2.654 2 9.635(2 0.5)10 21.0 2.766      Note that when calculating v1о, the values of ωо1 were taken unchanged, i.e. ωо1 = 0.5 kgfpts. Note that when calculating v1о, the values of ωо1 were taken unchanged, i.e. ωо1 = 0.5 kgfpts. g p The relative calculation error performed by formulas (2) and by formulas of elementary physics is equal to δv6s2о ≈ 4.51%, which is not enough. Let's calculate the rolling speed of the car after the dividing switch (S2) of the section of the slide according to the formula (2), taking into account the projection of the force of the tailwind Fwх, mps: 5 https://doi.org/10.1051/e3sconf/202346006024 E3S Web of Conferences 460, 06024 (2023) BFT-2023      2 c 6 -3 2 c 6 2 c 06 2 н6c2 6c2 )10 ( g 2 l w i v v x b 2 -3 2.654 2 9.635(5.15 2.2246)10 21.0 2.893      The relative calculation error performed by formulas (2) and by formulas of elementary physics is equal to δv6s2b ≈ 0.14%, which is negligible. Calculation example 3. Let's explore the section of the first sorting path (SP1) of the slide. 2 Example of performing hill calculations The initial datka of the SP1 section are as follows: vi7 = 3,154 – the accepted value of the speed of the car's entrance to the SP1 section of the slide after the car exits the switch zone (SZ), mps; g = 9.635 – acceleration of the free fall of the body taking into account the mass of the rotating parts, mps2; l7 = 59.18 – the length of the SP1 section of the slide, m; i7 = 1.6 – slope of the section of the slide SP1, ‰; Fo7 = ko7G = 0,001G = 0.908 – the force of the main resistance to the movement of the car on the section of the slide SP1 (where ko7 = 0.001 is a coefficient that takes into account the resistance from the rolling friction force with sliding of the wheelset taking into account the tailwind Fwx), кН; Fcur7 = kcur7G = 0,00067G = 0.061 – the resistance force during the transition of the curves of the sections paths (where kr7 = 0.00067 is the resistance from the curves), kN; Fr = krG = 0,0005G = 0.454 is the resistance force from the air and wind (where kr = 0.0005 is the resistance from the medium), kN; Fsn = ksnG = 0,00025G = 0.227 is the resistance force from air snow and frost (where ksn = 0.00025 is the resistance from snow and frost), kN. ( ) Calculation results [27]. 1) Calculate the total resistivity of the movement of the car according to the formula: |w7| = |ko7 + kcur7 + kr + ksn| = = – (0.001 + 0.000067 + 0.0005+ 0.00025) = – 0.001817 2) The rolling speed of the wagon v7 on the section of the slide SP1, calculated by the formula of elementary physics with the given initial data of the problem, is equal to: v7 = 3.711 mps. 2) The rolling speed of the wagon v7 on the section of the slide SP1, calculated by the formula of elementary physics with the given initial data of the problem, is equal to: v7 = 3.711 mps. 2 Example of performing hill calculations p g p 5) Calculate the rolling speed of the car on the section of the SP1 slide according to the formula (2), taking into account the projection of the force of the tailwind Fwx, mps: 𝑣𝑣𝑥𝑥7 = √ 𝑣𝑣i7 2 + 2𝑔𝑔′(𝑖𝑖𝑥𝑥7 + |𝑤 |)10-3 ⋅𝑙𝑙7 = 2 -3 3.154 2 9.635(1.6 1.817)10 59.18 3.702       2 -3 3.154 2 9.635(1.6 1.817)10 59.18 3.702      i7 𝑔𝑔(𝑖𝑥𝑥7 𝑤𝑤7 )𝑙 7 The relative calculation error performed according to formulas (2) and (9) in [13] is equal to δvx7 ≈ 0.25 %, which is negligible. 2 Example of performing hill calculations 3) We will calculate the rolling speed of the car on the section of the slide SP1 according to the formula (2) without taking into account the projection of the force of the tailwind Fwx, mps: 𝑣𝑣𝑔𝑖𝑤𝑙 𝑣𝑣7 = √𝑣𝑣i7 2 + 2𝑔𝑔′(𝑖𝑖7 + |𝑤𝑤7|)10-3 ⋅𝑙𝑙7 = 2 -3 3.154 2 9.635(1.6 1.817)10 59.18 3.114      Here, the resulting calculation result v7 = 3,114 mps is less than the initial velocity vi7 = 3,154 mps, since |w7| > i7. The relative calculation error performed by formulas (2) and by formulas of elementary physics is equal to δv7 ≈ 16.1%, which is almost 3 times higher than the accuracy of engineering calculations (≈ 5%). Hence, it becomes obvious that the calculations of the rolling speed of the car on the sections of the sorting hill with a small slope (i7 = 1.6 ‰) are erroneous without taking into account the projection of the force of the tailwind Fwx (see the second paragraph of the middle column on page 24 in [2]). If for the main specific resistance to movement, according to Table 4.2 in [15], take ωо1 = 0.5 kgfpts for a very good runner (VG), then the sliding speed of the wagon vо7 on the section of the slide SP1, mps: 6 6 https://doi.org/10.1051/e3sconf/202346006024 E3S Web of Conferences 460, 06024 (2023) BFT-2023 𝑣𝑣o7 = √𝑣𝑣i7 2 + 2𝑔𝑔′(𝑖𝑖7 + |𝑤𝑤o7|)10-3 ⋅𝑙𝑙7 = 2 -3 3.154 2 9.635(1.6 0.5)10 59.18 3.347       Note that when calculating vо7, the values of ωо7 were taken unchanged, i.e. ωо1 = 0.5 kgfpts. Note that when calculating vо7, the values of ωо7 were taken unchanged, i.e. ωо1 = 0.5 kgfpts The relative calculation error performed by formulas (2) and by formulas of elementary physics is equal to δvо7 ≈ 9.8%, which is almost 2 times higher than the accuracy of engineering calculations (≈ 5%). For this reason, the values w07 = ωо7 = 0.5 kgfpts are not recommended for performing practical calculations. 3 Conclusions Analyzing the results of calculations of the rolling speed of the car on the intermediate section (IN), the switch zone (SZ) after the second switch (S2), the first sorting path (SP1) of the slide (see examples of calculations 1 – 3), it can be concluded that it is inappropriate to use formula (2) in hill calculations as part of formula (2) (without incorrectly included subtractible) in [4] for all sections of the slide profile, since the relative error of calculations (with the same initial data) δv is compared with by the simplified calculation method of the authors of the article [3], when not taking into account the projection of the force of the tailwind Fwx reach from 4 to 16.1%. Based on this, the results of the calculations proved the inconclusiveness of the use of formula (2) in hill calculations, as part of formula (2) (without incorrectly included deductible) in [4], for all sections of the slide profile, since: firstly, in the intermediate section (IN) of the sorting slide to the dividing switch (S), the relative calculation error performed according to formulas (2) compared with the exact formula of elementary physics is equal to δv4 ≈ 10.3%, which is almost 2 times higher than the accuracy of engineering calculations (≈ 5%); secondly, in the section of the switch zone (SZ) of the sorting slide of the second dividing switch (S2), the relative calculation error performed according to formulas (2) and according to formulas of elementary physics is equal to δv6с2 ≈ 9.0%, which is almost 2 times higher than the accuracy of engineering calculations (≈ 5%); thirdly, on the section of the first sorting path (SP1), the relative calculation error performed according to formula (2) in comparison with the formula of elementary physics is equal to δv7 ≈ 16.1%, which is almost 3 times higher than the accuracy of engineering calculations (≈ 5%); Fourthly, on the section of the first sorting path (SP1), the relative calculation error performed according to formulas (2) and according to formulas of elementary physics at the value of the specific resistance to the movement of the car w07 = ωо7 = 0.5 kgfpts is equal to δvо7 ≈ 9.8%, which is almost 2 times higher than the accuracy of engineering calculations (≈ 5 %). 3 Conclusions 7 7 E3S Web of Conferences 460, 06024 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346006024 Based on this, it can be argued that the reasoning of the authors of the article [4] that the formula (2) in [4] can be used for calculations on any sections with a slope i of the sorting slides, taking into account the presence of specific values of resistance to movement w (see the first paragraph of the last column on page 36 in [4]) raise objections and/or fall under doubt. References 1. Kh.T. Turanov, A.A. Gordienko, Transport Information Bulletin 3(237), 29 - 36 (2015) 2. V.M. Rudanovsky, I.P. Starshov, V.A. Kobzev, Transport Information Bulletin 6(252), 19-28 (2016) 3. Kh.T. Turanov, A.A. Gordienko, Transport Information Bulletin 10(256), 19 - 24 (2016) 4. Yu.O. Pozoysky, V.A. Kobzev, I.P. Starshov, V.M. Rudanovsky, Transport Information Bulletin 2(272), 35-38 (2018) 5. Kh.T. Turanov, A.A. Gordienko, Transport of the Urals 2(57), 3–8 (2018) https://doi.org/10.20291/1815-9400-2018-2-3-8 6. Sh.U. Saidivaliev, AIP Conference Proceedings 2612, 060016 (2023) https://doi.org/10.1063/5.0115115 7. Sh. Saidivaliev, Sh. Djabborov, B. Abdullaev, Sh. Abduvakhitov, D. Juraeva, E3S Web of Conferences 389, 05023 (2023) https://doi.org/10.1051/e3sconf/202338905023 8. Sh. Saidivaliev, S. Sattorov, R. Tursunkhodjaeva, R. Abdullaev, E3S Web of Conf., 376, 04036 (2023) https://doi.org/10.1051/e3sconf/202337604036 9. Sh. Saidivaliev, S. Sattorov, D. Juraeva, E3S Web Conf., 371, 04033 (2023) https://doi.org/10.1051/e3sconf/202337104033 10. Kh. Turanov, A. Gordienko, MATEC Web of Conferences 216, 02027 (2018) 11. V.N. Obraztsov, Stations and nodes. Part II (Moscow, Transzheldorizdat, 1938) 12. S.V. Zemblinov, I.I. Strakovsky, Stations and nodes (Moscow, Transzheldorizdat, 1963) 13. V.P. Parfenov, M.M. Filipov, M.M. Uzdin, V.P. Pavlov, High-power sorting humps: A manual for coursework and diploma design (Leningad, LIIZhT, 1972) 14. V.M. Akulinichev, L.P. Kolodiy, Calculation and design of hump humps of large and medium capacity (Moscow, MIIT, 1981) 15. Rules and regulations for the design of sorting devices on 1,520 mm gauge railways (Moscow, TEKHINFORM, 2003) 16. N.V. Pravdin, V.G. Shubko, E.V. Arkhangelsky et.al, Railway stations and nodes (problems, examples, calculations) (Moscow, Route, 2005) 17. N.V. Pravdin, S.P. Vakulenko, A.K. Golovich et al. Design of railway transport infrastructure (stations, railway and transport hubs) (Moscow, Federal State Budgetary Educational Institution “Training and Methodological Center for Education in Railway Transport”, 2012) 18. V.I. Apattsev et al, Railway stations and junctions (Moscow, Federal State Budgetary Educational Institution “Training and Methodological Center for Education in Railway Transport”, 2014) 19. V.A. Kobzev, Technical means of sorting humps that ensure traffic safety. Part 1 (Moscow, MIIT, 2009) 8 8 E3S Web of Conferences 460, 06024 (2023) BFT-2023 https://doi.org/10.1051/e3sconf/202346006024 20. Instructions for calculating the maximum permissible cut length when unraveling at hump yards (Moscow, JSC Russian Railways, 2012) 21. A.A. Yablonsky, V.M. Nikiforova, Course of theoretical mechanics (St. Petersburg, Lan Publishing House, 1998) 22. S.M. Targ, A short course in theoretical mechanics: a textbook for colleges (Moscow, Higher school, 1998) 23. E.G. Makarov, Mathcad: Training course (+CD) (St. Petersburg, Peter, 2009) 9 9
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ATTITUDE OF PLUS TWO STUDENTS TOWARDS PUBLIC EXAMINATION IN RELATION TO PARENTAL PRESSURE
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ATTITUDE OF PLUS TWO STUDENTS TOWARDS PUBLIC EXAMINATION IN RELATION TO PARENTAL PRESSURE S. Vinolin Chrysolyte 1, Dr B. William Dharma Raja 2 1 M.Ed Student, Department of Education, Manonmanium Sundaranar University, Tirunelveli – 627 012, INDIA 2 Head, Department of Education, Manonmanium Sundaranar University, Tirunelveli – 627 012, INDIA DOI: 10.5281/zenodo.167761 DOI: 10.5281/zenodo.167761 Keywords: Parental Pressure, Public Examination, Students. Parental Pressure, Public Examination, Students. Cite This Article: S. Vinolin Chrysolyte, and Dr B. William Dharma Raja, “ATTITUDE OF PLUS TWO STUDENTS TOWARDS PUBLIC EXAMINATION IN RELATION TO PARENTAL PRESSURE” International Journal of Research – Granthaalayah, Vol. 4, No. 10: SE (2016): 9-13. ABSTRACT This study examines the attitude of plus two students towards public examination and its relationship with parental pressure. The sample of the study comprised of 386 plus two students from Tirunelveli district. The authors employed survey method for collecting the data and they were analysed using descriptive and inferential statistical techniques. It was found that there is a significant relation between the attitude of students towards +2 public examination and parental pressure. ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Chrysolyte et. al., Vol.4 (Iss.10: SE): October, 2016] Social 1. INTRODUCTION Parental pressure is an unknowing burden of a parent shown towards their children in order to make their lives in progress. The pressure maybe there in any type of family and the pressure may be in relation to school for achieving the highest marks. Parental pressure is normally experienced by children in school, sports, arts and careers. All parents love to see their children excelling in curricular and co-curricular activities and sometimes over expect from their children. The term ‘parental pressure’ refers to the efforts that parental behaviors have on children’s functioning (Watson & Skinner, 2004). Parental influence emerged as pressure and stressful. The children believe that their parents had high expectation on them. Generally, students fear to take an examination and doing examination normally students become nervous and stressful. It is Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [9-13] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Chrysolyte et. al., Vol.4 (Iss.10: SE): October, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) commonly believed that examinations are the test of merit. It is not true, always. Any have it is an essential evil. commonly believed that examinations are the test of merit. It is not true, always. Any have it is an essential evil. 2. SIGNIFICANCE OF THE STUDY Every parent wants their children to lead a very successful life. So they give pressure on children and it will affect the children emotionally and physically. When parents give pressure to their children in studies and compel them to attend special classes they have lot of stress. Parents’ unrealistic expectations with their wards put continuous pressure on them to do well in their academic activities. Parents want their children to get high marks in public examination. After the public examination in +2 they have to choose a promising course. When they don’t get the expected course, they get frustrated. Parents’ pressure on their children regarding their study builds an unwanted burden on them. TOOL USED In the present study, the authors prepared two self-constructed scales namely, ViWi’s Scale on parental pressure and ViWi’s attitude scale on +2 public examinations with three points and 20 items. For establishing the face validity, the tool was pruned with field experts. The reliability was established by split-half method and the reliability co efficient was found to be r = 0.767 3. OBJECTIVES OF THE STUDY 1) To find out significant difference, if any, in attitude of plus two students towards public examination with regard to select background variables; 1) To find out significant difference, if any, in attitude of plus two students towards public examination with regard to select background variables; 2) To find out significant difference, if any, in parental pressure on plus two students relating public examination with regard to select background variables; and 3) To find out significant relation, if any, between attitude of plus two students towards public examination and parental pressure. SAMPLE The population for the study was plus two students of Tirunelveli district. The sample size for this study was 386, selected by simple random sampling technique. 4. METHOD USED FOR THE STUDY Survey method was adopted for the study. ANALYSIS OF DATA The statistical techniques used for analysis of data are ‘t’test, ‘F’ test and pearson’s correlation. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [9-13] [Chrysolyte et. al., Vol.4 (Iss.10: SE): October, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) Table 1: Significant of Difference in Attitude towards Plus Two Public Examinations NS-Not Significant at 5% level ** Significant at 1% level Background variable N Mean SD t-value p-value Gender Male Female 226 160 71.932 74.614 9.350 9.301 2.783 .006** Locality of school Rural Urban 120 266 72.986 73.070 8.574 9.781 .081 .935NS Medium of instruction Tamil English 317 69 72.797 74.178 9.525 8.849 1.105 .270NS 1: Significant of Difference in Attitude towards Plus Two Public Examinations The above table shows that there is no significant difference in attitude of +2 students towards public examination with regard to locality of school and medium of instruction and there is significant difference in the attitude of students towards +2public examination with regard to gender. The mean scores show that the attitude of female higher secondary students is more towards the +2 public examinations than male higher secondary students. The above table shows that there is no significant difference in attitude of +2 students towards public examination with regard to locality of school and medium of instruction and there is significant difference in the attitude of students towards +2public examination with regard to gender. The mean scores show that the attitude of female higher secondary students is more towards the +2 public examinations than male higher secondary students. Table 2: Significant of Difference in Parental pressure on plus two students relatin Examination Table 2: Significant of Difference in Parental pressure on plus two students relating Public Examination Background variable N Mean SD t-value p-value Gender Male Female 226 160 65.744 67.218 12.479 12.583 1.139 .255NS Locality of school Rural Urban 120 266 67.347 65.908 11.966 12.768 1.044 .297NS Medium of instruction Tamil English 317 69 66.687 64.830 12.426 12.960 1.116 .265NS NS-Not Significant at 5% level Table 2: Significant of Difference in Parental pressure on plus two st gnificant of Difference in Parental pressure on plus two students relating Public Examination The above table shows that there is no significant difference in parental pressure on plus two students towards public examination with regard to gender, locality of school and medium of instruction. 6. RECOMMENDATIONS In the light of the findings of the study, the authors give the recommendation as below: In the light of the findings of the study, the authors give the recommendation as below: A statistics is given by Tamil Nadu government (http://www.tn.results-nic.in/hsc- results.html).The percentage of boys in 2015 plus two public examination is 87.05% which is less than 5.54% that of girls. The percentage of boys in 2016 plus two public examination is 84.45% which is less than 6.33% which is less than that of girls. So the parental pressure is needed for male children. The pressure be more psychological and practical, the children should be expected to achieve more than what they could. Since the children are at the adolescence stage, the deals with them should be accordance with that.Parents should act as a guide. They should not torture their children. They should motivate and make enjoying in their studies. Parents should not make their studies as a burden. Children should not know that they are pressurizing by their parent. Children should be under their control. Schools should periodically conduct awareness programme to the parents of public examination – facing students employing counselor and psychological-sound experienced teachers. The parents should know that marks are not the only weapon to win the life. 5. FINDINGS 1) The attitude of female higher secondary students is more favorable towards the +2 public examinations than male higher secondary students. 1) The attitude of female higher secondary students is more favorable towards the +2 public examinations than male higher secondary students. 2) There is no significant difference in attitude towards plus two public examinations with regard to locality of school and medium of instruction. 3) There is no significance difference in parental pressure on students rela examination with regard to gender, locality of school and medium of instructio 4) There is a positive relationship between higher secondary students’ attitude towards +2 public examinations and parental pressure. 4) There is a positive relationship between higher secondary students’ attitude towards +2 public examinations and parental pressure. ANALYSIS OF DATA Table 3: Relation between Attitude of plus two students (N = 386) towards public examination and parental pressure Variables Mean SD r value p value Parental Pressure And Attitude towards Public examination 66.355 73.044 12.527 9.411 0.372 0.000** ** Significant at 1% level Table 3: Relation between Attitude of plus two students (N = 386) towards public and parental pressure Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [9-13] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [Chrysolyte et. al., Vol.4 (Iss.10: SE): October, 2016] It is inferred from the above table that p value is lesser than 0.01. Hence the null hypothesis is not accepted at 1% level of significance. It reveals that there is a significant positive relationship between higher secondary students’ attitude towards +2 public examinations and parental pressure. [1] Deb, S., Strodl, E., & Sun, J. (2015). Academic stress, parental pressure, anxiety and mental health among Indian high school students. International Journals of Psychology and Behavioural Sciences,5(1),26-34. 7. REFERENCES [1] Deb, S., Strodl, E., & Sun, J. (2015). Academic stress, parental pressure, anxiety and mental health among Indian high school students. International Journals of Psychology and Behavioural Sciences,5(1),26-34. [1] Deb, S., Strodl, E., & Sun, J. (2015). Academic stress, parental pressure, anxiety and mental health among Indian high school students. International Journals of Psychology and Behavioural Sciences,5(1),26-34. and Behavioural Sciences,5(1),26 34. [2] Freedom from parental pressure. (n.d). Retrieved from https://sites.google.com/site/freedomfromparents/introduction-background-study [3] Golden, S. A. R. (2011). An Analysis Of Mental Stress In Heavy Alloy Penetrator Project, Tiruchirapalli. SELP Journal of Social Science, 13. [4] Golden, S. A. R. (2011). Problems and Prospectus of Distance Learning. Bharathidhasan University, 343, 344. [5] Golden, S. A. R. (2011). Strategy For Success Of Human Beings:-Time Management. Department Of BBA, St. Joseph’s College, Trichy, 388, 390. from [2] Freedom from parental pressure. (n.d). Retrieved from https://sites.google.com/site/freedomfromparents/introduction-background-study [3] Golden, S. A. R. (2011). An Analysis Of Mental Stress In Heavy Alloy Penetrator Project, Tiruchirapalli. SELP Journal of Social Science, 13. [4] Golden, S. A. R. (2011). Problems and Prospectus of Distance Learning. Bharathidhasan University, 343, 344. [5] Golden, S. A. R. (2011). Strategy For Success Of Human Beings:-Time Management. Department Of BBA, St. Joseph’s College, Trichy, 388, 390. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [9-13] ISSN- 2350-0530(O) ISSN- 2394-3629(P) [Chrysolyte et. al., Vol.4 (Iss.10: SE): October, 2016] ISSN- 2350-0530(O) ISSN- 2394-3629(P) IF: 4.321 (CosmosImpactFactor), 2.532 (I2OR) [6] https://www.bouddunan.com/articles/education/42-general/9268-examination-and-its- importance.html [6] https://www.bouddunan.com/articles/education/42-general/9268-examination-and-its- importance.html p [7] Monica, S. B., & Mary, A. P. (2016). Parental involvement and interpersonal intelligence of XI standard students. Research and Reflection on Education,14(3), 2-10. [8] Naveen. (2009). Examination and importance. Retrieved from [9] Regi, S. B., & Golden, S. A. R. (2014). A Study On Educational Loan Availed By Students In Trichy City. Journal Of International Academic Research For Multidisciplinary (Jiarm), 2 (1). [10] Tamil Nadu HSC Result 2016 Released Today, (2016). Retrieved from http://www.tn.results-nic.in/hsc-results.html [11] Watson., T. S., & Skinner., C. H. (2004). Encyclopedia of school psychology. New York : Klwer academic Publishers. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [9-13]
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Postneoadjuvant Therapy Stage I Esophageal Adenocarcinoma AJCC v8
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Postneoadjuvant Therapy Stage I Esophageal Adenocarcinoma AJCC v8 National Cancer Institute Qeios ID: 4HJWLK · https://doi.org/10.32388/4HJWLK Source National Cancer Institute. Postneoadjuvant Therapy Stage I Esophageal Adenocarcinoma AJCC v8. NCI Thesaurus. Code C133433. Stage I includes: T0-2, N0, M0. T0: No evidence of primary tumor. T1: Tumor invades the lamina propria, muscularis mucosae, or submucosa. T2: Tumor invades the muscularis propria. N0: No regional lymph node metastasis. M0: No distant metastasis. (AJCC 8th ed.) Qeios ID: 4HJWLK · https://doi.org/10.32388/4HJWLK 1/1
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Lung Topology Characteristics in patients with Chronic Obstructive Pulmonary Disease
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Lung Topology Characteristics in patients with Chronic Obstructive Pulmonary Disease Received: 13 December 2017 Accepted: 12 March 2018 Published: xx xx xxxx Francisco Belchi   1, Mariam Pirashvili1, Joy Conway2,3, Michael Bennett3,4, Ratko Djukanovic   3, & Jacek Brodzki   1 Francisco Belchi   1, Mariam Pirashvili1, Joy Conway2,3, Michael Bennett3,4, Ratko Djukanovic  & Jacek Brodzki   1 Quantitative features that can currently be obtained from medical imaging do not provide a complete picture of Chronic Obstructive Pulmonary Disease (COPD). In this paper, we introduce a novel analytical tool based on persistent homology that extracts quantitative features from chest CT scans to describe the geometric structure of the airways inside the lungs. We show that these new radiomic features stratify COPD patients in agreement with the GOLD guidelines for COPD and can distinguish between inspiratory and expiratory scans. These CT measurements are very different to those currently in use and we demonstrate that they convey significant medical information. The results of this study are a proof of concept that topological methods can enhance the standard methodology to create a finer classification of COPD and increase the possibilities of more personalized treatment. Chronic obstructive pulmonary disease (COPD) is a progressive lung disease, affecting more than 200 million people worldwide. COPD is the fourth leading cause of death in the world and is projected to be the third leading cause of death by 2020. There were more than 3 million deaths from COPD in 2012 worldwide. The global burden on health resources as a result of COPD is expected to rise1,2. COPD is characterized by chronic inflammation of the bronchi and the lung parenchyma, resulting in varying degrees of obstructive bronchitis and emphysema due to remodeling of the airways and destruction of the alveoli, respectively. Although its pathology is heterogeneous, in functional terms, all forms of COPD result in loss of lung function, which is usually quantified by measuring the forced expiratory volume in 1 second (FEV1) and the Forced Vital Capacity (FVC). While these spirometry measures are widely used in clinical practice, both to diagnose and stratify COPD by severity, they have important limitations, the main being that they are integrative measurements which, therefore, do not take into account the highly heterogeneous regional pathological changes of COPD3. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 13 December 2017 Accepted: 12 March 2018 Published: xx xx xxxx SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 Lung Topology Characteristics in patients with Chronic Obstructive Pulmonary Disease Furthermore, FEV1 correlates weakly with clinical outcomes and health status4–6 For the needs of COPD, lung function measurements are increasingly complemented by imaging methods as a means of visually quantifying regional ventilation and perfusion abnormalities, gas trapping, emphysema, and airway remodeling3. High-resolution computed tomography (HRCT) scans are the most widely used form of imaging, with MRI and nuclear medicine increasingly, but still less commonly, used. Technical advances have resulted in dramatic reductions in radiation dose of CTs, allowing repeat imaging in longitudinal studies. Assessment of bronchial wall and cross-section thickness is comparable to histological quantification and also enables estimation of the degree of small airways disease that are not directly visualized by CT7. Of note, CT imaging allows for detection of lung pathology, such as smoking-related inflammation of the small, distal bronchi (bronchiolitis), years before airflow limitation is detected by spirometry8. For example, CT-detected emphysema, assessed by the 15% percentile (Perc15) technique is able prospectively to identify rates of lung function decline, even in individuals in whom spirometry does not detect airway obstruction9. p y y Common CT measurements in COPD research include lung attenuation area, mean lung density, airway wall area percentage, Perc15, lung volume, airway wall thickness and airway lumen area10. There is, however, signifi- cant room for the development of radiomic features derived by data-characterization algorithms applied to large sets of quantitative features extracted from medical images to uncover characteristics that cannot be appreciated 1Mathematical Sciences, University of Southampton, Southampton, UK. 2Faculty of Health Sciences, University of Southampton, Southampton, UK. 3NIHR Southampton Respiratory and Critical Care Biomedical Research Centre. University of Southampton, Southampton, UK. 4Clinical and Experimental Science, Faculty of Medicine, University of Southampton, Southampton, UK. Correspondence and requests for materials should be addressed to J.B. (email: J.Brodzki@soton.ac.uk) SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 1 www.nature.com/scientificreports/ by the naked eye. In the current study, we have applied, to our knowledge for the first time ever, the technique of persistent homology to process lung CT data. We took advantage of the computational tool of persistent homol- ogy11–13 to create topological descriptors which capture the complexity of the lung structure; this also enabled computation of a measure of similarity between images. Using this approach, our study has introduced a novel set of descriptors computable from a chest CT scan, focusing on characteristics that are very different from those used at present. Resultsh The overall aim of this study was to develop a set of new radiomic features that can distinguish between healthy non-smokers as well as healthy smokers and patients with COPD. For this purpose, the following four study par- ticipant groups defined by smoking status and spirometry given by the GOLD guidelines6 were studied: healthy non-smokers and healthy smokers (both judged as healthy by spirometry showing FEV1 > 80% of predicted and FEV1/FVC > 0.75), mild COPD patients, consisting of GOLD stage 1 (with FEV1 ≥ 80% of predicted and FEV1/ FVC < 0.70) and moderate COPD patients, consisting of GOLD stage 2 (50% ≤ FEV1 < 80% of predicted and FEV1/FVC < 0.70). See Methods for cohort details and data used. ) In this paper, we made use of Topological Data Analysis (TDA), with emphasis on persistent homology, for the computation of our new radiomic features. In Supplementary Information, we explain what persistent homol- ogy is and how it works. In Methods, we explain the way we use this TDA tool to obtain each of our geometric signatures. Directional complexity. For this computation, we began by extracting a graph representing the bronchial tree from each inspiratory CT scan (see Methods). Starting from the top of the scan, we recorded the height at which a segment of the bronchial tree changes direction and starts pointing upwards or downwards. We com- puted a geometric summary of this information using TDA as described in Methods. This consisted of a single numerical output we call upwards complexity, which was obtained by counting the number of times a particular branch changes its trajectory to start stretching upwards and sum this number over all branches in the bronchial tree. Upwards complexity allowed us to stratify the inspiratory CT scans of our cohort into COPD groups that agree with those given by the GOLD guidelines. A boxplot illustrating this group-separation can be found in Fig. 1A and details of the pairwise Kolmogorov-Smirnov (KS) tests can be found in Fig. 2A. The table in Fig. 2A shows (with the notation introduced there) that upwards complexity was able to distinguish HNS from Mild (|KS| = 0.51, and = . × − p 2 14 10 2), HNS from Mod (|KS| = 0.7, = . × − p 4 95 10 4) and HS from Mod (|KS| = 0.53, = . × p 1 56 102). Lung Topology Characteristics in patients with Chronic Obstructive Pulmonary Disease Specifically, we started by considering three new radiomic features: upwards complexity, which quantifies the way branches stretch upwards, the length of the bronchial tree visible in an inspiratory CT scan, and the number of bifurcations in the same tree. We then showed that these three numerical values are very closely related and any of them can stratify the inspiratory CT scans of our cohort into groups that agree with those given by the GOLD guidelines of COPD. Of note, these stratification results are better than those obtained by other CT measurements, like the emphysema score, volume of the lumen or airway diameter. Apart from the upwards complexity, we also computed two additional numerical values related to the way branches stretch upwards. Using these, we could clearly distinguish between inspiratory and expiratory CT scans. Additionally, we observed that we can also classify our cohort into healthy individuals and COPD patients by quantifying and classifying the topological structure of the space between the lung periphery and the visible airways in an inspiratory CT scan. Finally, we developed a computable characteristic that describes how the branches in the bronchial tree curve towards one another and showed that this radiomic feature correlates with lung function more strongly when the computations are done using the expiratory CT scans rather than the inspiratory CT scans, a phenomenon that is also seen when using standard CT measurements. g We propose that the relation between lung diseases and the shape of the bronchial tree, including properties such as trajectory changes, are of value to advancing our understanding of the mechanisms of COPD. We also propose that further research that applies this method in prospective, longitudinal studies and interventional trials is justified. Resultsh We also studied how the branches in the bronchial tree bend in other directions, obtaining a different number for each direction. This directional complexity in directions other than upwards did not improve the group-separation results obtained by the combination of upwards complexity and bronchial tree length (intro- duced in the next section), hence our focus on the latter two measurements. More details on this are given in Methods. Length of the bronchial tree and number of branching points. To complement directional com- plexity, we measured the length of the entire bronchial tree observable in an inspiratory CT scan. The length of the bronchial tree was estimated from a graph representing the bronchial tree in the CT (see Methods) using the number of vertices in this graph as a proxy for the length of the bronchial tree. Using this measure, we could again stratify the inspiratory CT scans of our cohort into groups that agree with those given by the GOLD guidelines. For these group-separation results, see Fig. 1B for the boxplot and Fig. 2B for the results of the pairwise KS test. In particular, notice how the bronchial tree length separates the group of moderate COPD patients from the other three groups. g p Moreover, it is remarkable that the length of the bronchial tree was able to distinguish all groups, except for that of healthy smokers (HS) from those of healthy non-smokers (HNS) or mild COPD patients (Mild). To be precise, Fig. 2B shows that the bronchial tree length distinguishes HNS from Mild (with a Kolmogorov-Smirnov score of |KS| = 0.48, and a p-value of = . × − p 3 24 10 2), HNS from Mod (|KS| = 0.76, = . × − p 1 26 10 4) and HS from Mod (|KS| = 0.61, = . × − p 3 44 10 3), as upwards complexity did. In addition, bronchial tree length also separates Mild from Mod (|KS| = 0.63, = . × − p 4 59 10 3). Of note, these findings may indicate that the group of SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 2 ntificreports/ Figure 1. Differences between severity groups given by 6 radiomic features. In the boxplots, HNS = health non-smokers, Mild = mild COPD patients, Mod = moderate COPD patients and HS = healthy smokers. The + signs denote outliers. Resultsh The 6 radiomic features studied are (A) upwards complexity (see Methods for details), (B) bronchial tree length, (C) emphysema score (as percentage of low attenuation area), (D) volum of the airways (computed as the number of voxels inside the grey airway structure in Fig. 4A), (E) upwards complexity divided by participant’s height, (F) bronchial tree length divided by participant’s height. The combination of radiomic features A and B can distinguish all groups except for HS from HNS or from Mild which outperforms the combination of methods C and D. www.nature.com/scientificreports/ Figure 1. Differences between severity groups given by 6 radiomic features. In the boxplots, HNS = healthy non-smokers, Mild = mild COPD patients, Mod = moderate COPD patients and HS = healthy smokers. The + signs denote outliers. The 6 radiomic features studied are (A) upwards complexity (see Methods for details), (B) bronchial tree length, (C) emphysema score (as percentage of low attenuation area), (D) volume of the airways (computed as the number of voxels inside the grey airway structure in Fig. 4A), (E) upwards complexity divided by participant’s height, (F) bronchial tree length divided by participant’s height. The combination of radiomic features A and B can distinguish all groups except for HS from HNS or from Mild, which outperforms the combination of methods C and D. Figure 1. Differences between severity groups given by 6 radiomic features. In the boxplots, HNS = healthy Figure 1. Differences between severity groups given by 6 radiomic features. In the boxplots, HNS = healthy non-smokers, Mild = mild COPD patients, Mod = moderate COPD patients and HS = healthy smokers. The + signs denote outliers. The 6 radiomic features studied are (A) upwards complexity (see Methods for details), (B) bronchial tree length, (C) emphysema score (as percentage of low attenuation area), (D) volume of the airways (computed as the number of voxels inside the grey airway structure in Fig. 4A), (E) upwards complexity divided by participant’s height, (F) bronchial tree length divided by participant’s height. The combination of radiomic features A and B can distinguish all groups except for HS from HNS or from Mild, which outperforms the combination of methods C and D. healthy smokers is heterogeneous and intersects with the healthy non-smokers at one end and with the mild COPD patients at the other. This could not be established using FEV1 (% of predicted) and the ratio FEV1/FVC. Resultsh The 6 radiomic features analyzed are (A) upwards complexity (see Methods for details), (B) bronchial tree length, (C) emphysema score (as percentage of low attenuation area), (D) volume of the airways (computed as the number of voxels inside the airway structure in Fig. 4A), (E) upwards complexity divided by participant’s height, (F) bronchial tree length divided by participant’s height. The combination of radiomic features A and B can distinguish all groups except for HS from HNS or from Mild, which outperforms the combination of methods C and D. Figure 3. Analysis of topological characteristics. (A) Correlation between upwards complexity and bronchial tree length (Pearson correlation coefficient ρ = 0.97, p-value ρ = 1.56.10−41). Similar results were obtained using directional complexity in other directions. (B) Correlation between the inspiratory branch-to-branch proximity, which quantifies how branches of the inspiratory bronchial tree bend towards one another, and FEV1 (% of predicted) (ρ = 0.38, ρ = 0.040). (C) Expiratory counterpart of (b) (ρ = 0.57, ρ = 0.001). Notice that the correlation is stronger and more significant for expiratory scans than for inspiratory scans. Figure 3. Analysis of topological characteristics. (A) Correlation between upwards complexity and bronchial tree length (Pearson correlation coefficient ρ = 0.97, p-value ρ = 1.56.10−41). Similar results were obtained using directional complexity in other directions. (B) Correlation between the inspiratory branch-to-branch proximity, which quantifies how branches of the inspiratory bronchial tree bend towards one another, and FEV1 (% of predicted) (ρ = 0.38, ρ = 0.040). (C) Expiratory counterpart of (b) (ρ = 0.57, ρ = 0.001). Notice that the correlation is stronger and more significant for expiratory scans than for inspiratory scans. to be strongly related (see Fig. 3A for an illustration of this using upwards complexity). Moreover, as shown in Fig. 3A, this close relation was maintained across the four groups in the cohort. It should be noted that while one can expect directional complexity to be related to the bronchial tree length, the precise nature of this relationship is not at all clear a priori. Comparison with other analytical methods. Having observed significant separation between study groups using our new radiomic methods on inspiratory CT scans, we looked for similar differences between subject groups when using other CT measurements. Resultsh To investigate whether any part of the lung may be contributing more to the above findings, we computed the length of the bronchial tree starting from different airway generations. This showed that such thresholding does not improve the separation presented in Fig. 1B and Fig. 2B regardless of the generation from which the computa- tion began. In a separate computation, an almost identical separation to the one in Fig. 1B and Fig. 2B was repro- duced by using the total number of points where airways branch out instead of the length of the bronchial tree. Relationship between directional complexity and bronchial tree length. When investigating the irectional complexity in any given direction and the length of the bronchial tree, these two measures were found SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 3 www.nature.com/scientificreports/ Figure 2. Differences between severity groups given by 6 radiomic features. For each radiomic feature, we show the table with the pairwise Kolmogorov-Smirnov that compares HNS = healthy non-smokers, Mild = mild COPD patients, Mod = moderate COPD patients and HS = healthy smokers. The values in italics in yellow shaded boxes indicate the absolute value of the KS score and the values in roman type in blue shaded boxes indicate p-values. The 6 radiomic features analyzed are (A) upwards complexity (see Methods for details), (B) bronchial tree length, (C) emphysema score (as percentage of low attenuation area), (D) volume of the airways (computed as the number of voxels inside the airway structure in Fig. 4A), (E) upwards complexity divided by participant’s height, (F) bronchial tree length divided by participant’s height. The combination of radiomic features A and B can distinguish all groups except for HS from HNS or from Mild, which outperforms the combination of methods C and D. Figure 2. Differences between severity groups given by 6 radiomic features. For each radiomic feature, we show the table with the pairwise Kolmogorov-Smirnov that compares HNS = healthy non-smokers, Mild = mild COPD patients, Mod = moderate COPD patients and HS = healthy smokers. The values in italics in yellow shaded boxes indicate the absolute value of the KS score and the values in roman type in blue shaded boxes indicate p-values. Resultsh Specifically, we quantified emphysema using the standard measure of percentage area of low attenuation and we approximated the volume of the airway lumen as the num- ber of voxels inside the airways (see the grey airway structure in Fig. 4A). This showed that the differences between subject groups identified by our radiomic features were much more significant than the differences identified by the emphysema score and the volume of the lumen (compare the boxplots Fig. 1C and Fig. 1D with those in Fig. 1A and Fig. 1B, and the numerical results in Fig. 2C and Fig. 2D with those in Fig. 2A and Fig. 2B). Indeed, using the volume of the lumen, we did not find any difference between subject groups with a p-value < 0.05, and the emphysema score only found two such differences – namely, that between healthy smok- ers and moderate COPD patients ( KS  = 0.55, = . × − p 9 88 10 3) and that between healthy non-smokers and moderate COPD patients ( KS =0.48, = . × − p 4 16 10 2). These separations were weaker and less significant than the separation of the same groups obtained using the bronchial tree length (Fig. 1B and Fig. 2B). SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 4 www.nature.com/scientificreports/ Figure 4. MSCT analysis. (A) The Apollo software (Vida Diagnostics, Iowa, USA) was used to segment the lobes from the MSCT scans. The resulting contour of the right lung lobes is presented here using custom software written in Matlab (R2015b, MathWorks, Natick, MA 01760–2098, US). (B) Illustration of the extracted branch center lines, along with the segmented airway tree for one of the participants. The center lines are colored according to generation number. Note that for the purposes of illustration, the center lines are plotted between the branch points only. For all of the analysis described in this paper, the complete center line information was used, which captured the true shape of the airways as in Fig. 8. Figure 4. MSCT analysis. (A) The Apollo software (Vida Diagnostics, Iowa, USA) was used to segment the lobes from the MSCT scans. The resulting contour of the right lung lobes is presented here using custom software written in Matlab (R2015b, MathWorks, Natick, MA 01760–2098, US). (B) Illustration of the extracted branch center lines, along with the segmented airway tree for one of the participants. Resultsh The center lines are colored according to generation number. Note that for the purposes of illustration, the center lines are plotted between the branch points only. For all of the analysis described in this paper, the complete center line information was used, which captured the true shape of the airways as in Fig. 8. Figure 5. Spatial representation of similarities between lungs. These are obtained by describing the shape of each lung through a set of topological characteristics called barcodes (see Methods for details) and computing distances between the barcodes of individual subjects. The resulting space is represented in 2D through an MDS embedding. In the legends, Healthy = healthy smokers and non-smokers, COPD = mild and moderate COPD patients. (A) This representation uses degree-2 persistent homology of inspiratory data to infer the shape of the airways inside the cavity of the lobes and it shows a clear distinction between Healthy and COPD groups. The overlap between the groups suggests that our characteristics are on a continuous spectrum. The presence of two nominally healthy cases so deep in the COPD region suggests a potential undiagnosed problem. Interestingly enough, those two individuals were healthy smokers. Similarly, all 6 COPD points which lie in or just outside the healthy region correspond to mild COPD patients. (B) This representation takes into account how the airways bend upwards and shows that this topological feature clearly separates the inspiratory and expiratory stages of the bronchial tree. This analysis was not performed for the expiratory phase because the information about the lobe structure was not available. igure 5. Spatial representation of similarities between lungs. These are obtained by describing the shape of Figure 5. Spatial representation of similarities between lungs. These are obtained by describing the shape of each lung through a set of topological characteristics called barcodes (see Methods for details) and computing distances between the barcodes of individual subjects. The resulting space is represented in 2D through an MDS embedding. In the legends, Healthy = healthy smokers and non-smokers, COPD = mild and moderate COPD patients. (A) This representation uses degree-2 persistent homology of inspiratory data to infer the shape of the airways inside the cavity of the lobes and it shows a clear distinction between Healthy and COPD groups. The overlap between the groups suggests that our characteristics are on a continuous spectrum. Resultsh The presence of two nominally healthy cases so deep in the COPD region suggests a potential undiagnosed problem. Interestingly enough, those two individuals were healthy smokers. Similarly, all 6 COPD points which lie in or just outside the healthy region correspond to mild COPD patients. (B) This representation takes into account how the airways bend upwards and shows that this topological feature clearly separates the inspiratory and expiratory stages of the bronchial tree. This analysis was not performed for the expiratory phase because the information about the lobe structure was not available. Relation to height. When assessing standard lung function measurements, the values are typically normal- ized by the individual’s height. To study the effect of height on some of our new radiomic features, we divided the upwards complexity of each participant by the person’s height. We then examined the effect of normalization by height on group separation. To this end, we compared 2-sample Kolmogorov–Smirnov tests. Normalizing upwards complexity exhibited a clearer separation in 3 cases (using the notation in Fig. 1, those cases are the SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 5 www.nature.com/scientificreports/ Figure 6. Computing of branch-to-branch proximity. Consider the graph representing the bronchial tree as explained in Methods (A). This graph is called a tree since it contains no loops, i.e., no branches that bifurcate and then merge. Of note, there are many nodes (up to 264) between any two consecutive bifurcations, so the nodes appear dense in the graph representation. Centered at each node of this graph, we virtually set a ball of a fixed radius, thickening the construction. As we keep thickening more and more, by increasing the radius of those balls, at some point we will find that some branches merge, creating a loop (B). We record the radius r1 at which this happens. For a large enough radius r2, though, this loop will be filled in (C). If a merging of branches creates a loop that appears for the value r1 of the radius and disappears at r2, we represent this merging as the positive number r2 − r1. Summing up all these terms, we obtain a number we call branch-to-branch proximity. Figure 6. Computing of branch-to-branch proximity. Consider the graph representing the bronchial tree as explained in Methods (A). This graph is called a tree since it contains no loops, i.e., no branches that bifurcate and then merge. Resultsh Of note, there are many nodes (up to 264) between any two consecutive bifurcations, so the nodes appear dense in the graph representation. Centered at each node of this graph, we virtually set a ball of a fixed radius, thickening the construction. As we keep thickening more and more, by increasing the radius of those balls, at some point we will find that some branches merge, creating a loop (B). We record the radius r1 at which this happens. For a large enough radius r2, though, this loop will be filled in (C). If a merging of branches creates a loop that appears for the value r1 of the radius and disappears at r2, we represent this merging as the positive number r2 − r1. Summing up all these terms, we obtain a number we call branch-to-branch proximity. Figure 7. Calculations show that the lung function is better when more branches bend towards one another in the expiratory bronchial tree (such as the branches in the two circles on the left, in contrast with those in the circle on the right). See Fig. 3C. Figure 7. Calculations show that the lung function is better when more branches bend towards one another in the expiratory bronchial tree (such as the branches in the two circles on the left, in contrast with those in the circle on the right). See Fig. 3C. comparisons Mod-HNS, HS-Mod and Mod-Mild) and a less clear separation in 2 cases (Mild-HNS, HS-Mild) (compare Fig. 1E and Fig. 2E to Fig. 1A and Fig. 2A, respectively). We repeated the same normalization with the length of the bronchial tree and found that in 2 instances (HNS-Mild, HS-Mod), not normalizing by height provided a clearer separation between groups, and normaliz- ing did not improve the clarity of separation in any instance (compare Fig. 1F and Fig. 2F to Fig. 1B and Fig. 2B, comparisons Mod-HNS, HS-Mod and Mod-Mild) and a less clear separation in 2 cases (Mild-HNS, HS-M (compare Fig. 1E and Fig. 2E to Fig. 1A and Fig. 2A, respectively). SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 6 www.nature.com/scientificreports/ Figure 8. Explanation of upwards complexity. The color gradient indicates height. (A) To study upwards complexity, we slide a horizontal plane downwards. Resultsh The stronger correlation with expiratory scans is in keeping with our previous findings using standard CT measurements that mean lung density (MLD) during expiration correlated better with reduced lung function than inspiratory MLD14.i To address the first point, we considered again the height at which branches in the bronchial tree start or stop stretching upwards, as used in the computation of upwards complexity. We used the same input to compute a different topological summary (see Methods), which allowed us to compare the scans of different participants and plot them together. The output of our computations were two values per CT scan, which we used as coordinates of a point in the plane (see Fig. 5B). This showed a clear separation between the inspiratory and expiratory CT scans. To address the first point, we considered again the height at which branches in the bronchial tree start or stop stretching upwards, as used in the computation of upwards complexity. We used the same input to compute a different topological summary (see Methods), which allowed us to compare the scans of different participants and plot them together. The output of our computations were two values per CT scan, which we used as coordinates of a point in the plane (see Fig. 5B). This showed a clear separation between the inspiratory and expiratory CT scans. To quantify how branches curve towards one another in the bronchial tree graph, we introduced another radiomic feature, called branch-to-branch proximity. This was done by virtually thickening the visible airways and recording the thickness at which the airways begin to touch (see Fig. 6 and Fig. 7 for an illustration). Using this approach, we found that the branch-to-branch proximity observed in the expiratory phase correlated more strongly with FEV1 (% of predicted) than the branch to branch proximity observed in the inspiratory phase h To quantify how branches curve towards one another in the bronchial tree graph, we introduced another radiomic feature, called branch-to-branch proximity. This was done by virtually thickening the visible airways and recording the thickness at which the airways begin to touch (see Fig. 6 and Fig. 7 for an illustration). Using this approach, we found that the branch-to-branch proximity observed in the expiratory phase correlated more strongly with FEV1 (% of predicted) than the branch-to-branch proximity observed in the inspiratory phase (compare Fig. 3B and Fig. 3C). Resultsh Again, this was consistent with standard CT measurements, which also correlate better with FEV1 (% of predicted) when measured during expiration10,14. Small airways. Having shown that we can compute clinically meaningful topological features of the airways by using their tree structure, we showed that the shape of the space separating the lung periphery from the air- ways visible in an inspiratory CT scan is also related to the development of COPD. Our CT scan data consists of voxels which are cubes 0.7 mm long in each direction, giving a spatial resolution of 2.1 mm. This makes the small airways, which are defined as those with a diameter <2 mm, invisible in a CT scan. However, it is well known that small airways dysfunction plays a key role in COPD. Hence, what happens in the void between the visible airways and the lung periphery is crucial. To overcome the relatively low resolution of the standard CT scan, we provided numerical topological characteristics of the shape of the space between the airways and the boundary of the lobes. We demonstrated how the resulting radiomic feature can distinguish between healthy individuals and COPD patients. This was achieved by placing virtual balls centered within the visible structure (airways and lung periphery) and allowing them to expand until they fully occupied the space. This procedure uses thickening in a similar way to Fig. 6, see Methods for details. The output is a pair of real numbers (x, y) representing each CT scan. In Fig. 5A, we represent each CT scan as a point in the plane with the corresponding coordinates (x, y). This approach placed the healthy smoking and non-smoking individuals into one group that was distinct from the mild and moderate COPD patients who formed another group (see Fig. 5A). Resultsh If we denote by Xh the part of the tree that sits above the horizontal plane at distance h from the top of the image, then ⊆ ′ X X h h whenever ≤ ′ h h , obtaining a sequence of nested graphs approximating the bronchial tree more accurately as we increase h. (B) The right part of the panel shows the degree-0 barcode of the sequence of nested graphs in (A). In this picture, the correspondence between bars in the barcode and branches that change trajectory upwards becomes apparent. In particular, the length of a bar indicates for how long a branch follows that upwards trajectory. Figure 8. Explanation of upwards complexity. The color gradient indicates height. (A) To study upwards complexity, we slide a horizontal plane downwards. If we denote by Xh the part of the tree that sits above the horizontal plane at distance h from the top of the image, then ⊆ ′ X X h h whenever ≤ ′ h h , obtaining a sequence of nested graphs approximating the bronchial tree more accurately as we increase h. (B) The right part of the panel shows the degree-0 barcode of the sequence of nested graphs in (A). In this picture, the correspondence between bars in the barcode and branches that change trajectory upwards becomes apparent. In particular, the length of a bar indicates for how long a branch follows that upwards trajectory. espectively). This suggests that, unlike standard spirometry, the bronchial tree length may capture bronchia tructure information relevant in COPD in a way that is independent of height. Comparison of expiratory and inspiratory phase CTs. For 30 participants (8 healthy non-smokers, 9 healthy smokers, 8 mild COPD and 5 moderate COPD), both inspiratory and expiratory CT scans were obtained. This provided an opportunity to demonstrate first that our methodology can not only distinguish between healthy individuals and COPD patients but also detects differences in structure between the inspiratory and expiratory phases of the breathing cycle. Furthermore, we showed that the amount to which branches in the bronchial tree curve towards one another correlates with lung function more strongly in expiratory CT scans than in inspiratory ones. www.nature.com/scientificreports/ This could not be established using FEV1 (% of predicted) and the ratio FEV1/FVC.h ph g ( p ) Additionally, our comparison between inspiratory and expiratory phases can have various applications. The manner in which tissue inside the lung expands and contracts throughout the breathing cycle is known to be an indicator of disease, for example gas trapping in COPD. Comparison of the inspiratory and expiratory scans can therefore be exploited as a means of making localized measurements of disease32. Our proposed technique makes it possible to study how the shape of the bronchial tree changes during the breathing cycle and offers the potential to be a new method for the identification of localized areas of disease, such as gas trapping. Similarly, indices that are the subject of current, clinical, pulmonary CT research also include the Parametric Response Mapping (PRM) technique, which uses co-registration of paired inspiratory and expiratory scans to compare areas of low attenuation on a voxel to voxel basis33,34. Our methodology may further inform the PRM technique and could be the subject of future research.h j The precise physiological basis for our results is not completely clear at this stage. Upwards complexity counts the number of times a branch deflects upwards from the horizontal direction. Lower directional complexity means that there are fewer airways pointing upwards from a given plane. The same applies to downwards com- plexity, or indeed complexity evaluated in any direction in three dimensions. Our interpretation is that advancing smoking disease (fall in FEV1) is associated with increased distortion of the bronchial tree structure, which results in some of the airways pointing in a direction that is different from the other neighbouring airways, pos- sibly because of differences in air trapping. Thus, regional differences in the extent of lung damage (emphysema) could explain distortions that result in individual airways departing from the direction of the other neighbouring airways that have different degrees of air trapping. A natural pathophysiological explanation for the association between FEV1 and branch proximity is that it reflects the distortion (squashing) of the airways by hyperinflated lung parenchyma, also due to air trapping. As can be seen in Fig. 2C, this squashing is more pronounced in expi- ration, as shown by the stronger and more significant correlation in this respiratory phase (see p and p values, respectively). www.nature.com/scientificreports/ and virus evolution28. In this study, we took advantage of the ability of persistent homology to offer alternative ways of measuring global properties of complex objects through the use of topology. In respiratory imaging, this method represents a completely new way of taking measurements of the bronchial tree. In contrast to existing methods, such as measuring the dimensions of the airway walls and lumen, which look at airway branches individually, our approach condenses the topological properties of the entire bronchial tree into a small number of unique characteristics for each individual. Along with previous studies29,30, this creates major new opportunities for persistent homology to be used more widely in medicine, in particular in clinical radiology. This new methodology is especially applicable to studying the lung at the population level because of the manner in which it represents the complexity of the airway tree through a single low-dimensional data point that represents the entire bronchial tree. By collecting these data over a large number of subjects and combining them with other imaging, physiological and measurements of pathobiological biomarkers, we could build a pic- ture of how variations in the topological nature of the bronchial tree impact on the pathophysiology of people with a variety of respiratory diseases such as COPD, asthma and idiopathic pulmonary fibrosis (IPF). This is likely to have significant translational impact as a valuable tool for use in deep-phenotyping, which is central in stratified medicine and precision medicine31. i p We have created a set of novel radiomic features which capture the overall complexity of the lung structure and enable a quantitative comparison of the CT scan images. We have demonstrated that these properties are important in the context of a common respiratory disease. These measurements provided a more complete pic- ture of differences between the four groups in this study than standard CT measurements. In particular, there was a significant relationship between upwards complexity, the length of the bronchial tree, and COPD severity. Moreover, it is remarkable that the length of the bronchial tree was able to distinguish all groups, except for that of healthy smokers from those of healthy non-smokers or mild COPD patients, which may indicate that the group of healthy smokers is heterogeneous and intersects with the healthy non-smokers at one end and with the mild COPD patients at the other. SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 Discussion Since its creation, persistent homology, a key tool of TDA, has developed rapidly, both in terms of its mathemat- ical foundations15–17 and possible applications. Persistent homology has been used in fields as diverse as digital imaging18,19, sensor networks coverage20, materials science21,22, molecular modelling23–25, signal processing26,27 SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 7 www.nature.com/scientificreports/ Methods This software was designed to semi-automatically analyze pulmonary MSCT imaging data, including segmentation of the lungs, the airway tree and the lobes (see Fig. 4A). For the needs of the current study, only the lung, lobes and airway tree were of interest. In many cases, the Apollo soft- ware was able to achieve the desired results entirely automatically, but for some participants it was necessary to manually edit the results of the lobe segmentation to ensure that they were defined as accurately as possible. All segmentations were manually inspected for accuracy and completeness. In the case of the lobes, accuracy was checked by ensuring that the lobar boundary corresponded as closely as possible with the fissure, whilst for the airways the segmentation was inspected to ensure that the results were as complete as possible.t y g p p p Custom software written in Matlab (R2015b, MathWorks, Natick, MA 01760–2098, US) was used to extract the specific details of the center lines and branch points of the airway tree from the data output by the Apollo soft- ware. An example of the extracted center lines, along with the segmented airway tree is shown in Fig. 4B, where the center lines are colored according to generation number and are, for simplicity, plotted between the branch points only. For all of the analysis in this paper, the complete center line information was used, which captured the true shape of the airway branches. In particular, there can be up to 264 extra points describing the shape of the bronchial tree between two branch points. Persistent homology. Persistent homology11–13 has been designed to provide numerical information about the key features of an object under study at a range of scales, which can be regarded as variable resolution at which the object is viewed. A starting point of this process is a simplified approximation of the object, which grows as the scale parameter r is varied. In this study, we made use of two different approximations: one based on alpha complexes (see Supplementary Information) and one based on a notion of height function, explained in the direc- tional complexity section (below) and expanded in the Supplementary Information. We computed topological characteristics of the chosen approximation, using all scales at once. Methods All the procedures explained in the Results section can be formalized and computed efficiently through the tool of persistent homology, which is described in detail in the Supplementary Information. Study design and participants. The imaging data used for this study were acquired from two previous imaging studies performed in Southampton (manuscripts in preparation). Both studies focused on COPD and had identical inclusion and exclusion criteria. In both studies, participants were recruited into two COPD groups; GOLD stage 1 disease (FEV1/FVC ratio <0.70 and FEV1 ≥80% of predicted) and GOLD stage 2 disease (FEV1/ FVC ratio <70% and FEV1 50–79% of predicted), referred to as mild and moderate COPD, respectively. Both healthy smoker and healthy non-smoker groups had no clinical evidence of obstructive airways disease, and had spirometry results of FEV1/FVC ratio >0.75 and FEV1 >80% of predicted. In total, 64 participants were assessed (18 healthy non-smokers, 19 healthy smokers, 14 COPD GOLD-1 and 13 COPD GOLD-2). Both studies were approved by the Southampton and West Hampshire local research ethics committee (LREC number: 11/SC/0319 and 09/H0502/91). Written informed consent was obtained from all study participants and both studies were conducted according to all relevant guidelines and regulations. MSCT imaging. All CT scans were performed using a standardized protocol recommended for use with Apollo analysis software (Vida Diagnostics, Iowa, USA). Multi-Slice Computed Tomography (MSCT) scans were performed on a Siemens Sensation 64 CT scanner (Siemens Medical Solutions, Erlangen, Germany) using a high-resolution algorithm, with detector thickness 0.75 mm, pitch 1.0, effective mAs 90 and a tube voltage of 120 kV. The high-resolution algorithm was chosen to ensure the best visualization of the airway tree37. The scan- ning protocol was performed at maximal inspiration and expiration, with breath hold, for the duration of the scan (approximately 10–15 sec depending on the size of the thorax). All patients are coached beforehand and during the scan in a standardized manner, conforming with the Vida Diagnostics protocol and as used by other centers38,39. The images were reconstructed using a slice thickness of 0.75 mm, a reconstruction increment of 0.5 mm, and a sharp reconstruction algorithm. Additional reconstructions were also performed using several soft reconstruction kernels, including B30f and B35f, which were chosen to suit the recommended protocol in the Apollo analysis software. MSCT analysis. The Apollo software (Vida Diagnostics, Iowa, USA) was used to perform the analysis of the multi-slice computed tomography scans. www.nature.com/scientificreports/ p y Our approach to persistent homology is similar to that employed in30 to study cerebral vasculature, but our topological summaries, such as the directional complexity or branch-to-branch proximity, are different and have not been used before. As explained in Supplementary Information, the output from the persistent homology calculation is summarized in the so-called barcode. In the study of cerebral vasculature, Bendich et al.30 sim- plified the analysis by retaining the 100 longest bars from which summaries were produced using the Principal Component Analysis. In contrast, in our study this approach did not work as the number of bars can vary signifi- cantly between patients, which can be seen in Fig. 1A where some participants have about 160 bars in the upwards complexity barcode, whereas others exhibit only about 20 bars. For this reason, in our study we retained the entire barcode without thresholding. From this input data, we computed a measure of similarity between scans of indi- vidual patients. This can be used for visualization or to train classification models in a way similar to the approach by Adcock et al. on hepatic lesions29. y p We also created a new topological characteristic to circumvent the relatively low spatial resolution of CT scans. This is one of the main mathematical novelties of the paper as it provides a first instance where topology has been used to infer the structure of the object under study. We achieved this by incorporating the boundary of the lung lobes into the computation. This technique can be applied to any kind of imaging, for example, to 3-dimensional Magnetic Resonance Angiography images of the arterial tree within the brain30, where the new characteristics developed here can be used to enhance the analysis if the meninges are used in the same way we used the outer layer of the lobes in our study.ff y y Of note, we made use of persistent homology in degrees 0, 1 and 2 in different ways to obtain different kinds of clinical insight. In degree 0, it was used to define the directional complexity (a number that can distinguish sever- ity groups) and to characterize the distinction between the inspiratory and expiratory CT scans. www.nature.com/scientificreports/ Using persistent homology in degree 1, we showed that CT measurements correlate with FEV1 (% of predicted) more strongly SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 8 www.nature.com/scientificreports/ during the expiratory phase than in the inspiratory phase, which could be expected based on similar observa- tions in past CT studies10,14. As stated before, the degree 2 was used to overcome the limitation of the low spatial resolution of CT scans by including information of the outer boundary of the lobes. This led to a much clearer visualization of the difference between the healthy and COPD participants, which could not be recovered using the topological characteristics in degrees 0 and 1. Thus, our methodology provides one of the first significant uses of the second-degree persistent homology in applications. The other uses of degree 2 up to date are summarized in19,35,36. In summary, this study has shown that our analytical method can extract information from CT scans to pro- vide a new perspective on lung structure. Because this method can be readily applied to large CT datasets, we propose that it is of value for clinical research. Further studies are needed to assess its prognostic value in longi- tudinal and interventional studies. SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 www.nature.com/scientificreports/ known as bars, hence the name barcode for a collection of these. Intuitively, degree-0 gives information about the evolution of the connected components along the sequence of growing representations of the object under study. Similarly, degree 1 indicates the evolution of the loops or holes, and degree 2 captures the evolution of cavities or voids, etc. We compared the resulting barcodes using pseudo-distance functions, the Wasserstein and Bottleneck pseudo-distances being examples with important stability properties; see Supplementary Information. p g p p y p p pp y In summary, we used persistent homology to take a growing approximation of an object, compute the associ- ated degree-n barcode for some ≥ n 0, and compare the corresponding barcodes of different objects using the Bottleneck or Wasserstein distances. Directional complexity. We quantified the amount of changes in trajectory in a particular direction by defining a notion of directional complexity on the 3D graph representation of the bronchial tree described in the MSCT analysis subsection (above). To measure upwards complexity, we slid a horizontal plane downwards (see example in Fig. 8A). At any given distance h from the top of the imaginary box containing the bronchial tree, Xh was defined as the part of the tree that sits above the plane at that position. In this way, we obtained an approxi- mation of the bronchial tree that converged to the original tree as we increased the distance h from the top (see Fig. 8A).h The degree-0 barcode corresponding to this sequence of growing graphs has the following interpretation: a bar of the form (h1, h2) in this barcode indicates that there is a connected component C in the graph Xh1 which is not present in Xh for any < h h1. Additionally, the following holds for h2 but it does not hold for any < h h2: in the graph Xh2, the component represented by C will merge with another component of Xh2 which was present in Xh for some < h h1. 1 In Fig. 8B, we represent each bar of the form (h1, h2) in the degree-0 barcode as a vertical line, with the starting point at distance h1 from the top and end point at distance h2 from the top. In this representation, every bar corre- sponds to a branch changing trajectory to start stretching upwards. www.nature.com/scientificreports/ We called upwards complexity the number of vertical lines in such a representation, i.e., the number of upwards changes of trajectory of the airways. p p g j y y To compute directional complexity in other directions, we rotated the bronchial tree, slid the plane to bottom and counted the number of finite bars in the corresponding degree-0 barcode. i p g g As mentioned in the Results section, directional complexity in other directions did not improve the group-separation results obtained by the combination of upwards complexity and bronchial tree length, hence our focusing on the upwards direction. For instance, by rotating 10°, 220° and 0° around the X, Y and Z axes, respectively, following the right-hand rule, directional complexity produced no group separation at all. However, using instead the angles 20°, 40° and 0°, respectively, the group-separation results given by directional complexity were very similar to those of the bronchial tree length.t y g To generate these barcodes, we used the publicly available software package TDATools40. To compute the bar- code of one of these graphs in a 3D box, we used the function ‘rca1mfscm’ of this package, which requires the definition of a function F on each vertex and edge of the graph. For instance, to compute the upwards complexity, we assigned to each vertex its distance to the top of the box, and to each edge, the maximum of the values of F attained at the two vertices it connects. Note that all barcodes in this study were computed with coefficients in the field of two elements, 2. Length of the bronchial tree. The length of the bronchial tree was estimated from the 3D graph rep- resentation of the bronchial tree described in the MSCT analysis subsection above. We used the number of ver- tices in this graph (that include not only the branch points but also the many vertices connecting consecutive branch points) as a proxy for the length of the bronchial tree. As stated in the Results section, a separate com- putation with only the branch points was performed and the results were similar to those in Fig. 1B and Fig. 2B. Methods These are numerical invariants obtained by computing homology groups Hn of the approximation, and tracing the life-span of features as they appear and disappear with the changing scale. For each degree ≥ n 0, this information is represented in the form of a collec- tion of intervals with multiplicities, called the degree-n barcode explained in detail in the Supplementary Information. These intervals have the form (r1,r2) for different values of the changing parameter r, and are also SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 9 www.nature.com/scientificreports/ Length of the bronchial tree. Small airways. For the computation of the representation in Fig. 5A, we started with a 3D array of binary voxels representing the luminal surface of the airways together with the surface of the lobes as in Fig. 4A. For each binary voxel image, we constructed a point cloud in 3 by including the coordinates of every voxel with value 1 and then built the alpha complex filtration (see Supplementary Information) on these points. The degree-2 bar- code of this filtration gave information about how the airways fill the cavity of the lobes. The alpha complex filtra- tions and their barcodes were computed using the GUDHI library41.h Next, we computed the bottleneck distances between all the degree-2 barcodes. This gave a measure of dis- tance between the lung scans by proxy, giving us a pseudo-metric on the set of lungs. The bottleneck distances were computed using the Hera software42. Due to computational constraints, we made use of the software’s approximate bottleneck calculation. If one supplies a relative error, then the software computes an approximate distance which satisfies the inequality − < d d d relative error / , exact approx exact where dexact is the exact bottleneck distance and dapprox is the computed approximation, as described in the docu- mentation of 42. We used a relative error of 10−4. After measuring the pairwise distances between all barcodes, we used Multi-Dimensional Scaling (MDS) to obtain a 2D representation shown in Fig. 5A. where dexact is the exact bottleneck distance and dapprox is the computed approximation, as described in the docu- mentation of 42. We used a relative error of 10−4. After measuring the pairwise distances between all barcodes, we used Multi-Dimensional Scaling (MDS) to obtain a 2D representation shown in Fig. 5A. Expiratory CT analysis. For 30 participants (8 healthy non-smokers, 9 healthy smokers, 8 mild COPD and 5 moderate COPD), both inspiratory and expiratory CT scans were obtained. Recall that the upwards complexity was computed as the number of vertical lines in Fig. 8B. This was, in turn, the number of bars in the degree-0 barcode constructed by considering the part of the bronchial tree graph that sits on top of a horizontal plane that we slide downwards. SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 10 www.nature.com/scientificreports/ In order to compute the representation shown in Fig. 5B, we used the same degree-0 barcode in a different way. Length of the bronchial tree. We computed such barcodes for both the inspiratory and expiratory bronchial tree graphs and compared those 60 barcodes (corresponding to the inspiratory and the expiratory bronchial tree of the 30 patients) using the Wasserstein2 distance (see Supplementary Information). The Wasserstein computations were done with the software package Hera 42. After calculating the distances between all these barcodes, the final representation in Fig. 5B was obtained using a 2D MDS projection. g g p j In a separate computation, we quantified how branches bend towards one another on the tree graph (See Fig. 6 and Fig. 7 for an intuitive illustration of this computation). We used the alpha complex filtration (see Supplementary Information) built on the nodes of this graph. Next, we computed the degree-1 barcode, which consisted of points of the form r r ( , ) 1 2 . Finally, we defined the branch-to-branch proximity as the sum of the num- bers − r r 2 1 corresponding to all such points. We performed this for both the inspiratory and expiratory tree graphs of each participant. The alpha complex filtrations and their barcodes were computed using the GUDHI library41. Data and materials availability. The data that support the findings of this study are available on request from the corresponding author (J. B.). 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SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 References Am Journal Cri Care Met 194(1), 794–806 (2016). 40. Harer, J. et al. TDAtools, Available at https://github.com/ksian/ML2015FP/tree/master/3TDATools (2014).h 41. The_GUDHI_Project, GUDHI User and Reference Manual, Available at http://gudhi.gforge.inria.fr/doc/late b & A A l bl h //b b k / /h ( ) 41. The_GUDHI_Project, GUDHI User and Reference Manual, Available at http://gudhi.gforge.inria.fr/doc/latest/ (2015). 42 Kerber, M , Morozov, D & Nigmetov, A , Hera, Available at https://bitbucket org/grey narn/hera (2017) 41. The_GUDHI_Project, GUDHI User and Reference Manual, Available at http://gudhi.gforge.inria.fr/doc/latest/ (2015) 42 Kerber M Morozov D & Nigmetov A Hera Available at https://bitbucket org/grey narn/hera (2017) Acknowledgementsh g This work was partially supported by the EPSRC grant EPSRC EP/N014189/1, and by the National Institute for Health Research (NIHR) through support of the NIHR Southampton Respiratory Biomedical Research Unit, specifically funding Dr. Michael Bennett and the acquisition of CT data. Professor Djukanovic is a NIHR Senior Investigator. Author Contributions J.B., M.B. and R.D. designed the study with contributions from F.B. and M.P. The CT data were created and preprocessed by M.B. and J.C. The computations were designed by F.B., J.B., and M.P., and carried out by F.B. and M.P. The results were analyzed by F.B., M.B., R.D. and J.B. The manuscript was written by F.B., M.B., R.D., and J.B. The Supplementary Information was written by M.P., F.B., and J.B. 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Quantitative Computed Tomography Assessment of Airway Wall Dimensions: Current Status and Potential Applications for Phenotyping Chronic Obstructive Pulmonary Disease. Proceedings of the American Thoracic Society 5, 940–945 (2008). ( ) 8. Chen, Y. et al. Validation of human small airway measurements using endobronchial optical coherence tomography. Resp Med 109 1446–1453 (2015). ( ) 39. Sieren, J. P. SPIROMICS Protocol for Multicenter Quantitative Computed Tomography to Phenotype the Lungs. Am Journal Crit Care Met 194(1), 794–806 (2016). 40 H J t l TDA l A il bl h // i h b /k i /ML2015FP/ / /3TDAT l (2014) 9. Sieren, J. P. SPIROMICS Protocol for Multicenter Quantitative Computed Tomography to Phenotype the Lungs. SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 © The Author(s) 2018 Supplementary information accom Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23424-0. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23424-0. Competing Interests: The authors declare no competing interests. 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 Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Quantified Hypoxia and Anoxia in Lakes and Reservoirs
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Quantified Hypoxia and Anoxia in Lakes and Reservoirs Gertrud K. Nürnberg Freshwater Research, 3421 Hwy. 117, RR 1, Baysville, Ontario, P0B 1A0, Canada; Tel.: (705) 767-3718 Gertrud K. Nürnberg Freshwater Research, 3421 Hwy. 117, RR 1, Baysville, Ontario, P0B 1A0, Canada; Tel.: (705) 767-3718 E-mail: gkn@fwr.on.ca E-mail: gkn@fwr.on.ca Received October 31, 2003; Revised January 19, 2004; Accepted January 20, 2003; Published February 26, 2004 Received October 31, 2003; Revised January 19, 2004; Accepted January 20, 2003; Published February 26, 2004 Received October 31, 2003; Revised January 19, 2004; Accepted January 20, 2003; Published Febr Hypoxia and anoxia occur frequently in freshwater systems and have biological and chemical implications. Anoxia can be expressed and quantified as the anoxic factor; hypoxia, for a specific level of oxygen depletion, can be expressed as the hypoxic factor in lakes, reservoirs, and river sections. These methods summarize information of individual dissolved oxygen profiles as annual values or factors that facilitate comparison between and within lakes. Therefore, these factors are useful in the formulation and testing of hypotheses related to the dissolved oxygen status in water bodies. yp yg Methods of calculating different factors for different oxygen levels and water layers, including those applying separately to the epilimnion and hypolimnion, are presented in detail. Proven and potential applicability include: (1) the quantification of relationships with lake water quality variables and lake classification (trophic state), (2) the evaluation of restoration techniques with respect to their effects on hypolimnetic oxygen depletion, (3) the determination of internal phosphorus loading in stratified and polymictic lakes, (4) the exploration of habitat constraints due to hypoxia (e.g., fish species richness and winterkill), (5) forecasting potential effects of climatic change on oxygen content and internal phosphorus loading, and (6) the establishment and examination of criteria and guidelines with respect to hypoxia by custom-made definitions. KEYWORDS: oxygen depletion, anoxia, hypoxia, eutrophication, freshwater systems, climatic effects, method DOMAINS: freshwater systems, environmental modeling, environmental chemistry DOMAINS: freshwater systems, environmental modeling, environmental chemistry Research Article TheScientificWorldJOURNAL (2004) 4, 42–54 ISSN 1537-744X; DOI 10.1100/tsw.2004.5 Research Article TheScientificWorldJOURNAL (2004) 4, 42–54 ISSN 1537-744X; DOI 10.1100/tsw.2004.5 ©2004 with author. INTRODUCTION Hypoxia and anoxia occur frequently in freshwater systems[1], and increases in organic and nutrient loading and changes in water flow have increased oxygen depletion in lakes[2] and reservoirs[3]. Oxygen depletion is so widespread that methods of remediation have been proposed and applied in Europe and North America for many years[4,5,6]. Ways to quantify oxygen depletion in lakes were introduced before including oxygen depletion rates[7], the probability of anoxia[8], anoxic factor (AF)[1], and hypoxic factor (HF)[9]. Oxygen 42 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors depletion rates are determined from decreasing dissolved oxygen (DO) concentrations before the onset of anoxia. They do not describe the extent of anoxia in lakes, but only the speed of acquiring anoxia and therefore do not quantify anoxia itself. The probability of anoxia was developed from regression analysis of 55 stratified lakes with oxic, slightly anoxic, and considerably anoxic hypolimnia using lake characteristics pertaining to morphometry, hydrology, and nutrient load. This measure is based on an empirical model and therefore is restricted by the characteristics of the lakes for which the model was formulated; it appears to underestimate anoxia in pristine, oligotrophic lakes that may be anoxic for reasons other than high productivity, as noted in [1]. depletion rates are determined from decreasing dissolved oxygen (DO) concentrations before the onset of anoxia. They do not describe the extent of anoxia in lakes, but only the speed of acquiring anoxia and therefore do not quantify anoxia itself. The probability of anoxia was developed from regression analysis of 55 stratified lakes with oxic, slightly anoxic, and considerably anoxic hypolimnia using lake characteristics pertaining to morphometry, hydrology, and nutrient load. This measure is based on an empirical model and therefore is restricted by the characteristics of the lakes for which the model was formulated; it appears to underestimate anoxia in pristine, oligotrophic lakes that may be anoxic for reasons other than high productivity, as noted in [1]. In comparison, the anoxic or hypoxic factors quantify extent and duration of anoxia or hypoxia because they are computed from numerous measured DO profiles taken throughout the year[1,9]. The annual values of the factors are comparable between and within lakes and hence facilitate the formulation and testing of hypotheses related to lake oxygen. DEFINITIONS AND COMPUTATION The anoxic and hypoxic factors quantify the extent and duration of anoxia and hypoxia. They are based on a series of measured oxygen profiles and morphometric (hypsographic) data and can be computed for any water body (lake, reservoir, river, estuary, or marine area). The difference between HF and AF lies in the application of a different minimum or threshold DO concentration in the calculation procedure. The AF is based on the oxycline depth or its approximation (i.e., the depth at which 1 or 2 mg L–1 DO is observed), while the HF is based on a defined threshold that indicates the hypoxic criterion of choice for that particular water body, e.g., 6.5 mg L–1 DO. Both factors are computed for the hypothetical lake in Fig. 1 according to the following steps: 1. For AF, the oxycline is determined at 1 or 2 mg L–1 DO concentration from (DO) profiles. The choice of the threshold values depends on the method of DO measurement. For example, 2 mg L–1 measured by a DO probe about 1 m above the sediment usually coincides with anoxic conditions at the sediment surfaces located at that depth. For HF, the depth of the DO concentration threshold in question, e.g., 6.5 mg L–1, is determined. 2. The periods (total number n) are established for which the DO levels are at approximately the same depth according to the chosen threshold. 2. The periods (total number n) are established for which the DO levels are at approximately the same depth according to the chosen threshold. 3. The duration of each period of constant DO levels (ti, in days) is multiplied by the corresponding area (ai) and divided by the total surface area (Aoi) for the period (i). Especially in reservoirs where volumes and therefore areas change, it is important to use the surface area specific for the period. In water bodies without large volume changes, the average surface area Ao can be used instead. Correction for surface area is done to render this index comparable across waters similar to other areal measures, e.g., areal nutrient loads and fish yield. g y 4. These n terms, numbers of periods at different oxyclines, are then added up over the season or year, according to Eq. 1. 4. These n terms, numbers of periods at different oxyclines, are then added up over the season or year, according to Eq. 1. INTRODUCTION Furthermore, the factors are useful in setting DO criteria and guidelines because the quantification of hypoxia is based flexibly on individual threshold values and can be adapted to various DO levels and spatial restrictions (e.g., mixed surface layer only). Presented here is a comprehensive summary of the methods used to calculate both factors, examples of their proven and potential applicability, and their flexibility in dealing with differing DO thresholds and criteria. DEFINITIONS AND COMPUTATION ∑ = ⋅ = ⋅ + + ⋅ + ⋅ + ⋅ = n i oi i i on n n o o o A a t A a t A a t A a t A a t HF or AF 1 3 3 3 2 2 2 1 1 1 ... (1) (1) Expressed this way, AF or HF is the sum of ratios and represents the number of days in a year or season that a sediment area equal to the (lake) surface area is anoxic or hypoxic. Expressed this way, AF or HF is the sum of ratios and represents the number of days in a year or season that a sediment area equal to the (lake) surface area is anoxic or hypoxic. 43 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors a1 a2 a3 FIGURE 1. The sketch indicates different areas of a hypothetical lake that become anoxic sequentially with time, where a1 becomes anoxic first, then a2, etc. FIGURE 1. The sketch indicates different areas of a hypothetical lake that become anoxic sequentially with time, where a1 becomes anoxic first, then a2, etc. Seasonal Factors When AF and HF are determined for the stratification periods separately, a winter (e.g., AFwin in d winter–1) or summer factor (e.g., AFsum in d summer–1) is specified. When there is no winter anoxia, AFwin is zero and AFsum is equal to AF in d yr–1. Epilimnetic Factors To consider the mixed surface layer separately, epilimnetic factors (AFepi, HFepi) can be computed. These factors are determined by subtracting, for each period i, terms corresponding to anoxia or hypoxia in the seasonal stratified area below the thermocline (athermo_i) from those for the whole water column before summation, according to Eq. 2. ⎟⎟ ⎠ ⎞ ⋅ − ⎜⎜ ⎝ ⎛ ⋅ = ∑ = i o i thermo i n i i o i i epi epi A a t A a t HF or AF _ _ 1 _ (2) (2) (2) Such spatially distinct factors are especially useful in determining certain habitat requirements and for the support of guidelines specific to the mixed zone. For example, occasional epilimnetic anoxia may lead to a complete fish kill, while hypolimnetic anoxia may (only) prohibit the occurrence of certain fish species (especially coldwater species like Salmonidae, Gadidae, and Corigonidae). Method Accuracy The estimates are more accurate if they are based on DO profiles from more than the deepest location. Nürnberg[11] observed that in two lakes with long complicated shapes, anoxia started and ended earlier at the shallower sites. Spatial within-lake variability is discussed in more detail in the section on Application. End of Summer Stratification In stratified lakes, much of the oxygen depletion occurs in the fall when DO profiles may not be available. In this case, the endpoint at fall turnover can be predicted by a model based on summer average hypolimnetic temperature, mean depth, and latitude from 92 worldwide lakes according to Eq. 3, R2 = 0.47, p < 0.0001[10]. Log (fall turnover date) = 2.62 – 0.116 log (T) + 0.042 log (z) – 0.002 Latitude (3) (3) Log (fall turnover date) = 2.62 – 0.116 log (T) + 0.042 log (z) – 0.002 Latitude (3) where: fall turnover date, Julian day of the year; T, average July–August temperature at ca. 1 m above the bottom at the deepest location of the lake (Cº); z, mean depth, i.e., lake volume/lake surface area (m). 44 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors Method Precision The precision of the AF calculations was estimated by comparing AF computations by two different individuals for weekly to monthly DO profiles over 88 (accumulated) years of seven lakes, and their long- term averages of up to 11 years per lake. The error was 8% of the mean for the individual years, but differences between computations were not significant for the long-term averages. Spatial Within-Lake Variability In large and morphometrically distinct water bodies, the factors can be estimated separately for different sections. For example, AF was quite different between the two basins in small (77 ha) Cinder Lake, Ontario, at 12 and 40 d summer–1. Similarly pronounced were differences for the 100-km long, narrow (maximum 1-km wide) reservoir, Brownlee Reservoir on the Snake River, Idaho–Oregon[12]. The total reservoir had an AF of 68 d yr–1 on average between 1962 and 2001, ranging from 53–90 d yr–1. But AF of the 48-km long, deep lacustrine section was higher, 88 d yr–1 on average, ranging from 72–118 d yr–1, because this section is stratified all summer long (Fig. 3). On the other hand, AF for the 52-km long, shallow riverine section was only 10 d yr–1 in 1999 and 14 d yr–1 in 2000, because it is often mixed and reaerated with atmospheric oxygen. Epilimnetic factors were smaller than factors for the whole water column, as expected (Fig. 3). Severe anoxia in this reservoir is apparent since the long-term average lacustrine AFepi was 10 d yr–1, indicating that overall, an area in the lacustrine epilimnion equal to the lacustrine surface area is overlain by water below 2 mg L–1 DO for 10 d summer–1. The average HFepi of 58 d yr–1 indicates that the Oregon State criterion of 6.5 mg L–1 DO for the water column was exceeded in the lacustrine epilimnion for an equivalent of ca. 2 months on average. Determination of AF from One End-of-Summer Sampling Event The redox potential can be used to indicate the severity of anoxia once the water is anoxic, although its exact value is largely influenced by the amount of iron. Consequently, the redox potential measured within 1 m above the sediment surface at the end of the anoxic summer period and a variable related to iron (iron concentration of the sediment or lake water) was highly significantly correlated with AF in several Ontario lakes; it predicted AF of the previous summer reasonably well (n = 19, R2 = 0.76 or n = 52, R2 = 0.50[1]). Therefore, such relationships may be useful in estimating AF values from one sampling effort in remote and poorly studied lakes. APPLICATION Applications for methods of quantifying oxygen depletion are obvious. Because anoxic and hypoxic conditions can be expressed as a single annual value and be compared between and within lakes for different years, the formulation and testing of hypotheses related to lake oxygen is immensely facilitated. Since AF and HF are based on a large quantity of observed and measured data, they are more robust and exact than other estimates like rates of oxygen depletion, modeled measures (e.g., probability of anoxia and end-of-summer DO profiles), and simple DO criteria (i.e., threshold concentrations) as discussed in Nürnberg[1]. Nonetheless, the AF can be predicted with reasonable accuracy from models containing nutrient and morphometric variables. The following applications have been found most useful. Example Brownlee Reservoir Fig. 2 presents DO isopleths for the deep section of the Snake River Brownlee Reservoir in 1999 and 2000. The factors computed from these data were as follows: AF was 84 and 106 d yr–1 in 1999 and 2000, while HF was 135 and 175 d yr–1[12]. Epilimnetic factors were 7 and 8 d yr-1 AFepi and 43 and 60 d yr-1 HFepi for 1999 and 2000. 7/15/99 5/26/99 9/3/99 10/23/99 12/12/99 1/31/00 3/21/00 5/10/00 6/29/00 8/18/00 10/7/00 620 600 580 640 Elevation (m) 560 0 1 2 3 4 5 6 7 7 8 9 10 11 12 DO (mg L-1) FIGURE 2. DO isopleths in Brownlee Reservoir for 1999 and 2000 near the dam in the lacustrine section. Broken lines represent thresholds at 2.0 mg L–1 as used for the anoxic factor and 6.5 mg L–1 for the hypoxic factor. (From [9], reprinted with permission.) DO (mg L-1) FIGURE 2. DO isopleths in Brownlee Reservoir for 1999 and 2000 near the dam in the lacustrine section. Broken lines represent thresholds at 2.0 mg L–1 as used for the anoxic factor and 6.5 mg L–1 for the hypoxic factor. (From [9], reprinted with permission.) 45 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors Annual Variability Year-to-year variability can be quite large and ranged from 72–118 d yr–1 AF and 102–215 d yr–1 HF in lacustrine Brownlee Reservoir between 1962 and 2001 (Fig. 3); from 45–68 d yr–1 AF in a small urban kettle lake, Lake Wilcox, between 1987 and 2002; and from 0–10, 9–30, and 10–50 d yr–1 AF in three small glacial lakes on the Precambrian shield[Nürnberg, unpublished data,1,5,9]. Physical characteristics, especially hydrology in flow-managed reservoirs, have been found to control a significant portion of the between-years variance of DO depletion. For example, several annual and seasonal hydrological variables were significantly (p < 0.01) correlated with AF, HF, AFepi, and HFepi in Brownlee Reservoir. In particular, spring (April–May), summer inflow (July–Sept), and late fall (Oct– 46 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors 70 80 90 100 110 120 AF (days yr-1) 0 50 100 150 200 250 Factors (days yr-1) 1991 2001 1992 2000 1996 1994 1999 1993 1962 1997 1963 1995 70 80 90 100 110 120 0 50 100 150 200 250 FIGURE 3. Comparison of AF (x-axis) with several other factors for the lacustrine section of Brownlee Reservoir, Idaho–Oregon: HF (circle, years are indicated), HFepi, (triangle), and AFepi (arrow). All regressions are significant at least at p < 0.04 for n = 12 (lines are shown). Data from [9] and Nürnberg unpublished. 70 80 90 100 110 120 0 50 100 150 200 250 Factors (days yr-1) 1991 2001 1992 2000 1996 1994 1999 1993 1962 1997 1963 1995 70 80 90 100 110 120 0 50 100 150 200 250 FIGURE 3. Comparison of AF (x-axis) with several other factors for the lacustrine section of Brownlee Reservoir, Idaho–Oregon: HF (circle, years are indicated), HFepi, (triangle), and AFepi (arrow). All regressions are significant at least at p < 0.04 for n = 12 (lines are shown). Data from [9] and Nürnberg unpublished. Dec, Fig. 4 A, B) flushing rate or its inverse, the water residence time, were strongly correlated with the factors, so that they were smallest during periods of high inflow or flushing[9]. Such a good relationship of the fall outflow or residence time with anoxia and hypoxia can be expected, because severe DO depletion happens mostly in the summer and fall; low flow rates out of the dam and high residence times would stabilize stratification in the lacustrine section, leading to increased hypoxia. Annual Variability On the other hand, a strong inverse correlation for spring inflow with hypoxia can be explained by a delay in the onset of stratification. Evaluation of Lake Restoration Techniques Aiming at the Reduction of Oxygen Depletion Hypolimnetic oxygenation and aeration were used for 3 years, in an effort to improve water quality of Lake Wilcox[13]. Although fall turnover was earlier during treatment years than ever recorded before, AF was as high as and higher than in seven pretreatment and two post-treatment years, rendering the benefit of the aeration treatment questionable. Hypolimnetic withdrawal, the preferential discharge of hypolimnetic water by damming the surface water, has been used to enhance the export of nutrients and reduced substances out of lakes. The oxygen conditions, expressed as AF, indicate that the decrease in hypolimnetic anoxia was slower than the decrease of epilimnetic nutrient concentration and algal biomass. A positive effect on AF was evident only after at least four treatment years, while epilimnetic phosphorus concentration decreased already in the first year after operation in Lake Wononscopomuc, Connecticut[14]. 47 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors 100 150 200 250 HF(days yr-1) 1997 1996 1999 1995 1992 2001 1991 1994 2000 1963 1993 1962 30 40 50 60 70 Fall water residence time (days) 30 40 50 60 70 80 Epilimnetic HF(days yr-1) 1997 1996 1999 1995 1992 2001 1991 1994 2000 1963 1993 1962 1999 1997 1996 1962 2001 1992 1994 1993 1963 1995 2000 1991 -1 0 1 2 PNI 1999 1997 1996 1962 2001 1992 1994 1993 1963 1995 2000 1991 B D C A FIGURE 4. Lacustrine HF (top) and HFepi (bottom) of Brownlee Reservoir vs. fall (October to December) water residence time (total of 92 days, left panel) and the Pacific Northwest Climate Index (PNI, right panel). Regressions are significant; at least at p < 0.005 for the log-transformed water residence time and at p < 0.04 for the PNI relationships (n = 12, regression lines are shown). Revised from [9] including unpublished data. 100 150 200 250 HF(days yr-1) 1997 1996 1999 1995 1992 2001 1991 1994 2000 1963 1993 1962 30 40 50 60 70 Fall water residence time (days) 30 40 50 60 70 80 Epilimnetic HF(days yr-1) 1997 1996 1999 1995 1992 2001 1991 1994 2000 1963 1993 1962 1999 1997 1996 1962 2001 1992 1994 1993 1963 1995 2000 1991 -1 0 1 2 PNI 1999 1997 1996 1962 2001 1992 1994 1993 1963 1995 2000 1991 B D C A C C A B 2 FIGURE 4. Lacustrine HF (top) and HFepi (bottom) of Brownlee Reservoir vs. Evaluation of Lake Restoration Techniques Aiming at the Reduction of Oxygen Depletion fall (October to December) water residence time (total of 92 days, left panel) and the Pacific Northwest Climate Index (PNI, right panel). Regressions are significant; at least at p < 0.005 for the log-transformed water residence time and at p < 0.04 for the PNI relationships (n = 12, regression lines are shown). Revised from [9] including unpublished data. Lake-to-Lake Variation Anoxia can vary extensively between lakes and quantification of anoxia by the AF has been proven useful in explaining lake-to-lake variation. Even in a geographically close area like the Muskoka–Haliburton region on the Precambrian shield in southern Central Ontario, AF ranged from 0–70 d yr–1 and at least 50% had AF > 10 d yr–1. These lakes are relatively pristine, often colored, and usually small, but deep. Occasional anoxia in many of these remote lakes is probably due to a combination of morphometry and high organic acid content due to natural humic and fulvic acids exported from surrounding wetlands. Mesotrophic and eutrophic lakes in more fertile regions, like in the St. Lawrence–Great Lakes region had consistently high AF between 40 and 70 d yr–1. Such differences in lakes and their AF reflect differing water and catchment chemistries and indicate the need for diverse management strategies[5]. Of all tested physical and chemical variables (including dissolved organic carbon as measure of organic acids that contributed only 17% to the variance of AF[1]), a dependency of anoxia on nutrient concentration and morphometry was found the most important. Accordingly, multiple regressions of long-term average AF on average lake TP and a morphometric ratio, measured as z/A0.5 (m km–1), were highly significant in North American lakes[1] (Table 1, Eq. A, B). Recent analysis with data from Nürnberg[15], showed a similarly strong relationship with long-term average summer total nitrogen (Fig. 5, Table 1, Eq. C). These long-term relationships can be used to model (or predict) AF in lakes where DO 48 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors TABLE 1 Relationships and Models Involving AF in North American Stratified Lakes, Where Data of Equations E to G are Based on Central Ontario Lakes Eq. Lake-to-Lake Variation Predicted1 Regression Eq.1 N R2 Source A AFsum –35.4 (5.1) + 44.2 (4.3) log (TPannual) + 0.950 (0.187) z/Ao 0.5 73 0.65 [1] B AFsum –36.2 (5.2) + 50.1 (4.4) log (TPsummer) + 0.762 (0.196) z/Ao 0.5 70 0.67 [15] C AFsum –173 (22) + 73.0 (8.6) log (TNsummer) + 0.925 (0.272) z/Ao 0.5 54 0.62 This study D AFsum –39.9 (9.7) + 27.0 (4.0) log (TP-Load) 17 0.76 [1] E Fish species number1 0.97 (2.42) – 1.53 (0.49) log (AFsum+1) + 5.38 (1.02) log (Ao) 52 0.51 [20] F Fish species number1 4.92 (1.38) – 6.12 (1.44) log (AFwin+1)* + 0.56 (0.11) z 32 0.71 [20] G Winterkill- AFwin –1 + 10 (0.091 z + 0.804) — — Based on Eq. F Note: All regressions and partial probabilities are highly significant at p < 0.001, except in Eq. F where noted with *, p < 0.01. Standard errors of the regression coefficients are given in parenthesis. 1 Variables: AFsum, summer AF (d summer–1); AFwin, winter AF (d winter–1); Winterkill-AFwin, value of AFwin above which a fish winterkill is likely for given z; z, mean depth (m); Ao, lake surface area (km2); TPannual, annual water-column average TP (µg L–1); TPsummer, epilimnetic summer TP (µg L–1); TNsummer, epilimnetic summer TN (µg L–1); Fish species number, also called fish species richness, number of species of all fish present in specific lake. TABLE 1 essions and partial probabilities are highly significant at p < 0.001, except in Eq. F where noted < 0.01. Standard errors of the regression coefficients are given in parenthesis. Note: All regressions and partial probabilities are highly significant at p < 0.001, except in Eq. F where noted with *, p < 0.01. Standard errors of the regression coefficients are given in parenthesis. 1 Variables: AFsum, summer AF (d summer–1); AFwin, winter AF (d winter–1); Winterkill-AFwin, value of AFwin above which a fish winterkill is likely for given z; z, mean depth (m); Ao, lake surface area (km2); TPannual, annual water-column average TP (µg L–1); TPsummer, epilimnetic summer TP (µg L–1); TNsummer, epilimnetic summer TN (µg L–1); Fish species number, also called fish species richness, number of species of all fish present in specific lake. 100 1000 Average summer total nitrogen (µg L-1) 0 20 40 60 80 Summer AF (days yr-1) FIGURE 5. Summer AF in 54 dimictic lakes of northeastern North America vs. Internal load = Release rate · AFsum where: (summer) internal load, mg total phosphorus (TP) per lake surface area (m2) per summer, and phosphorus release rate representing a summer average, in mg TP released per anoxic sediment area per day of the anoxic summer period (mg m–2 d–1). If there is phosphorus release under ice in the winter, a release rate representative for the winter and AFwin are substituted in Eq. 4 to estimate winter internal load. where: (summer) internal load, mg total phosphorus (TP) per lake surface area (m2) per summer, and phosphorus release rate representing a summer average, in mg TP released per anoxic sediment area per day of the anoxic summer period (mg m–2 d–1). If there is phosphorus release under ice in the winter, a release rate representative for the winter and AFwin are substituted in Eq. 4 to estimate winter internal load. Modeling and prediction of future internal load and subsequently lake phosphorus concentration and trophic state can be accomplished by predicting one or both of its components, AF and phosphorus release rates, according to Eq. 4. For example, a sequence of models and empirical equations predicted future water quality after the potential increase in lakeshore development of a seasonal community in the reach of the metropolitan Toronto (ca. 500 lakes in the District of Muskoka[16]). In particular, future TP concentrations based on increased external loading (i.e., a minimum estimate of TP concentration since internal load is not being considered) were used to predict future anoxic factors (from equations in Table 1 and release rates based on regional release rate - TP relationships). Postdevelopment internal load could then be computed as the product of these variables (Eq. 4) and used to predict annual average lake TP concentration and other water quality variables like chlorophyll and Secchi transparency[17]. As mentioned previously, of particular usefulness is the application of the AF model to shallow lakes with intermittent stratification. The direct determination of internal load in these polymictic lakes is almost impossible without major technical effort (determination of detailed hydrologic and phosphorus mass balance, including down and upward fluxes throughout the summer), since external mix with internal phosphorus inputs frequently and untraceably throughout the summer[18]. However, a modeled AF can be used to determine internal load according to Eq. Trophic State Classification Because of the dependency on nutrient concentration, the anoxic factor was used to establish limits for trophic state classification with respect to anoxia[15]. Because colored lakes often have anoxic hypolimnia despite low algal biomass, the classification of data including 70 North American lakes indicates that oligotrophic lakes can have an AF of up to 20 d yr–1. Consequently, when classified with respect to trophic state, an AF below 20 d yr–1 indicates oligotrophic conditions, 20–40 d yr–1 are usually found in mesotrophic lakes, 40–60 d yr–1 represent eutrophic conditions, and above 60 d yr–1 is typical for hypereutrophic conditions. A more exact measure of trophic state would include the morphometric ratio, because the deeper the lake compared to its area (the larger z/A0.5), the larger the AF. From Eq. B, Table 1, a chart was drawn to assist the evaluation of a specific trophic state with respect to anoxia (Fig. 6). Lake-to-Lake Variation long-term averages of summer total nitrogen concentration. Regression line of Eq. C in Table 1 is shown, where one outlier was removed and only AF above zero was included. 100 1000 Average summer total nitrogen (µg L-1) 0 20 40 60 80 Summer AF (days yr-1) FIGURE 5. Summer AF in 54 dimictic lakes of northeastern North America vs. long-term averages of summer total nitrogen concentration. Regression line of Eq. C in Table 1 is shown, where one outlier was removed and only AF above zero was included. 100 1000 0 20 40 60 80 Summer AF (days yr-1) Average summer total nitrogen (µg L-1) FIGURE 5. Summer AF in 54 dimictic lakes of northeastern North America vs. long-term averages of summer total nitrogen concentration. Regression line of Eq. C in Table 1 is shown, where one outlier was removed and only AF above zero was included. FIGURE 5. Summer AF in 54 dimictic lakes of northeastern North America vs. long-term averages of summer total nitrogen concentration. Regression line of Eq. C in Table 1 is shown, where one outlier was removed and only AF above zero was included. 49 Nürnberg: Anoxic and Hypoxic Factors TheScientificWorldJOURNAL (2004) 4, 42–54 TheScientificWorldJOURNAL (2004) 4, 42–54 data are not available or difficult to obtain, like in polymictic lakes. Since AF describes the sediment surface that is overlain by anoxic water in stratified lakes, it can be hypothesized that its predicted value resembles anoxic sediment surfaces in polymictic lakes. Such a modeled variable is useful in determining internal load for shallow lakes as described below, where release rates may be available but not the extent of anoxic sediment areas. data are not available or difficult to obtain, like in polymictic lakes. Since AF describes the sediment surface that is overlain by anoxic water in stratified lakes, it can be hypothesized that its predicted value resembles anoxic sediment surfaces in polymictic lakes. Such a modeled variable is useful in determining internal load for shallow lakes as described below, where release rates may be available but not the extent of anoxic sediment areas. Internal Load Calculation The anoxic factor was originally developed to determine internal phosphorus load in stratified lakes as the product of anoxic areal release rates (experimentally determined or predicted from sediment phosphorus concentration) and AF according to Eq. 4[11]. (4) Internal load = Release rate · AFsum (4) (4) Internal load = Release rate · AFsum Fish Species Richness Fish species richness was found to be correlated with AF in Central Ontario lakes after variability due to lake area was taken into account, and winter kill could be predicted by using an AFwin that quantifies anoxia under ice, and mean depth (Table 1, E–G). In particular, cold water species including Salmonidaes, Coregonidae, and Gadidae were sensitive to summer and winter anoxia and they only occurred when AFsum was below 32 d summer–1 or AFwin was below 4.4 d winter–1[20]. Internal load = Release rate · AFsum 4 in shallow, polymictic lakes, where release rates may be measured in the laboratory or predicted from sediment phosphorus content[19], but the extent and duration of sediment anoxia is unknown. 50 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors (m km-1) o m e h 0 20 40 60 80 100 AF (d/summer) 0 10 20 30 40 50 z/A0 5 AFsum (days summer-1) FIGURE 6. Summer AF (AFsum) and the morphometric variable z/Ao0.5 can be used to classify lakes with respect to their trophic state. Observed values from 70 North American lakes are presented[15]. Symbol sizes indicate the four trophic states oligotrophy, mesotrophy, eutrophy, and hypereutrophy (from small to large) and lines indicate the trophic state boundaries based on TPsummer in Eq. B, Table 1. (m km-1) o m e h 0 20 40 60 80 100 0 10 20 30 40 50 z/A0.5 o FIGURE 6. Summer AF (AFsum) and the morphometric variable z/Ao0.5 can be used to classify lakes with respect to their trophic state. Observed values from 70 North American lakes are presented[15]. Symbol sizes indicate the four trophic states oligotrophy, mesotrophy, eutrophy, and hypereutrophy (from small to large) and lines indicate the trophic state boundaries based on TPsummer in Eq. B, Table 1. Establishment of Criteria and Guidelines Hypoxia causes species changes in fish[20,22] and in macro benthos[23,24], so that water quality objectives have been established by governmental agencies to restrict the acceptable hypoxic level. Typically, a threshold DO concentration is set under which a water body is considered to be “impaired” and a plan is initiated to attain the guidelines, including the process of determining a total maximum daily load (TMDL) in the U.S.[25]. In the case of Brownlee Reservoir as presented here, this threshold is 6.5 mg L–1. However, these levels vary for different water bodies, agencies, and intended usage between 4.8 and 10.5 mg L–1[9], so that a useful quantification of hypoxia must be flexible and adaptable respective these thresholds, as can be accomplished by specific definitions of the hypoxic factor. Climatic Effects Since the factors quantify annual trends, climatic effects on DO depletion can be investigated. For example, the Pacific Northwest Index (PNI) was significantly correlated with HF and HFepi in Brownlee Reservoir (Fig. 4 C, D)[9]. The PNI is a normalized index based on three terrestrial climate variables for the western North American continent: the air temperature at San Juan Islands, the total precipitation at Cedar Lake in the Cascade Mountains, and snowpack depth on Mount Rainier, Washington State[21]. 51 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors CONCLUSIONS It can be concluded that the quantification of anoxia and hypoxia leads to increased knowledge of the controlling mechanisms of oxygen depletion in water bodies. Although it has not been applied to rivers other than run-of-the-river reservoir sections, it should provide a useful quantification of hypoxia and anoxia there as well, since hypoxia is widespread in these freshwater systems[3,31,32]. The concept could be especially useful in estuarine and marine systems, since hypoxic conditions are widely spread in saline environments. Especially in recent years, anthropogenic impacts have led to severe increases in estuarine and coastal anoxia (e.g., Gulf of Mexico[33], European coast lines[34]). For example, Diaz[35] describes 44 marine areas of moderate to severe hypoxia world-wide. Additional Applications AF helped to compare methods to measure denitrification rates in lakes and determine the most appropriate one[26] and reveal the dependency of hypolimnetic anoxia on depth and volume in tropical African reservoirs[27]. The anoxic factor and its relationship with phosphorus and morphometry were combined with information on fossil midge (Chironomidae) assemblages in paleolimnological studies to establish training sets and hindcast hypolimnetic anoxia for the past[23,28,29]. In this way specific hypotheses could be supported, for example, that AF is correlated to the average end-of-summer hypolimnetic oxygen concentration and that the damming of lakes during European settling affected the oxygen status of lakes[30]. ACKNOWLEDGMENTS Helpful comments by the editor of Freshwater Systems, Karl Havens, and several referees including Jack Jones, are acknowledged. Bruce LaZerte provided constructive and supportive critique. The concept of the hypoxic factor was developed while working with Brown and Caldwell for the City of Boise, ID on the Snake River Hells Canyon TMDL (total maximum daily load). 1. Nürnberg, G.K. (1995) Quantifying anoxia in lakes. Limnol. Oceanogr. 40, 1100–1111. 2. Schertzer, W.M. and Sawchuk, A.M. (1990) Thermal structure of the Lower Great Lakes in a warm year: implications for the occurrence of hypolimnion anoxia. Trans. Am. Fish. Soc. 119, 195–209. 3. Gordon, J.A. (1993) Dissolved oxygen in streams and reservoirs. Water Environ. Res. 65, 571–573. 4. Cooke, G.D., Welch, E.B., Peterson, S.A., and Newroth, P.R. (1993) Restoration and Management of Lakes and Reservoirs. Lewis Publishers, Ann Arbor, MI. , ( ) yg , 4. Cooke, G.D., Welch, E.B., Peterson, S.A., and Newroth, P.R. (1993) Restoration and Management of Lakes and Reservoirs. Lewis Publishers, Ann Arbor, MI. ( ) Q y g g , W.M. and Sawchuk, A.M. (1990) Thermal structure of the Lower Great Lakes in a warm year: implications urrence of hypolimnion anoxia Trans Am Fish Soc 119, 195–209 1. Nürnberg, G.K. (1995) Quantifying anoxia in lakes. Limnol. Oceanogr. 40, 1100–1111. occurrence of hypolimnion anoxia. Trans. Am. Fish. Soc. 119, 195–209. yp , , J.A. (1993) Dissolved oxygen in streams and reservoirs. Water Environ. Res. 65, 571–573. g, ( ) Q y g g , 2. Schertzer, W.M. and Sawchuk, A.M. (1990) Thermal structure of the Lower Great Lakes in a warm year: implications for the occurrence of hypolimnion anoxia. Trans. Am. Fish. Soc. 119, 195–209. 3 Gordon J A (1993) Dissolved oxygen in streams and reservoirs Water Environ Res 65 571 573 2. Schertzer, W.M. and Sawchuk, A.M. (1990) Thermal structure of the Lower Great Lakes in a warm for the occurrence of hypolimnion anoxia. Trans. Am. Fish. Soc. 119, 195–209. REFERENCES 1. Nürnberg, G.K. (1995) Quantifying anoxia in lakes. Limnol. Oceanogr. 40, 1100–1111. 2. Schertzer, W.M. and Sawchuk, A.M. (1990) Thermal structure of the Lower Great Lakes in a warm year: implications for the occurrence of hypolimnion anoxia. Trans. Am. Fish. Soc. 119, 195–209. 3. Gordon, J.A. (1993) Dissolved oxygen in streams and reservoirs. Water Environ. Res. 65, 571–573. 4. Cooke, G.D., Welch, E.B., Peterson, S.A., and Newroth, P.R. (1993) Restoration and Management of Lakes and Reservoirs. Lewis Publishers, Ann Arbor, MI. 52 TheScientificWorldJOURNAL (2004) 4, 42–54 Nürnberg: Anoxic and Hypoxic Factors 5. Nürnberg, G.K. (1997) Coping with water quality problems due to hypolimnetic anoxia in Central Ontario Lakes. Water Qual. Res. J. Can. 32, 391–405. 5. Nürnberg, G.K. (1997) Coping with water quality problems due to hypolimnetic anoxia in Central Ontario Lakes. Water Qual. Res. J. Can. 32, 391–405. 6 Gächter R and Wehrli B (1998) Ten years of artificial mixing and oxygenation: no effect on the internal P loading hter, R. and Wehrli, B. (1998) Ten years of artificial mixing and oxygenation: no effect on the internal P loa wo eutrophic lakes. Environ. Sci. Technol. 32, 3659–3665. 7. Hutchinson, G.E. (1938) On the relation between the oxygen deficit and the productivity and typology of lakes. Int. Rev. Gesamten Hydrobiol. 36, 336–355. y , 8. Reckhow, K.H. (1979) Empirical lake models for phosphorus: development, applications, limitations and uncertainty. Perspectives in Lake Ecosystem Modeling. Scavia, D. and Robertson, A., Eds. pp. 193–221. w, K.H. (1979) Empirical lake models for phosphorus: development, applications, limitations and uncertainty. ti i L k E t M d li S i D d R b t A Ed 193 221 , ( 9 9) p p p p , pp , Perspectives in Lake Ecosystem Modeling. Scavia, D. and Robertson, A., Eds. pp. 193–221. Perspectives in Lake Ecosystem Modeling. Scavia, D. and Robertson, A., Eds. pp. 193–221. 9. Nürnberg, G.K. (2002) Quantification of oxygen depletion in lakes and reservoirs with the hypoxic factor. Lake Reserv. Manage. 18, 298–305. rg, G.K. (1988) A simple model for predicting the date of fall turnover in thermally stratified lakes. Limnol gr. 33, 1190–1195. g 11. Nürnberg, G.K. (1987) A comparison of internal phosphorus loads in lakes with anoxic hypolimnia: laboratory incubations versus hypolimnetic phosphorus accumulation. Limnol. Oceanogr. 32, 1160–1164. incubations versus hypolimnetic phosphorus accumulation. Limnol. Oceanogr. 32, 1160–1164. 12. REFERENCES Freshwater Research, Brown and Caldwell (2001) Assessment of Brownlee Reservoir Water Quality. Technical Report for the City of Boise, ID. yp p p 12. Freshwater Research, Brown and Caldwell (2001) Assessment of Brownlee Reservoir Water Quality. Technical Report for the City of Boise, ID. 13. Nürnberg, G.K., LaZerte, B.D., and Olding, D.D. (2003) An artificially induced Planktothrix rubescens surface bloom in a small kettle lake in southern Ontario compared to blooms world-wide. Lake Reserv. Manage. 19, 307–322. 13. Nürnberg, G.K., LaZerte, B.D., and Olding, D.D. (2003) An artificially induced Planktothrix rubescens surface bloom in a small kettle lake in southern Ontario compared to blooms world-wide. Lake Reserv. Manage. 19, 307–322. 14. Nürnberg, G.K., Hartley, R., and Davis, E. (1987) Hypolimnetic withdrawal in two North American lakes with anoxic 14. Nürnberg, G.K., Hartley, R., and Davis, E. (1987) Hypolimnetic withdrawal in two North American lakes with anoxic phosphorus release from the sediment. Water Res. 21, 923–928. Nürnberg, G.K. (1996) Trophic state of clear and colored, soft- and hardwater lakes with special consider nutrients, anoxia, phytoplankton and fish. Lake Reserv. Manage. 12, 432–447. p y p g , 16. Nürnberg, G.K. and LaZerte, B.D. (2004) Modeling the effect of development on internal phosphorus load in nutrient-poor lakes. Water Resour. Res. 40, W01105, doi:10.1029/2003WR002410. 17. Nürnberg, G.K. and LaZerte, B.D. (2001). Predicting lake water quality. In Managing Lakes and Reservoirs. Holdren, C., Jones, W., and Taggart, J., Eds. North American Lake Management Society and Terrene Institute, Madison, WI in cooperation with the U.S. Environmental Protection Agency. pp. 139–163. p g y pp 18. Søndergaard, M., Jensen, J.P., and Jeppesen, E. (2001) Retention and internal loading of phosphorus in shallow, eutrophic lakes. TheScientificWorldJOURNAL 1, 427–442. p f 19. Nürnberg, G.K. (1988) Prediction of phosphorus release rates from total and reductant-soluble phosphorus in anoxic lake sediments. Can. J. Fish. Aquat. Sci. 45, 453–462. 20. Nürnberg, G.K. (1995) Anoxic factor, a quantitative measure of anoxia and fish species richness in Central Ontario lakes. Trans. Am. Fish. Soc. 124, 677–686. 21. Petersen, J.H. and Kitchell, J.F. (2001) Climate regimes and water temperature changes in the Columbia River: bioenergetic implications for predators of juvenile salmon. Can. J. Fish. Aquat. Sci. 58, 1831–1841. 22. Stefan, H.G., Hondzo, M., Fang, X., Eaton, J.G., and McCormick, J.H. (1996) Simulated longterm temperature and dissolved oxygen characteristics of lakes in the north-central United States and associated fish habitat limits. Limnol. Oceanogr. 41, 1124–1135. TheScientificWorldJOURNAL (2004) 4, 42–54 REFERENCES Quinlan, R., Smol, J.P., and Hall, R.I. (1998) Quantitative inferences of past hypolimnetic anoxia in south Ontario lakes using fossil midges (Diptera: Chironomidae). Can. J. Fish. Aquat. Sci. 55, 587–596. 24. Rabalais, N.N., Smith, L.E., Harper, D.E., and Justic, D. (2001) 12. Effects of seasonal hypoxia on continental shelf benthos. In Coastal Hypoxia, Consequences for Living Resources and Ecosystems. Rabalais, N.N. and Turner, R.E., Eds. American Geophysical Union, Washington, D.C. pp. 211–240. p y g pp EPA (1999) Protocol for Developing Nutrient TMDLs. 841-B-99-007. U.S. Environmental Protection A Washington, D.C. 26. Molot, L.A. and Dillon, P.J. (1993) Nitrogen mass balances and denitrification rates in Central Ontario lakes. Biogeochemistry 20, 195–212. 27. Townsend, S.A. (1999) The seasonal pattern of dissolved oxygen, and hypolimnetic deoxygenation, in two tropical Australian reservoirs. Lake Reserv. Res. Manage. 4, 41–53. 28. Clerk, S., Hall, R., Quinlain, R., and Smol, J.P. (2000) Quantitative inferences of past hypolimnetic anoxia and nutrient levels from a Canadian Precambrian Shield lake. J. Paleolimnol. 23, 319–336. 29. Little, J.L. and Smol, J.P. (2001) A chironomid-based model for inferring late-summer hypolimnetic oxygen in 28. Clerk, S., Hall, R., Quinlain, R., and Smol, J.P. (2000) Quantitative inferences of past hypolimnetic anoxia and nutrient levels from a Canadian Precambrian Shield lake. J. Paleolimnol. 23, 319–336. 29. Little, J.L. and Smol, J.P. (2001) A chironomid-based model for inferring late-summer hypolimnetic oxygen in southeastern Ontario lakes. J. Paleolimnol. 26, 259–270. Quinlan, R. and Smol, J.P. (2002) Regional assessment of long-term hypolimnetic oxygen changes (Canada) Shield lakes using subfossil chironomids. J. Paleolimnol. 27, 249–260. 31. Quinn, J.M. and McFarlane, P.N. (1989) Epilithon and dissolved oxygen depletion in the Manawuta River, New Zealand: simple models and management implications. Water Res. 23, 825–832. p g p , 32. Chambers, P.A., Brown, S., Culp, H.M., and Lowell, R.B. (2000) Dissolved oxygen decline in ice-covered rivers of northern Alberta and its effects on aquatic biota. J. Aquat. Ecosys. Stress Recov. 8, 27–38. 33. Johannessen, T. and Dahl, E. (1996) Declines in oxygen concentrations along the Norwegian Skagerrak Coast, 1927– 1993: a signal of ecosystem changes due to eutrophication? Limnol. Oceanogr. 41, 766–778. 53 Nürnberg: Anoxic and Hypoxic Factors TheScientificWorldJOURNAL (2004) 4, 42–54 TheScientificWorldJOURNAL (2004) 4, 42–54 34. Rabalais, N.N., Turner, R.E., and Wiseman, W.J. (2001) Hypoxia in the Gulf of Mexico. J. Environ. Qual. 30, 320– 329. 35. Diaz, R.J. (2001) Overview of hypoxia around the world. J. Environ. Qual. 30, 275–281. 34. Rabalais, N.N., Turner, R.E., and Wiseman, W.J. (2001) Hypoxia in the Gulf of Mexico. J. Environ. Qual. 30, 320– 329. 35. Diaz, R.J. (2001) Overview of hypoxia around the world. J. Environ. Qual. 30, 275–281. TheScientificWorldJOURNAL (2004) 4, 42–54 REFERENCES 34. Rabalais, N.N., Turner, R.E., and Wiseman, W.J. (2001) Hypoxia in the Gulf of Mexico. J. Environ. Qual. 30, 320– 329. 35. Diaz, R.J. (2001) Overview of hypoxia around the world. J. Environ. Qual. 30, 275–281. This article should be referenced as follows: Nürnberg, G.K. (2004) Quantified hypoxia and anoxia in lakes and reservoirs. TheScientificWorldJOURNAL 4, 42–54. Handling Editor: Karl E. Havens, Principal Editor for Freshwater Systems — a domain of TheScientificWorldJOURNAL. This article should be referenced as follows: G.K. (2004) Quantified hypoxia and anoxia in lakes and reservoirs. TheScientificWorldJOURNAL 4, 42–54. Karl E. Havens, Principal Editor for Freshwater Systems — a domain of TheScientificWorldJOURNAL. Karl E. Havens, Principal Editor for Freshwater Systems — a domain of TheScientificWorldJOURNAL. 54
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VIOLÊNCIA DOMÉSTICA: CONCEITO E ATUAÇÃO POLICIAL
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PALAVRAS-CHAVE: Violência Doméstica, Atuação Policial, Lei Maria da Penha PALAVRAS-CHAVE: Violência Doméstica, Atuação Policial, Lei Maria da Penha VIOLÊNCIA DOMÉSTICA: CONCEITO E ATUAÇÃO POLICIAL LIMA, Humberto Santos RESUMO Mostrar o que realmente é a violência doméstica, as consequências que podem causar as vítimas, e como deve ser a atuação policial, é de grande relevância, ao modo em que, evidencia o assunto, trazendo debates a fim de combater em nosso meio social, haja vista que a violência doméstica é grande óbice que sola a sociedade, sendo totalmente imparcial, podendo ser qualquer mulher vítima deste tipo de violência. E o artigo objtiva definir o que é a violência contra a mulher e doméstica, identificando os tipos de violência que a mulher sofre, descrevendo as possíveis motivações de aceitarem a condição de violência, identificando as causas e efeitos psicológicos e físicos que resultam na vítima e, por fim, demonstrando como deve ser a atuação policial. CONCEITUAÇÃO DE VIOLÊNCIA CONTRA A MULHER E DOMÉSTICA Violência contra a mulher e doméstica, são termos muito usados, mas como definir essa violência que a mulher sofre? Usando o dispositivo legal, a Lei Maria da Penha, obtemos a definição de violência doméstica no Art.5º da Lei 11340/06, como “Para os efeitos desta Lei, configura violência doméstica e familiar contra a mulher qualquer ação ou omissão baseada no gênero que lhe cause morte, lesão, sofrimento físico, sexual ou psicológico e dano moral ou patrimonial” (BRASIL, 2006). A conceituação de violência contra a mulher entendida como a relação violenta, do gênero masculino para com o feminino, seja física, psicológica, até mesmo ameaçar verbalmente conforme visto na apostila Atendimento Policial a vítimas de violência doméstica (SENASP, 2007, p. 08) “a violência contra a mulher constitui qualquer ação ou conduta, baseada no gênero, que cause morte, dano ou sofrimento físico, sexual ou psicológico à mulher, tanto no âmbito público como no privado”, em virtude do elevado índice dessa modalidade de violência ocorrer no espaço privado passou a ser conhecida com violência doméstica. INTRODUÇÃO Em pleno século 21, onde temos a maior globalização jamais vista, cujo gênero feminino se faz a cada dia mais presente em papeis importantes na sociedade, sejam eles ramos profissionais, políticos, antropólogos e afins, mostrando o enaltecimento da mulher perante seus pares, onde mídias, sejam elas sociais ou de meios de comunicação, fazem questão, e obviamente por merecimento, demonstrar que não existe distinção entre o potencial masculino e feminino, que acaba ocorrendo violência contra mulher, principalmente no meio doméstico, seja ela física, psicológica ou social. E muitas destas mulheres não sabem ao menos que violência psicológica também é violência, não sabem como proceder em situações assim, a quem buscar ajuda, os trâmites legais, os direitos que possuem a metodologia de atuação em crimes dessa natureza, a que órgão pedir ajuda. A Lei nº 11.340 (Maria da Penha) criada em 2006, teve como objetivo de sua criação a criminalização da violência doméstica, criar mecanismo de atendimento à vítima e operacionalidade policial. Versando sobre a Violência Doméstica: Conceito e Atuação Policial. Trazendo essa temática para aplicação no âmbito policial, almejando compilar tanto a experiência policial no atendimento dessas ocorrências com o conhecimento teórico e metodológico, alcançado pelo artigo. Diante do exposto, se faz necessário demonstrar a sociedade e principalmente a estas mulheras, o papel fundamental do policial em sua obrigatoriedade de zelar por sua integridade Policial Militar, Graduado em Tecnologia em Segurança Pública – Instituto Federal do Paraná – IFPR e Pós Graduando em Segurança Pública- Faculdade UNINA, sl.humberto@gmail.com 2 como pessoa haja vista seu respaldo legal no Art. 5º Inciso I e III e Art. 3º Inciso IV da Constituição Federal que dizem: “homens e mulheres são iguais em direitos e obrigações, nos termos desta Constituição”; “ninguém será submetido à tortura nem a tratamento desumano ou degradante”; “promover o bem de todos, sem preconceitos de origem, raça, sexo, cor, idade e quaisquer outras formas de discriminação” (BRASIL, 1988), respectivamente. Mostrando que através dos policiais existem caminhos que podem ser adotados para que se consiga enfrentar a violência doméstica e as medidas assistenciais que estes órgãos ofertam e designam para estas vítimas, enaltecendo os aspectos legais que norteiam as forças de segurança nos atendimentos de ocorrências dessa natureza, que, infelizmente, é desconhecida pela sociedade. Violência patrimonial: (ações que envolvam dano financeiro); Violência patrimonial: (ações que envolvam dano financeiro); Violência moral (Difamar, caluniar e injuriar) onde novamente Dias (2007, p.54) colabora dizendo que “a violência moral encontra proteção penal nos delitos contra a honra: calúnia, difamação e injúria. São denominados delitos que protegem a honra, mas cometidos em decorrência de vínculo de natureza familiar ou afetiva, configuram violência moral”. TIPOS DE VIOLÊNCIA Pode-se dizer que há cinco formas violência, baseados no Art. 7º da Lei Maria da Penha (Lei nº 11.340, de 07 de agosto de 2006), são elas: Violência física (ação de agredir, através de chute, socos, e afins que ofenda a integridade física); Violência psicológica: qualquer ação que viole e resulte em danos emocionais, desde um xingamento, até a humilhação; Violência psicológica: qualquer ação que viole e resulte em danos emocionais, desde um xingamento, até a humilhação; 3 3 Violência sexual: (forçar a presenciar, ter, ver as relações). Reforçado por Dias (2007 p.50) que diz “delito de assédio sexual, que está ligado às relações de trabalho, pode constituir violência doméstica quando, além do vínculo afetivo familiar, a vítima trabalha para o agressor”; DE VIOLÊNCIA Visto que muitas mulheres aceitam permanecerem nessa condição violentas por alguns fatores, podendo ser a: Preocupação com os filhos- (Um dos fatores mais ouvidos nos atendimentos nesses tipos de situação cujas vítimas); A vítima, oriunda de uma família onde teve uma criação agressiva (Principalmente por ter uma imagem paternal violenta contra a maternal). Sousa et al. (2013, p.426) diz que “a violência doméstica normalmente segue um ciclo, denominado “ciclo da violência”, representada por fases que se repetem ritualisticamente”. Pela falta de conhecimento legal (Achando que pra ser "Maria da Penha" ela tem que aparecer na delegacia com um olho roxo), conforme demonstrado IPEA (2001, p.39), “apenas 9,4% das mulheres negras acreditavam que esse tipo de agressão não era importante a ponto de demandar uma ação policial”. Por comodidade (Por mais absurdo que pareça, muitas mulheres submetem-se a serem humilhadas e agredidas por questão financeira). Por uso de entorpecentes- Sejam alcoólicos ou psicoativos, onde ambos utilizam juntos tais entorpecentes, e agridem-se mutualmente. Vinculo Afetivo (as mulheres alegaram amarem o companheiro do jeito que são, mesmo que isso lhe renda algumas "surras" semanais). Onde os motivos supracitados corroboram com o pensamento de Sousa et al. (2013, p. 426) “a primeira destas fases é formada por humilhação, intimidação, provocações mútuas, seguida pelo uso de estratégias de ameaças como a separação, o impedimento de participação na vida dos filhos, entre outras, finalizando o conflito em agressão física”. 4 É um quadro alarmante, pois muitos dessas situações supracitadas culminam num atendimento de local de morte. É um quadro alarmante, pois muitos dessas situações supracitadas culminam num atendimento de local de morte. CAUSAS E EFEITOS DA VIOLÊNCIA Mas por que ocorrem esses tipos de violência e quais efeitos causam nessas mulheres? Há vários tipos de causas que podem ser apurada na situação da violência doméstica, alternando do racismo, pela falta de aquisição financeira da vítima, xenofobia, até desigualdade de gênero em si, como afirma Blanch (2001, p. 7), essa violência ocorre em "um contexto de relações de poder, em uma determinada ordem social e cultural, sustentada por uma ideologia (pseudolegitimadora dessa ação)". Ou seja, pela supremacia masculina para com a feminina. As principais consequências sofridas pelas mulheres que passam por situação de violência estão os sentimentos de aniquilação, tristeza, desânimo, solidão, estresse, baixo autoestima, incapacidade, impotência, ódio e inutilidade. E entre as doenças, estão: obesidade, síndrome do pânico, gastrite, doenças inflamatórias e imunológicas, mutilações, fraturas e lesões. E por fim, mudanças comportamentais, como: insegurança no trabalho, dificuldade de relacionamento familiar, dificuldades sexuais e obstétricas, desenvolvimento do hábito de fumar, maior propensão a acidentes, vindo a entrar em concordância com o que diz Fonseca et al. (2012, p.308) “correlacionam à violência distúrbios gastrointestinais, lesões, doenças sexualmente transmissíveis, gravidez não desejada, sentimento de culpa, baixa autoestima, depressão, ansiedade, suicídios”. No Brasil, existe a LEI N° 11.340\06, de grande importância para os direitos das mulheres, principalmente as que estão sofrendo violências. Todos os atos criminosos que anteriormente a esta Lei era tratados como crime de menor potencial ofensivo, conforme a Lei 9.099, de 26 de setembro de 1995, onde o autor saia praticamente impune e as vítimas sofriam retaliação posteriormente as denúncias. E no dia 20 de setembro de 2006 em diante passou a ser inafiançável, sendo crime comum e apurado por meio de inquérito policial. Onde então foram criados diversos mecanismos a fim de prevenir tais violências e coibí-las. Citemos: A criação dos Juizados de Violência Doméstica e Familiar contra a Mulher; As integrações operacionais do Poder Judiciário, Ministério Público e da Defensoria Pública com as áreas da segurança pública; Maior rigor as infrações penais praticadas contra a mulher; Estabelece todas as diretrizes das políticas públicas que objetivam prevenir a violência doméstica e familiar; Criação dos juizados de violência doméstica e familiar. CAUSAS E EFEITOS DA VIOLÊNCIA No artigo 17 da Lei nº 11.340 de 07 de Agosto de 2006 reintera, “é vedada à aplicação, nos casos 5 de violência doméstica e familiar contra a mulher, de penas de cesta básica ou outras de prestação pecuniária, bem como a substituição de pena que implique o pagamento isolado de multa” (BRASIL, 2006). ÓRGÃOS DE SEGURANÇA PUBLICA ATUANTES NA QUESTÃO E os órgãos que acabam por realizar o cumprimento da legislação supracitada, são as Guardas Municipais: (a GM atendem situações envolvendo violências domésticas bem como outras demandas, sendo de grande ajuda na prevenção e prisão de ocorrências de natureza violenta ao gênero feminino); Polícias Civis (a elas incumbem encaminharem aos fóruns os pedidos de medidas protetivas de urgência, ofertar a guia de corpo de delito, dar continuidade no processo e procedimentos em parceria com o judiciário); Polícias Militares (é o órgão que fará o primeiro atendimento nos casas de violência doméstica e acaba por assumir tanto a competência de agir administrativamente, criminalmente quanta nos atos preparatórios para a condenação efetiva do agressor, assistencialmente, e posteriormente ao encaminhamento das partes, a fim de averiguar se as medidas protetivas estão sendo cumpridas). No início, foi compreendida a grande valia da conceituação do que é violência doméstica, seu entendimento legal, seu real significado, e quais são os tipos de violência que a mulher pode vir a sofrer. O policial irá prestar atendimento, tendo como base teórica o que realmente é a violência doméstica, e todas as suas variações de violência. Onde foram criados mecanismos para coibir a violência doméstica e familiar contra a mulher, nos termos da Lei nº 11.340/06 Lei Maria da Penha, teve por seu objetivo criar mecanismos jurídicos para coibir e prevenir a violência doméstica e familiar contra a mulher. Quando o procedimento a ser adotado pelo policial visa ser em um caso da Lei Maria da Penha há alguns dispositivos legais que norteiam a atuação policial, contudo sem distinguir qual dos órgãos de segurança pública policial será o responsável, porém conforme Anjos (2008, p.16) “a Polícia Militar, inserida neste contexto como agente encarregado de garantir a segurança pública, é a primeira instituição a ser solicitada nestes casos, e legalmente deve agir, principalmente procurando resguardar a integridade física das vítimas”. E nessas ocasiões de violência doméstica independe da representação da vítima, o Policial Militar prende em flagrante de delito o autor, em concordância com o que Lemos (2010, p.148) diz, “é permitido ao policial militar prender o agressor em flagrante sempre que houver qualquer das formas de violência doméstica e familiar contra a mulher”. ÓRGÃOS DE SEGURANÇA PUBLICA ATUANTES NA QUESTÃO 6 Normalmente a vítima almeja mais a solução para sua vida do que a punição do autor, e o policial investido da legalidade e representatividade do Estado na atuação de proteger e servir irá adotar procedimentos preventivos e assistencialistas de forma imediata para auxiliar a vítima, corroborando para o que diz Jesus (2010, p.71) “de acordo com o art. 10, caput, da Lei n. 11.340/2006, na hipótese da iminência ou da prática de violência doméstica ou familiar contra a mulher, a autoridade policial que tomar conhecimento da ocorrência adotará, de imediato, as providências legais cabíveis”. PROCEDIMENTOS POLICIAIS FRENTE ÀS SITUAÇÕES DE VIOLÊNCIAS Ao analisar da Lei nº 11.340/06, e no entendimento de vários autores observados à frente, afirmam que o procedimento policial nos casos de violência contra mulher e doméstica deve ser conforme preconiza os dispositivos desta Lei. Onde este agente de segurança pública irá adotar as medidas cabíveis, tanto pela questão de manutenção de ordem pública quanto pela proteção de vítima, jamais sendo negligente, como ignorar a versão da vítima ou não encaminhar o autor, ao contrário, deverá cumprir a Lei Maria da Penha na sua totalidade. Adotando os procedimentos previstos no Art.11 desta mesma Lei (Lei nº 11.340/06 Lei Maria da Penha) zelando pela lisura física das vítimas. Os policiais além de demonstrar empatia, respeito, cuidados necessários, irão atuar: Dando garantia de proteção à mulher, algo primordial no atendimento as essas vítimas principalmente de acordo com Lemos (2010, p.165) “os agentes policiais, como autores envolvidos no ciclo da violência, são os responsáveis por impedir ou reproduzir a perpetuação de violência”. Encaminhando a ofendida ao hospital, ou Instituto Médico Legal, inclusive para realização de exame de corpo de delito; priorizando sempre a integridade física da vítima antes de qualquer procedimento documental. Fornecendo transporte para abrigo seguro; longe do agressor, visando seu bem estar, evitando que tenha contato com quem lhe ofereceu o mau. Indiferente do local a ser encaminhada. Acompanhando a ofendida para assegurar a retirada de seus pertences do local da ocorrência ou do domicílio familiar, onde a vítima devido o anseio de fuga acaba por evadir- se do local, esquece-se de acionar o policial e também de pegar seus objetos pessoais, onde Cunha e Pinto (2008, p. 89) expõe que ”no calor da violência, a fuga da mulher, fisicamente mais frágil, é quase sempre a única alternativa que lhe resta, muitas vezes sem que tenha, inclusive, oportunidade para retirada de seus objetos pessoais”. 7 Lavrando o boletim de ocorrência; a função básica de qualquer ocorrência policial, mas muito importante, onde será neste documento que será descrito a ocorrência em si, bem como utilizado de base para demais procedimentos judiciário. Tomando a representação da ofendida, colhendo as provas, ouvindo o agressor e a vítima. Informando à ofendida dos direitos a ela conferida pela Lei Maria da Penha, o policial terá que usar todo seu conhecimento legal, da lei em questão, e entendimento dos procedimentos que deve tomar nessas situações para orientar de melhor maneira possível à vítima. PROCEDIMENTOS POLICIAIS FRENTE ÀS SITUAÇÕES DE VIOLÊNCIAS Mostrando a real necessidade e valia de estudos e pesquisas voltadas para este tema, devido o entendimento da violência doméstica e a atuação policial eficiente no cumprimento da lei supracitada. Encaminhamento dos autores em situações de descumprimento de medidas protetivas e afastamento do lar (sendo triviais tais procedimentos que garanta o afastamento do agressor da vítima, trazendo maior segurança para o seio familiar), confirmado por Dias (2008, p. 85) quando diz que a “proibição de contato, ao impedir a interação do agressor com a ofendida, seus parentes e testemunhas, por quaisquer meios de comunicação, mostra-se como uma restrição extremamente fundamental e benéfica, pois gera a paz e tranquilidade mental da vítima”. Contudo em situações que o ex-casal possua filhos, a medida deve ser feita de forma temporária para que os filhos não sofram com a ausência paterna, como é dito por Dias (2015, p. 149) “para que os filhos não percam a referência paterna, a medida deve ser temporária, perdurando apenas enquanto houver ameaça de reiteração dos atos de violência”. Mostrando e encaminhando até a casa de abrigo para mulheres; indicando para a agredida os meios e locais que esta possa a vir ficar caso haja necessidade; E conforme a Secretaria Especial de Políticas para as Mulheres através da cartilha Enfrentando a Violência contra a Mulher - Orientações Práticas para Profissionais e Voluntários (as) Soares (2005, pg.45) diz que os policiais e ou agentes de segurança não devem Paternalizar; Infantilizar a situação; Culpabilizar a vítima; Ser incapaz de escutar, ignorar a vítima; Generalizar histórias individuais, Agir sem parcialidade; Envolver-se ou Distanciar-se em excesso com situação. REFERÊNCIAS ANJOS, Rosângela Rita Alves Fernandes dos. A Polícia Militar e a Lei Maria da Penha: reflexões necessárias sobre sua atuação a partir do 3º CPA/NORTE. Cuiabá: UFMT, 2008. BLANCH, J. M. Violencia social e interpersonal. "Dossier de Lecturas" Del Máster Interdisciplinar de Estúdio e Intervención em Violencia Domestica. Barcelona: Universidad Autónoma de Barcelona, 2001. BRASIL. Constituição da Republica Federativa do Brasil. Promulgada em 5 de outubro de 1988, 2019. CONCLUSÃO O presente artigo proporciona o melhor entendimento sobre a conceituação da violência contra mulher, que quando passa a ser em âmbito familiar (espaço restrito) tem o entendimento de violência doméstica, e que essa violência pode ser divididas em 5 formas: 8 Violência física, psicológica, sexual, patrimonial e moral, ou seja, não caracterizando somente violência quando a mulher apresenta hematomas. A importância deste artigo está ligada também na diminuição de dúvidas que pairam quanto às motivações de muitas mulheres aceitarem continuar vivendo nesses cenários violentos, onde teve grande surpresa quando visto que as mulheres alegaram amarem o companheiro do jeito que são, mesmo que isso lhe renda algumas "surras" semanais, ficando nestas condições violentas por vínculos afetivos. Ao entender as causas dos homens as tratarem desta maneira, e como isso ira afetar estas mulheres fisicamente e mentalmente. Impactando ao descobrir que como consequência, estas mulheres podem cometer uma brutalidade contra sua própria vida, o suícidio. Foi permitido conhecer como os policiais atuam nessas questões, usando os aspectos legais da Lei nº 11.340/2006-Lei Maria da Penha, resultando numa melhor atuação policial e demais órgãos, buscando contribuir na diminuição das ditas violências prezando pela preservação da integridade física, mostrando que devem ser encaminhados os autores da agressão e nos descumprimentos de medidas protetivas, contudo em situações que o ex-casal possua filhos, a medida deve ser feita de forma temporária para que os filhos não sofram com a ausência paterna. Informado também às formas de agir e não agir com a vítima, tendo destaque para a não generalização de situações individuais, não culpabilizando a vítima pela agressão sofrida, e que a atuação policial deve ser pautada na imparcialidade. Onde então, de acordo com o que foi examinado, observou-se que é muito importante conhecer o que realmente e a violência doméstica, as sua definições e subdivisões de violência, as motivações, causas e efeitos que essa violência provocam, o entendimento legal da Lei Maria da Penha, e principalmente a atuação policial, pautadas nesta legislação, zelando pela lisura física das vítimas, demonstrando que a atuação polícia deve agir com empatia, respeito, cuidados necessários. Diante do exposto, este artigo pode vir a ser usado como material de nivelamento, formação, especialização, instrução, e definição do “Modus Operandi” da equipe policial no atendimento de situações envolvendo violência doméstica. 9 9 BRASIL. Constituição da Republica Federativa do Brasil. Promulgada em 5 de outubro de 1988, 2019. BRASIL. Lei Maria da Penha. Lei n. 11.340/2006. Coíbe a violência doméstica e familiar contra a mulher. Brasília: Secretaria Especial de Políticas para Mulheres / P residência da Republica, 2006b. CUNHA, Rogério Sanches; PINTO, Ronaldo Batista. Violência doméstica: Lei Maria da Penha (11.340/2006), comentada artigo por artigo. 2 ed., rev., atual. E ampl. - São Paulo: Editora Revista dos Tribunais, 2008. DIAS, Maria Berenice. A Lei Maria da Penha na justiça: a efetividade da Lei 11.340/2006 de combate a violência doméstica e familiar contra a mulher. São Paulo: Editora Revista dos Tribunais, 2007. ______. Lei Maria da Penha na Justiça: A Efetividade da Lei 11.340/2006 de combate à Violência Doméstica e Familiar Contra a Mulher.2. Ed. –São Paulo: Editora Revista dos Tribunais, 2008. ______. Lei Maria da Penha: A Efetividade da Lei 11.340/2006 de combate à Violência Doméstica e Familiar Contra a Mulher.4. Ed. rev., atual. e ampl. –São Paulo: Editora Revista dos Tribunais, 2015. FONSECA, Denire Holanda da; RIBEIRO, Cristiane Galvão; LEAL, Noêmia Soares Barbosa; Violência doméstica contra mulher: Realidades e representações sociais. Rev.Psicologia e sociedade. Belo Horizonte/MG, 2012. Disponível em: http://www.scielo.br/pdf/psoc/v24n2/07.pdf acessado em 30 de abril de 2021. IPEA, Instituto de Pesquisa Econômica Aplicada em Retratos da Desigualdade de gênero e raça, Retrato das Desigualdades de Gênero e Raça, 2001. Disponível em: https://www.ipea.gov.br/retrato/pdf/revista.pdf acessado em 30 de abril de 2021. JESUS, Damásio de. Violência contra a mulher: aspectos criminais da Lei n. 11.340/2006. São Paulo: Saraiva, 2010. LEMOS, Marilda de Oliveira. Um estudo sobre a interpretação e aplicação da Lei Maria da Penha nas delegacias de defesa da mulher e distritos policiais da seccional de polícia de Santo André – São Paulo. 2010. 307 f. Tese (Doutorado em Sociologia) – Faculdade de Filosofia, Letras e Ciências Humanas, Universidade de São Paulo, São Paulo, 2010. 10 SENASP, Secretaria Nacional de Segurança Publica. Atendimento policial a vitimas de violência domestica Apostila de curso 2007. SENASP, Secretaria Nacional de Segurança Publica. Atendimento policial a vitimas de violência domestica Apostila de curso 2007. SOARES, Bárbara M. Cartilha ENFRENTANDO A VIOLÊNCIA CONTRA A MULHER, Secretaria Especial de Políticas para as Mulheres, Brasília 2005, Disponível em: http://www12.senado.leg.br/institucional/omv/entenda-a-violencia/pdfs/enfrentando-a- violencia-contra-a-mulher-orientacoes-praticas-para-profissionais-e-voluntarios acessado em 15 de abril de 2021. SOUSA, Ane karine Alkmim de; NOGUEIRA, Denismar Alves; GRADIM, Clicia Valim Cortês; Perfil da violência doméstica e familiar contra mulher em um município de Minas Gerais, Brasil. Cad. Rev.Saúde. Rio de Janeiro/RJ. 2013. BRASIL. Constituição da Republica Federativa do Brasil. Promulgada em 5 de outubro de 1988, 2019. Disponível em: http://www.scielo.br/pdf/cadsc/v21n4/v21n4a11.pdf acessado em 30 de abril de 2021.
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A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro”
Diálogo com a Economia Criativa
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RESUMO Neste fragmento de nossa pesquisa de mestrado, discutimos de que forma a memória discursiva é mobilizada em discursos midiáticos sobre beleza e saúde. Para tanto, analisamos textos verbais e não verbais do discurso da campanha “Silicone Seguro”, da Johnson & Johnson, a partir dos di­ tos e não ditos que convocam o sujeito neoliberal feminino ideal (DARDOT E LAVAL, 2016; ELIAS, GILL, SCHARFF, 2017; PRADO, 2013). Defendemos que o labor estético exigido às mulheres para serem consideradas bem-sucedidas na contemporaneidade é ressignificado em “Silicone Seguro” como empoderamento feminino, por meio de uma estratégia discursiva que mobiliza afetos po­ sitivos e aspectos dos feminismos para inculcar a mentalidade neoliberal do empreendedorismo de si, utilizando a força e a potência de agência das mulheres como catalisadores de individua­ lismo e desempenho (ELIAS, GILL, SCHARFF, 2017). Neste discurso, a mulher é posicionada como empoderada à medida que se conforma com antigas normas de feminilidade e de conduta, ainda presentes na memória discursiva de uma sociedade considerada machista. Palavras-chave: comunicação e consumo; memória discursiva; sujeito neoliberal feminin 1 Mestre pelo Programa de Pós-graduação em Comunicação e Práticas do Consumo – PPGCOM ESPM (bolsista CAPES, modalidade taxa), publicitária graduada pela ESPM-SP e integrante do Grupo de Pesquisa BIOCON – Comunicação, Discursos e Biopolíticas do Consumo, certificado pelo CNPq. E-mail: sabi­ na.lovato@gmail.com. 1 INTRODUÇÃO N o Brasil, um país em que a beleza corporal guarda a promessa de um futuro melhor, as cirurgias plásticas são legitimadas pela medicina e divulgadas pe­ los dispositivos midiáticos como procedimentos seguros cujos benefícios vão desde o aumento da autoestima até a mobilidade social (JARRÍN, 2017; SANT’ANNA, 2014). São mais de 1.400.000 de cirurgias estéticas ao ano, o que coloca o Brasil como segundo lugar no ranking mundial - atrás dos Estados Unidos -, sendo a de implantes mamários a mais realizada, com mais de 230.000 procedimentos (ISAPS, 2017). Desde a popularização da prática nos anos 1960, impulsionada pela figura emblemática do cirurgião carioca Ivo Pitanguy, que oferecia o «direito à beleza» àqueles que até então não podiam arcar com os custos dos procedimentos estéticos, os discursos sobre beleza e saúde em nossa cultura contemporânea são imbricados de afeto e racionalidade e constituem uma rica memória social por conta das narra­ tivas midiáticas em que se fazem presentes. N DOSSIÊ Dito isto, em nossa pesquisa de mestrado, analisamos as interações discursivas em discursos midiáticos sobre beleza e saúde, com foco nas cirurgias plásticas de im­ plantes de mama no contexto brasileiro. Para este artigo, retomamos parte de nossa pesquisa com o objetivo de compreender de que forma o conceito de memória dis­ cursiva é mobilizado para convocar o sujeito neoliberal feminino em textos verbais e não verbais da campanha Silicone Seguro, da Johnson & Johnson. Com o aporte teórico-metodológico da Análise do Discurso Francesa (ADF), tratamos do texto em seu processo discursivo mais amplo por conta da noção de interdiscurso, que remete a algo que já foi dito na ordem discursiva, viabilizando a leitura do enunciado. Segundo Orlandi (2015, p. 29), “é o que chamamos de me­ mória discursiva: o saber discursivo que torna possível todo dizer e que retorna sob a forma do pré-construído, o já-dito que está na base do dizível, sustentando cada tomada da palavra”. Um determinado discurso é sustentado por outros, pelo que já foi dito, e seu sentido acontece não por causa das palavras por si próprias, mas pelo que estas remetem à sua interpretação. Nenhum discurso significa sem que haja sentidos anteriores que irão constituir os novos significados e, conforme a conjuntu­ ra sócio histórica se transforma, os discursos circulantes acompanham-na. ABSTRACT h f In this fragment of our Master’s research, we discuss how the concept of discursive memory happens in me­ dia discourses about beauty and health. To do this, we analyse verbal and nonverbal texts of the discourse in the “Safe Silicone” campaign by Johnson & Johnson considering the sayings and nonsayings that convoke the ideal feminine neoliberal subject (DARDOT AND LAVAL, 2016; ELIAS, GILL, SCHARFF, 2017; PRADO, 2013). We defend that the demand for women to perform aesthetic labour in order to be considered suc­ cessful in our society today is ressignified in “Safe Silicone” as feminine empowerment through a discursive strategy that mobilize positive affects and feminisms aspects to inculcate the neoliberal mentality of self entrepreneurship, using the women’s strength and power of agency as catalysts for individualism and per­ formance. In this discourse, women are positioned as empowered as long as they compromise with old rules of femininity and conduct still present in the discursive memory of a society that is considered misogynist. Keywords: communications and consumption; discursive memory; neoliberal feminine subject. 1 Mestre pelo Programa de Pós-graduação em Comunicação e Práticas do Consumo – PPGCOM ESPM (bolsista CAPES, modalidade taxa), publicitária graduada pela ESPM-SP e integrante do Grupo de Pesquisa BIOCON – Comunicação, Discursos e Biopolíticas do Consumo, certificado pelo CNPq. E-mail: sabi­ na.lovato@gmail.com. 72 Diálogo com a Economia Criativa ESPM Rio Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 1 INTRODUÇÃO O discurso publicitário é um campo fértil para analisarmos o funcionamento da memória discursiva, pois incorpora os valores sociais da época e se reestrutura para se adequar a eles. Sabemos que a mídia tem função pedagógica, pois ensina valores e modos de ser, e hoje é considerada a instituição mais relevante neste as­ pecto (PRADO, 2013; SILVERSTONE, 2014; SODRÉ, 2013). A publicidade, assim como outras narrativas do consumo, a partir do repetível no enunciado e com dizeres que se renovam e se atualizam ao longo do tempo, constrói os mundos possíveis das mar­ cas, representações ideais da realidade, acessíveis por meio do consumo (SEMPRINI, 2006). Ao identificarmos os interdiscursos nos enunciados, podemos perceber de que forma discursos prejudiciais à emancipação feminina mantêm-se recorrentes na sociedade, ainda que sob outras formas de dizer. 73 Sabina Lovato Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Elias, Gill e Scharff (2017) afirmam que as mulheres, principalmente as jovens, são frequentemente posicionadas no discurso como sujeitos neoliberais ideais por sua suposta força de agência e transformação social e, também, pela capacidade de maximizar as novas oportunidades que lhes aparecem. Este sujeito neoliberal, segundo Dardot e Laval (2016), é produzido nos discursos circulantes da gestão neo­ liberal que hoje governa o mundo, uma racionalidade econômica e política que tem o princípio da concorrência como norma de conduta. A lógica de mercado perpassa relações sociais e os modos de ser e estar no mundo globalizado, excedendo a es­ fera dos negócios. O sujeito deste discurso é convocado a se comportar com uma empresa, tornando-se o empreendedor de si, responsável por sua própria vigilância e desenvolvimento. DOSSIÊ De acordo com Scharff (2015), o sujeito neoliberal se caracteriza por sua com­ petência em consumir, o que privilegia ainda mais o feminino devido à histórica associação entre mulher e consumo, intensificada pelas narrativas midiáticas. A in­ citação à transformação de si também é um ponto relevante, à medida que estas convocações são mais frequentemente direcionadas às mulheres, principalmente quando se trata de controle do corpo e sexualidade. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 1 INTRODUÇÃO Segundo a autora, a ligação entre feminilidade, transformação de si e corpo é chave de entendimento da inter­ -relação entre gênero e subjetividade na era neoliberal. Dito isto, conforme as mulheres alcançam posições mais relevantes na esfera pública e passam a contestar nas mídias sociais as representações midiáticas que ob­ jetificam e hipersexualizam o gênero feminino, o mercado responde a estas trans­ formações. Elias, Gill e Scharff (2017) explicam que, a partir dos anos 2010, começou a ocorrer uma mudança de paradigma na publicidade voltada ao público feminino, impulsionada pelos feminismos de terceira e quarta onda, este com alta mobiliza­ ção nos meios digitais. Grandes marcas internacionais como Dove, Always, Mattel e, no Brasil, Avon, C&A e Natura deixaram de lado o discurso focado nas imperfeições da mulher para as convocarem a se amarem e a se sentirem lindas como são. A este tipo de discurso as autoras chamam de “love your body” (LYB), ou “ame seu corpo” e, embora possam ser percebidos como um avanço em um primeiro momento, para as autoras, são também uma nova forma de regulação e controle. Estes discursos LYB estão ligados à noção de pós-feminismo, entendida como “neoliberalismo sob uma visão de gênero” por Elias, Gill e Scharff (2017). O pós-fe­ minismo é uma sensibilidade que percorre grande parte da cultura contemporânea, sendo, ao mesmo tempo, discursiva, ideológica, afetiva e psicossocial. A ressonância com o neoliberalismo opera em três níveis: pelo individualismo que ignora pressões externas e toma o lugar de noções políticas e sociais; a relação do sujeito neoliberal com o sujeito pós-feminista, ambos ativos, de livre escolha e que estão sempre se reinventando; e os discursos de autogestão e disciplina que, historicamente, são mais direcionados às mulheres para governarem todos os aspectos de suas vidas. Isto nos leva à dimensão psíquica do regime de beleza contemporâneo que vai além da aparência física. Hoje, não basta ser bonita, é preciso sentir-se bonita. A es­ sência dos discursos LYB está em produzir afetos positivos e afirmativos para inculcar 74 ESPM Rio ESPM Rio Diálogo com a Economia Criativa a lógica da autorregulação, partindo da dimensão física do corpo para a psíquica, mantendo seus objetivos de mercado (ELIAS; GILL; SCHARFF, 2017). Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 1 INTRODUÇÃO O uso da palavra “autocuidado” e suas variantes na comunicação de marcas do segmento da beleza é um exemplo de como estas práticas de consumo vêm sendo ressignificadas, passan­ do de uma necessidade imposta por pressões sociais para rituais quase terapêuticos de amor próprio (Figura 1), ainda que mais extensos e complicados do que nunca. A convocação ao consumo é a mesma, sendo os resultados o labor estético e a trans­ formação de si - o que muda é apenas a maneira de dizê-lo. Figura 1: Campanha da marca de cosméticos The Body Shop, veiculada no Instagram com influencia­ doras digitais (na imagem, Nátaly Neri) para divulgar nova linha de hidratantes para o corpo. Fonte: Instagram Stories patrocinado, capturado pela autora. DOSSIÊ Figura 1: Campanha da marca de cosméticos The Body Shop, veiculada no Instagram com influencia­ doras digitais (na imagem, Nátaly Neri) para divulgar nova linha de hidratantes para o corpo. Fonte: Instagram Stories patrocinado, capturado pela autora. A campanha Silicone Seguro segue nesta direção, mobilizando afetos positi­ vos e otimistas para convocar a consumidora a se amar, a valorizar seu corpo, a se libertar, tudo isto por meio da cirurgia de implantes de mama. A mulher não mais se submete ao implante porque odeia seu corpo, pelo contrário: ela se ama tan­ to que os implantes são uma forma de demonstrar esse amor. Diferente de anún­ cios publicitários de outras marcas de próteses de silicone que pesquisamos, como Silimed, Eurosilicone e Allergan, nos quais as imagens são visivelmente retocadas digitalmente e mostram repetidamente o corpo modelado artificialmente, exibido por modelos vestindo biquíni, o discurso de Silicone Seguro apresenta modelos em poses casuais, com pouca exposição do corpo, como vemos em nossa análise. Esta interrupção na regularidade do discurso asséptico e racionalista da indústria far­ macêutica indica a extensão da sensibilidade pós-feminista que posiciona a mulher como este sujeito neoliberal ideal. A Mentor, fabricante de implantes mamários subsidiada da Johnson & Johnson, é uma das líderes de mercado no Brasil. Foi fundada em 1969 nos Estados Unidos 75 Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” e, segundo seu site oficial, “é a principal fornecedora de produtos médicos para o mercado global de medicina estética”. Em uma estratégia de relações públicas, a Johnson & Johnson Medical Devices lança a campanha “Silicone Seguro”, em 2016, veiculada online. 1 INTRODUÇÃO Segundo o manifesto publicado no site oficial da mesma: Neste site, você encontra informações valiosas sobre próteses de silicone e se informa sobre aspectos de extrema importância, da segurança relacio­ nada à cirurgia e aos produtos utilizados até a qualidade dos diferentes tipos de implantes disponíveis hoje em dia. Definimos a campanha Silicone Seguro como objeto empírico pelo seu foco na paciente-consumidora final e também por se tratar de uma iniciativa direcionada a segurança dos implantes, a única dentre as marcas líderes de mercado no Brasil. Por trazer em seu bojo a dinâmica discursiva entre consumo, beleza e saúde, considera­ mos a campanha um exemplo exemplar do ambiente mercadológico dos implantes, tendo em vista a construção do sujeito neoliberal feminino. DOSSIÊ Neste artigo, apresentamos parte de nossa análise dos ditos e não ditos que convocam o sujeito neoliberal feminino na campanha Silicone Seguro para ilustrar­ mos o conceito de memória discursiva em ação neste discurso. Primeiro, coletamos o material no site oficial da campanha e em sua página no Facebook, no período de 31 de agosto de 2017 a 23 de novembro de 2018, totalizando 70 postagens. Em seguida, mapeamos este material, separando por temáticas, para então defi­ nirmos as categorias de análise. Estas, por sua vez, foram inspiradas em caracte­ rísticas do pós-feminismo elencadas por Elias, Gill e Scharff (2017) sendo que, das nove categorias definidas, abordamos neste artigo quatro delas, ainda que em parte: Empreendedorismo Empoderador; Novas Feminilidades; Palavras Desviadas, Informações Omitidas; e Ausência da Figura Masculina. 2 “I apply a feminist discursive Foucauldian-influenced approach, a key concept being that of‘governmentality’, namely the contact between the (objectifying) technologies of domination of others and the (subjectifying) technologies of the self” (FAVARO, 2017, p. 284). Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 2 O EMPREENDEDORISMO EMPODERADOR N a campanha “Silicone Seguro”, em consonância com a sensibilidade pós-fe­ minista e seus discursos, as modelos estão posicionadas como agentes de si mesmas e não tanto como objetos à mercê da vontade do outro. Este movi­ mento, no entanto, não constitui uma libertação, mas um novo modo de controle baseado no tipo de poder que Foucault denomina como “governamentalidade”. Isto é, “o contato entre as tecnologias de dominação do outro (objetificantes) e as tecnologias do self (subjetificantes)” (FOUCAULT, 1998 apud FAVARO, 2017, p. 284, tradução nossa)2. Em outras palavras, o controle do corpo é exercido pelo próprio sujeito e não mais por instituições disciplinares. N Nesta construção discursiva, o desejo pela transformação de si não é conse­ quência de estruturas sociais que medeiam relações de ser e estar no mundo, mas partem apenas do indivíduo isolado. Além disto, a força de agência conquistada por décadas de feminismo é canalizada para o benefício individual e não para engen­ drar lutas pela igualdade de gênero. As mulheres empoderadas neste cenário são 76 Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. ESPM Rio Diálogo com a Economia Criativa as que empreendem a gestão de si, no caso, as que tomam a atitude de realizar a cirurgia de implantes. as que empreendem a gestão de si, no caso, as que tomam a atitude de realizar cirurgia de implantes. Figura 2: Post “Meu peito, minhas regras Texto do post: “E tenho dito!” Fonte: SILICONE Seguro. 29 ago. 2018. Facebook: Siliconeseguro. Disponível em tinyurl.com/y6ftv4ok. Acesso em: 15 fev. Figura 2: Post “Meu peito, minhas regras!” Texto do post: “E tenho dito!” Fonte: SILICONE Seguro. 29 ago. 2018. Facebook: Siliconeseguro. Disponível em: http:// tinyurl.com/y6ftv4ok. Acesso em: 15 fev. 2019. Figura 2: Post “Meu peito, minhas regras!” Texto do post: “E tenho dito!” Fonte: SILICONE Seguro. 29 ago. 2018. Facebook: Siliconeseguro. Disponível em: http:// tinyurl.com/y6ftv4ok. Acesso em: 15 fev. 2019. DOSSIÊ McRobbie (2015) atesta o movimento do capitalismo em cooptar partes do fe­ minismo em seu próprio discurso, como observamos na Figura 2. A pose da modelo, flexionando os músculos dos braços, em uma relação de interdiscurso faz alusão à luta feminista, ainda que amenizada pela estética heteronormativa de feminilidade, composta pelos óculos escuros, os cabelos longos e ondulados, a maquiagem forte e os lábios franzidos. 3 Série de protestos que fazem parte do movimento internacional denominado Slut Walk, original de Toronto no Canadá, quando um oficial segurança, em palestra na Universidade de Toronto, orientou que as mulheres “não se vestissem como vadias” para evitarem ser estupradas. A fala causou revolta nas mulheres e mais de 3 mil marcharam em protesto nas ruas de Toronto. No Brasil, a Marcha representa, além do repúdio a culpabilização das vítimas de estupro, a luta geral pelo fim da violência doméstica, física, simbólica e sexual. (BOGADO, 2018) 2 O EMPREENDEDORISMO EMPODERADOR O título “Meu peito, minhas regras” mobiliza a memória discursiva do públi­ co feminino pelo jogo de palavras com o lema feminista “My body, my rules”, ou “Meu corpo, minhas regras”. Como explica Dosekun (2017), o pós-feminismo, como objeto intensamente midiatizado e imbricado de significados consumistas, percorre a mídia transnacional e as relações de consumo. Ao transpor este imaginário para a construção de seu mundo possível, a Johnson & Johnson sugere que as mulheres que optam pelos implantes de silicone podem sofrer alguma espécie de opressão por conta de sua escolha, ou seja, colocar próteses de silicone se torna um ato de resistência e coragem. A perversidade desta apropriação e esvaziamento de signifi­ cado fica ainda mais aparente quando lembramos que um dos símbolos da Marcha das Vadias3, uma das manifestações responsáveis pela popularização do lema, é, justamente, os seios desnudos simbolizando a reivindicação feminina do próprio corpo (Figura 3). 77 Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Figura 3: Militante da Marcha das Vadias. Fonte: ANGELO, Mario. 2015. Disponível em: https://tinyurl.com/y2o46saf. Acesso em: 23 mai. 2019. A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Sabina Lovato Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Figura 3: Militante da Marcha das Vadias. Fonte: ANGELO, Mario. 2015. Disponível em: https://tinyurl.com/y2o46saf. Acesso em: 23 mai. 2019. Figura 3: Militante da Marcha das Vadias. Fonte: ANGELO, Mario. 2015. Disponível em: https://tinyurl.com/y2o46saf. Acesso em: 23 mai. 2019. DOSSIÊ Para Dosekun (2017), as mulheres, ao exercerem a subjetividade neoliberal empreendedora que determina a beleza como o maior investimento em capital hu­ mano que pode ser feito, estão constantemente gerenciando os riscos que estas práticas oferecem. Por exemplo, a saúde das unhas fica debilitada com o uso cons­ tante de unhas acrílicas; pode ocorrer a queda de cabelo com o atrito e pressão dos apliques de cabelo; ao retirar os cílios postiços, os verdadeiros podem ser involunta­ riamente arrancados no processo. Estas micropráticas de autogoverno interconectam-se com a gestão bio­ política de mulheres via tecnologias de poder/saber cada vez mais centra­ 4 “These micro-practices of self-government interconnect with the biopolitical management of women via apparatuses of power/knowledge increasingly centred on inserting positive affect within infrastructures of measurement, discipline and exploitation.” (FAVARO, 2017, p. 297) 5 “Easy, enjoyable girlish fun.” (LAZAR, 2017, p. 52). 2 O EMPREENDEDORISMO EMPODERADOR Segundo a autora, estas práticas se apresentam às mulheres como obrigatórias para serem percebidas como femininas no ambiente de trabalho, naturalizando a conformidade à norma e, portanto, constituindo uma forma de governamentalidade que esvazia a potência do questionamento crítico e da resistência. Do mesmo modo, o discurso de “Silicone Seguro”, ao trazer a opção das próteses, naturaliza tanto os implantes quanto a insatisfação com o próprio cor­ po, enquanto a saúde fica em segundo plano. A diferença crucial aqui é que, dife­ rentemente de unhas, cílios e cabelo falsos, não existe a opção de retirar as próteses tão facilmente quando estas passam a causar incômodo. As práticas de embelezamento antes consideradas extremas, como as cirurgias plásticas, são agora ressignificadas, com o auxílio de campanhas como a “Silicone Seguro”, como práticas simples de autogoverno. Posicionando as mulheres como agentes transformadoras de si mesmas, estes discursos naturalizam práticas e en­ gendram na população as tecnologias do self. Segundo Favaro (2017, p. 297, tradu­ ção nossa): Estas micropráticas de autogoverno interconectam-se com a gestão bio­ política de mulheres via tecnologias de poder/saber cada vez mais centra­ 78 Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Diálogo com a Economia Criativa ESPM Rio das na inserção de afetos positivos nas infraestruturas de controle, disci­ plina e exploração.4 A campanha “Silicone Seguro” convoca o sujeito a empreender labor estético ao ressignificá-lo como empoderamento. O “direito à beleza” é, neste discurso, um direito “feminista”. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 3 NOVAS FEMINILIDADES As novas feminilidades compreendidas por Gill e Scharff (2011) referem-se às subjetividades que emergem no contexto pós-feminista e neoliberal. São novas for­ mas hegemônicas de ser e estar no mundo enquanto mulher que, no caso, referem­ -se à subjetividade empreendedora. Embora haja a presença de corpos diferentes (HOFF, 2016) nos discursos midiáticos pós-feministas, o ideal de beleza do sujeito neoliberal é heteronormativo, ou seja, hiperfeminino e, por sua vez, exige labor estético, ressignificado na campanha como divertimento de menina. DOSSIÊ Lazar (2017) aponta que o esperado para as mulheres performarem sua femi­ nilidade heterossexual é de que empreendam extenso labor estético voltado aos seus corpos, envolvendo gastos de tempo e dinheiro, aprendizagem de habilidades práticas (maquiagem, cuidados com o cabelo, técnicas de depilação etc.), desconfor­ to físico até riscos à saúde. Este labor não é um fenômeno recente em si, mas a au­ tora defende que, no pós-feminismo, ele se intensifica devido à maior abrangência do escopo e a escala. Neste sentido, o sujeito neoliberal feminino não é posicionado neste discurso como vítima, mas como participante ativo, que enxerga as práticas de beleza como um empreendimento que lhe renderá frutos. Para que esta modalização seja alcançada, o discurso midiático reformula as práticas de beleza como “fácil, prazeroso divertimento feminino” (LAZAR, 2017, p. 52)5. Mas este divertimento feminino, adverte Lazar (2017), no pós-feminismo não se coloca como ingênuo, passivo e inexperiente. De fato, é uma nova proposta de feminilidade que apresenta uma subjetividade ativa, dotada de conhecimento e expertise e que visa a um propósito até nas horas de lazer. A subjetividade feminina é apresentada neste discurso como um direito da mulher ao prazer, contrapondo-se ao feminismo tradicional que “reprime” as mulheres de se divertirem. Quando não utiliza modelos, a marca faz uso de elementos infantis na compo­ sição das peças, como o sorriso, a tipografia estilizada e o uso de cores de tons aber­ tos e saturados (Figura 4 e Figura 5). Na Figura 5, ainda temos o lembrete “Valorize seu corpo” como convocação à vigilância e trabalho constante que se impõe sobre a mulher. Valorizar o corpo, na memória discursiva pós-feminista, é não “descuidar”. 79 Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 3 NOVAS FEMINILIDADES Sabina Lovato Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Figura 5: Cartaz “Valorize o seu corpo” Fonte: SILICONE Seguro. Disponível em: https:// www.siliconeseguro.com. Acesso em: 13 fev. 2019. Figura 4: Cartaz “Porque beleza é...” Fonte: SILICONE Seguro. Disponível em: https:// www.siliconeseguro.com. Acesso em: 13 fev. 2019. DOSSIÊ Figura 4: Cartaz “Porque beleza é...” Fonte: SILICONE Seguro. Disponível em: https:// www.siliconeseguro.com. Acesso em: 13 fev. 2019. Figura 4: Cartaz “Porque beleza é...” Figura 5: Cartaz “Valorize o seu corpo” Fonte: SILICONE Seguro. Disponível em: https:// www.siliconeseguro.com. Acesso em: 13 fev. 2019. A linguagem utilizada na campanha remete à da publicidade de cosméticos e maquiagem, nivelando a cirurgia de implantes de mama com práticas de beleza me­ nos radicais. Tudo isto corrobora para o discurso de que os implantes são seguros, basta uma cirurgia simples para uma vida mais colorida e alegre. Os enunciados também convidam as consumidoras a interagirem com suas ami­ gas, marcando-as nos comentários do post, como o exemplo da Figura 6. Segundo Lazar (2017), esta estratégia discursiva objetiva levar à percepção de que as práticas de labor estético são divertidas, algo que as mulheres fazem para elas mesmas e que lhes dá prazer. Ademais, o uso de ironia e ambiguidade atesta a um pós-feminismo que não se leva muito a sério e se apresenta como leve. Nos posts contemplados nesta categoria, não há menção ao empoderamento nem à aceitação do corpo; é como se fosse uma pausa do trabalho “chato” de ser feminista, em que as mulheres podem fofocar ao telefone, fazer gracinhas, aproveitar a vida, sem ter que pensar em questões “sérias”. Ainda sobre o post representado na Figura 6, o enunciatário assume a pri­ meira pessoa (“Um beijo para minhas amigas do peito”), representando a “amiga”, papel que remete à memória de revistas voltadas ao público feminino adolescente, notáveis em exercer a pedagogia de gênero. Tanto a linguagem verbal quanto a visual, com o uso de uma ilustração e as cores lilás e cor-de-rosa contribuem para infantilizar o gênero feminino e ressignificar os implantes como diversão “de meni­ na”. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 3 NOVAS FEMINILIDADES Na Figura 7, a imagem da modelo falando ao telefone e o tom humorístico do 80 Diálogo com a Economia Criativa ESPM Rio ESPM Rio texto verbal nos remete aos tempos das revistas adolescentes. Hoje, no entanto, não se fala de ídolos da telinha, mas sobre implantes de silicone. texto verbal nos remete aos tempos das re se fala de ídolos da telinha, mas sobre im Ao infantilizarem a linguag nais de uma sociedade patriarcal, nos q Figura 6: Post “Um beijo para minhas amigas do peito” Texto do post: “Quem por aqui teve a sorte de passar o final de semana com as amigas do coração? ❤ Marque a sua melhor amiga!” Texto do post: DOSSIÊ Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: http://tinyurl. com/yxs3pd4o.Acesso em:15 fev. 2019. Texto do post: “Se você não tem, cuidado, essa amiga pode ser você! ;)” Texto do post: “Se você não tem, cuidado, essa amiga pode ser você! ;)” Texto do post: “Se você não tem, cuidado, essa amiga pode ser você! ;)” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: http://tinyurl. com/y5cwu378 Acesso em: 15 fev. 2019. Ao infantilizarem a linguagem, reafirmam papéis de gênero tradicio­ nais de uma sociedade patriarcal, nos quais se considera a mulher ser ingênuo e infantil e, logo, carente de proteção e inábil para certas funções. Isto corrobora o que Elias, Gill e Scharff (2017) atestam como o retorno das noções de “diferenças se­ xuais naturais”, uma das características do pós-feminismo. A estratégia de “Silicone Seguro” caracteriza a mulher como infantilizada, assim como outras marcas do seg­ mento da beleza que tratam mulheres como crianças ao lançarem coleções de ma­ quiagens temáticas com personagens da Disney (MAC), sereias (Anna Sui e Tarte) ou unicórnios (Too Faced e Tarte), ou ainda, quando utilizam linguagem informal exagerada em suas campanhas e embalagens, com muitos pontos de exclamações, gírias e expressões idiomáticas. 81 Sabina Lovato Sabina Lovato Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Ademais, ao repetir continuamente estas imagens infantilizadas, a campanha neutraliza a potência transformadora da mulher lembrando-as de como é divertido conversar com as amigas e imaginar seus seios “turbinados”. As novas feminilidades constituem, de fato, um retorno a uma representação de mulher romântica, cujo círculo social se restringe a uma figura de autoridade (o pai, o marido ou o médico, neste caso) e as amigas. Os assuntos de suas conversas se limitam a práticas de be­ leza e outros assuntos inofensivos, e a atuação na esfera pública é inexistente, pelo menos no mundo de “Silicone Seguro”. DOSSIÊ Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Texto do post: O que acontece é a formação de uma cápsula muscular em torno da prótese mamária, ação natural do seu corpo para se proteger de um objeto que ele considera estranho. Em alguns casos, essa cápsula pode se tornar muito den­ sa, causando desconforto, dor, mudança no formato da prótese e, em situações extremas, o rompimento. Por isso, é muito importante seguir todas as orientações médicas depois da cirurgia e fazer o acompanhamento com um profissional de confiança, relatando qualquer tipo de dor ou desconforto. Pode ser necessário fazer a substituição do implante! DOSSIÊ Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: http://tinyurl. com/y2ok9vjo. Acesso em: 15 fev. 2019. A formação discursiva da qual este discurso faz parte não permite a exposição clara dos riscos decorrentes dos implantes. No contexto neoliberal e pós-feminista da sociedade de consumo contemporânea, colocar silicone é uma negociação de riscos e gratificações, e o gerenciamento desta relação recai sobre o indivíduo. A mulher faz por ela mesma, mas também deve arcar sozinha com os problemas de­ correntes. E, no mundo possível que a Johnson & Johnson construiu, onde mulheres podem voltar a sentir-se como meninas enquanto exercem sua subjetividade empre­ endedora, a dor e o prejuízo são indizíveis. 4 PALAVRAS DESVIADAS, INFORMAÇÕES OMITIDAS C omo ensina Orlandi (2015), existe um não dito em todo e qualquer discurso, que se refere à memória discursiva de um “já dito” ausente. O silêncio tam­ bém significa, pois todo dito está relacionado com o não dito, ou a memória discursiva. Os não dizeres são formas implícitas de dizer, e podem estar pressupostos (não dito mas presente) ou subentendidos (depende do contexto). Segundo a auto­ ra, “o não dito é subsidiário ao dito. De alguma forma, o complementa, acrescenta­ -se. De todo modo, sabe-se por aí que, ao longo do dizer, há toda uma margem de não ditos que também significam” (ORLANDI, 2015, p. 81). C Carrascoza e Hoff (2015), por sua vez, explicam que à margem dos dizeres da comunicação mercadológica há um universo de não dizeres que são silenciados: 1) pela impossibilidade natural de serem ditos se, em seu lugar, algo já o foi; e 2) pela estratégia discursiva adotada por seu enunciador, que privi­ legia determinados ditos, em detrimento de outros que não lhe convém mover por diversos motivos – o principal deles, certamente, porque não promovem tão bem a mercadoria anunciada quanto os dizeres escolhidos. (CARRASCOZA; HOFF, 2015, p. 40). Existem diversos riscos relacionados às próteses de silicone, alguns já mais co­ nhecidos pelo público geral – e que os discursos favoráveis exaltam como “raros” –, outros, nem tanto. Nos enunciados de “Silicone Seguro”, identificamos alguns destes silenciamentos. O texto do post representado na Figura 8 desvia da pergunta e não a responde, pois o corpo pode, sim, rejeitar as próteses, já que são corpos estranhos invadindo o organismo. A pergunta “Meu corpo pode rejeitar o silicone?” pede uma resposta de “sim” ou “não”, mas o enunciador explica as cápsulas musculares. O texto não eluci­ da, no entanto, que estas cápsulas são o resultado do corpo tentando “expulsar” os implantes. E também não chega a nomear esta condição, pois é um dos riscos – essa palavra também é evitada – mais comuns dos implantes: a contratura capsular. Por fim, não há menção do explante de próteses, mas a troca destas, e tampouco se há chance de reincidência de contratura capsular após uma segunda operação. 82 Diálogo com a Economia Criativa ESPM Rio ESPM Rio Figura 8: Post “Meu corpo pode rejeitar o silico­ ne?” Texto do post: Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 5 AUSÊNCIA DA FIGURA MASCULINA É Além disso, os exames laboratoriais e de imagem são fundamentais para a colocação das próteses de silicone, pois atestam as suas condições de saú­ de para passar pela anestesia e pela cirurgia.” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: http://tinyurl. com/y62c5yjk.Acesso em: 15 fev. 2019. 5 AUSÊNCIA DA FIGURA MASCULINA É 5 AU É É interessante notar a importância dada à amizade entre mulheres na constru­ ção do mundo possível da Johnson & Johnson Evita-se contrapor uma figura masculina, já que isso interromperia o conceito pós-feminista da campanha. Deste modo, ignora-se a origem do imaginário em torno das próteses, vindo da es­ tética de atrizes pornográficas que, por sua vez, estão a serviço do olhar masculino. O mundo das cirurgias plásticas é dominado pelo gênero masculino, princi­ palmente na figura do cirurgião, o qual se encontra numa posição superior à da paciente nesta relação de poder. Ele participa ativamente do processo ao opinar sobre o corpo da mulher, explicando porque seus seios não são harmoniosos e jus­ tificando a necessidade da cirurgia. Além disto, é o médico a autoridade final que viabiliza uma cirurgia plástica (desde implantes de silicone até cirurgias estéticas da vulva, chamadas de ninfoplastia ou labioplastia) em adolescentes menores de 18 anos, atestando se ela é madura o suficiente para isto por meio de um julgamento subjetivo. Na Figura 9 temos uma mulher posando como médica, mas a realidade é que 79,4% dos cirurgiões plásticos são homens (Sociedade Brasileira de Cirurgia Plástica, 2018) enquanto 85,6% de seus pacientes ao redor do mundo são mulheres (International Society of Aesthetic Plastic Surgery, 2017). Por mais que a campanha tenha como objetivo trazer informações para au­ xiliar tomadas de decisão mais seguras e por mais que se repita que esta decisão é 83 Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” da própria consumidora, denotamos que o lugar da mulher no mundo de “Silicone Seguro” é de um sujeito passivo. A receita de amor próprio sugerida pela campanha não permite desvios: o destino final é a cirurgia de implante, à medida que omite quaisquer informações minimamente desfavoráveis ao silicone e legitima o cirur­ gião plástico como figura de autoridade máxima. A autonomia adquirida sobre seu corpo é ilusória, pois é uma escolha com apenas uma opção: conformar-se ao ideal heteronormativo de beleza, ainda que prejudique a própria saúde no processo. DOSSIÊ “Informações sobre a sua saúde, estilo de vida e expectativas influenciam muito na forma como o médico irá conduzir o procedimento e são essenciais para garantir sua segurança. Figura 9: Post “Converse com o seu médico” Texto do post: “Informações sobre a sua saúde, estilo de vida e expectativas influenciam muito na forma como o médico irá conduzir o procedimento e são essenciais para garantir sua segurança. Além disso, os exames laboratoriais e de imagem são fundamentais para a colocação das próteses de silicone, pois atestam as suas condições de saú­ de para passar pela anestesia e pela cirurgia.” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: http://tinyurl. com/y62c5yjk.Acesso em: 15 fev. 2019. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Figura 11: Post “Onde o silicone vai ser posicio­ nado no meu corpo?” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: https://tinyurl. com/y4ndpsko. Acesso em: 21 mai. 2019. 5 CONSIDERAÇÕES FINAIS S e os feminismos até a primeira década do século XXI eram pautados por de­ mandas sociais, à medida que o neoliberalismo avança globalmente, o social se desloca para o individual. Aquilo que antes era luta – a mulher alterando suas relações de poder na esfera pública –, com o neoliberalismo estimulando o su­ jeito individualizado, o que passa a ter relevância é o querer, são os imperativos do desejo e não mais as ações voltadas ao bem-estar coletivo. Assim, o pós-feminismo, ao mobilizar a memória discursiva das lutas feministas para convocar o sujeito neo­ liberal, é, na verdade, a continuação de relações de poder hegemônicas, das quais a figura masculina, branca e heterossexual é a maior beneficiária. Por sua vez, a mulher é infantilizada e tem sua esfera de atuação restringida ao universo da subje­ tividade e das emoções. A contradição do discurso, sugerindo um retorno a antigos papéis de gênero enquanto enaltece a individualidade e agência feminina quando se trata do consumo, é uma característica do pós-feminismo. Os seios já foram considerados sagrados por serem fontes de vida, mas esta memória se perdeu em um ponto da história, quando foram ressignificados como objeto erótico a serviço do olhar masculino. Já a medicalização dos seios pode ser observada desde a Grécia Antiga, quando não passavam de marcadores da inferio­ ridade do sexo feminino frente ao masculino. Por ser uma das fontes primordiais da nutrição humana, não faltaram práticas medicinais para toda e qualquer mazela que ameaçasse secá-la. Pelo mesmo motivo, os seios jovens sempre tiveram mais va­ 84 Diálogo com a Economia Criativa ESPM Rio ESPM Rio lor diante da sociedade do que os mais velhos, desprovidos de sua função primária. (YALOM, 1997) Ainda assim, o alto índice de compartilhamento de uma imagem (Figura 10) representando as glândulas mamárias dos seios humanos, que causou estranhamen­ to e espanto nos internautas por conta do desconhecimento sobre este aspecto de nossa anatomia, atesta o quão longe fomos em silenciar o feminino no discurso mé­ dico-científico, o qual prioriza a figura humana masculina como modelo anatômico. Na própria campanha Silicone Seguro, por exemplo, os dutos não são representados (Figura 11), demonstrando a regularidade com que este silenciamento ocorre. DOSSIÊ Figura 10: Ilustração das glândulas mamárias Fonte: BBC News Brasil. Disponível em: https:// www.bbc.com/portuguese/geral-48060887. Acesso em: 3 mai. 2019. Figura 10: Ilustração das glândulas mamárias Fonte: BBC News Brasil. Disponível em: https:// www.bbc.com/portuguese/geral-48060887. REFERÊNCIAS BOGADO, Maria. Rua. In: HOLLANDA, Heloisa Buarque De. Explosão Feminista: arte, cultura, políti­ ca e universidade. São Paulo: Companhia das Letras, 2018. BOLTANSKI, Luc; CHIAPELLO, Ève. O novo espírito do capitalismo. São Paulo: Martins Fontes, 2009. BRANDÃO, Helena H. Nagamine. Introdução à análise do discurso. Campinas: Editora da Unicamp, 2012. BUITONI, Dulcilia H. S. Revistas femininas: ainda somos as mesmas, como nossas mães. Communicare, v. 14, p. 36-45, 2014. CARRASCOZA, João; HOFF, Tania. Ditos e não-ditos: o Brasil e as práticas de consumo nos auto anúncios das agências de publicidade nos anos 1950. Revista Organicom, v. 1, p. 39-45, 2015. DOSSIÊ CENSO 2018 – Situação da cirurgia plástica no Brasil. Sociedade Brasileira de Cirurgia Plástica, 2018. Disponível em: <http://www2.cirurgiaplastica.org.br/wp-content/uploads/2019/08/ Apresentac%CC%A7a%CC%83o-Censo-2018_V3.pdf>. Acesso em: jan. 2019. CONTRATURA Capsular: Como Isso Pode Alterar o Resultado da Sua Cirurgia de Prótese de Silicone? Dream Plastic, 2017. Disponível em: https://www.plasticadosonho.com.br/blog/contratura-capsular. Acesso em: fev. 2019. DARDOT, Pierre; LAVAL, Christian. A nova razão do mundo: ensaio sobre a sociedade neoliberal. São Paulo: Boitempo, 2016. DOSEKUN, Simidele. The Risky Business of Postfeminist Beauty. In: ELIAS, Ana Sofia; GILL, Rosalind; SCHARFF, Christina. Aesthetic Labour: Rethinking Beauty Politics in Neoliberalism. Londres: Palgrave Macmillan, 2017. ELIAS, Ana Sofia; GILL, Rosalind; SCHARFF, Christina. Aesthetic Labour: Rethinking Beauty Politics in Neoliberalism. Londres: Pagrave Macmillan, 2017. FAVARO, Laura. Just Be Confident Girls: Confidence Chic as Neoliberal Governmentality. In: ELIAS, Ana Sofia; GILL, Rosalind; SCHARFF, Christina. Aesthetic Labour: Rethinking Beauty Politics in Neoliberalism. Londres: Palgrave Macmillan, 2017. GILL, Rosalind; SCHARFF, Christina. New femininities: Postfeminism, neoliberalism and subjectivity. Basingstoke: Palgrave Macmillian, 2011. GREGOLIN, Maria do Rosario de Fatima. Análise do discurso e mídia: a (re) produção de identidades. Comunicação mídia e consumo, v. 4, n. 11, p. 11-25, 2007. HOFF, Tania. Comunicação publicitária: dos regimes de visibilidade do corpo diferente às biossocia­ bilidades do consumo. In: HOFF, Tania. Corpos discursivos: dos regimes de visibilidade às biossociabi­ lidades do consumo Recife: Editora UFPE, 2016, p. 19-40. ISAPS International Survey on Aesthetic/Cosmetic Procedures Performed in 2017. International Society of Aesthetic Plastic Surgery, 2018. Disponível em: <https://tinyurl.com/y2fagj4v>. Acesso em: jan. 2019. JARRÍN, Alvaro. The Biopolitics of Beauty: Cosmetic Citizenship and Affective Capital in Brazil. Oakland: University of California Press, 2017. LAZAR, Michele M. Seriously Girly Fun!’: Recontextualising Aesthetic Labour as Fun and Play in Cosmetics Advertising. In: ELIAS, Ana Sofia; GILL, Rosalind; SCHARFF, Christina. Aesthetic Labour: Rethinking Beauty Politics in Neoliberalism. Londres: Palgrave Macmillan, 2017. MANIFESTO. Silicone Seguro. 5 CONSIDERAÇÕES FINAIS Acesso em: 3 mai. 2019. Figura 11: Post “Onde o silicone vai ser posicio­ nado no meu corpo?” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: https://tinyurl. com/y4ndpsko. Acesso em: 21 mai. 2019. Figura 11: Post “Onde o silicone vai ser posicio­ nado no meu corpo?” Figura 11: Post “Onde o silicone vai ser posicio­ nado no meu corpo?” Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: https://tinyurl. com/y4ndpsko. Acesso em: 21 mai. 2019. Para avançarmos em termos de igualdade de gênero, sugerimos olharmos para trás, reivindicando a antiga potência feminina, antes dos seios terem sido apo­ derados pelos homens ainda na mitologia grega, e recriarmos a memória de nossos antepassados pré-históricos, quando os seios eram adorados por serem fontes de alimento, nutrição e vida. A partir daí, poderemos criar algo novo, algo verdadeira­ mente nosso, respeitando a diversidade de seios, mulheres e femininos. Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: https://tinyurl. com/y4ndpsko. Acesso em: 21 mai. 2019. Fonte: SILICONE Seguro. Facebook: Siliconeseguro. Disponível em: https://tinyurl. com/y4ndpsko. Acesso em: 21 mai. 2019. Para avançarmos em termos de igualdade de gênero, sugerimos olharmos para trás, reivindicando a antiga potência feminina, antes dos seios terem sido apo­ derados pelos homens ainda na mitologia grega, e recriarmos a memória de nossos antepassados pré-históricos, quando os seios eram adorados por serem fontes de alimento, nutrição e vida. A partir daí, poderemos criar algo novo, algo verdadeira­ mente nosso, respeitando a diversidade de seios, mulheres e femininos. 85 Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. Sabina Lovato Sabina Lovato A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” A memória discursiva na construção do sujeito neoliberal feminino: ditos e não ditos da campanha “Silicone Seguro” Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. REFERÊNCIAS Disponível em: https://www.siliconeseguro.com/manifesto.html. Acesso em: dez. 2018. MCROBBIE, Angela. Notes on the Perfect: Competitive Femininity in Neoliberal Times. Australian Feminist Studies, v. 30, p. 3-20, 2015. MENTOR. Disponível em: https://www.mentorimplantes.com.br. Acesso em: ago. 2019. ORLANDI, Eni P. Análise de discurso: princípios e procedimentos. Campinas: Pontes Editores, 2015. Diálogo com a Economia Criativa, Rio de Janeiro, v. 4, n. 12, p. 72-87, set./dez. 2019. 86 Diálogo com a Economia Criativa ESPM Rio ESPM Rio PRADO, José Luiz Aidar. Convocações biopolíticas dos dispositivos comunicacionais. São Paulo: EDUC- Editora da PUC-SP, 2013. SANT’ANNA, Denise B. História da Beleza no Brasil. São Paulo: Contexto, 2014. SCHARFF, Christina. The Psychic Life of Neoliberalism: Mapping the Contours of Entrepeneurial Subjectivity. Theory, Culture & Society, v. 33, n. 6, p. 107-122, 2015. SEMPRINI, Andrea. A marca pós-moderna: poder e fragilidade da marca na sociedade contemporâ­ nea. São Paulo: Estação das Letras, 2006. SILVERSTONE, Roger. Por que estudar a mídia? São Paulo: Edições Loyola, 2014. SODRÉ, Muniz. Antropológica do espelho: uma teoria da comunicação linear e em rede. Petrópolis Editora Vozes, 2013. DOSSIÊ WEATHERFORD, Ashley. Why Are So Many Makeup Companies Treating Women Like Children? The Cut, 2017. Disponível em: https://www.thecut.com/2017/04/unicorn-makeup-trend-treats-women-li­ ke-children.html?mid=facebook_thecutblog. Acesso em: jan. 2019. YALOM, Marilyn. A history of the breast. Lexington, EUA: Ballantine, 1997. 87
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https://europepmc.org/articles/pmc3235710?pdf=render
English
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The Effect of Traditional Cupping on Pain and Mechanical Thresholds in Patients with Chronic Nonspecific Neck Pain: A Randomised Controlled Pilot Study
Evidence-based complementary and alternative medicine
2,012
cc-by
9,533
Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 429718, 10 pages doi:10.1155/2012/429718 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 429718, 10 pages doi:10.1155/2012/429718 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 429718, 10 pages doi:10.1155/2012/429718 Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 429718, 10 pages doi:10.1155/2012/429718 Romy Lauche,1 Holger Cramer,1 Claudia Hohmann,1 Kyung-Eun Choi,1 Thomas Rampp,1 Felix Joyonto Saha,1 Frauke Musial,2 Jost Langhorst,1 and Gustav Dobos1 1Chair of Complementary and Integrative Medicine, University of Duisburg-Essen, Knappschafts-Krankenhaus, Am Deimelsberg 34a, 45276 Essen, Germany Romy Lauche,1 Holger Cramer,1 Claudia Hohmann,1 Kyung-Eun Choi,1 Thomas Rampp,1 Felix Joyonto Saha,1 Frauke Musial,2 Jost Langhorst,1 and Gustav Dobos1 1Chair of Complementary and Integrative Medicine, University of Duisburg-Essen, Knappschafts-Krankenhaus, Am Deimelsberg 34a, 45276 Essen, Germany Department of Community Medicine, The National Research Centre in Complementary and Alternative Medicine (N aculty of Health Science, University of Tromsø, 9037 Tromsø, Norway Correspondence should be addressed to Romy Lauche, r.lauche@kliniken-essen-mitte.de Correspondence should be addressed to Romy Lauche, r.lauche@kliniken-essen-mitte.de Received 18 August 2011; Revised 7 September 2011; Accepted 13 September 2011 Received 18 August 2011; Revised 7 September 2011; Accepted 13 September 2011 Academic Editor: Gerhard Litscher Copyright © 2012 Romy Lauche et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction. Cupping has been used since antiquity in the treatment of pain conditions. In this pilot study, we investigated the effect of traditional cupping therapy on chronic nonspecific neck pain (CNP) and mechanical sensory thresholds. Methods. Fifty CNP patients were randomly assigned to treatment (TG, n = 25) or waiting list control group (WL, n = 25). TG received a single cupping treatment. Pain at rest (PR), pain related to movement (PM), quality of life (SF-36), Neck Disability Index (NDI), mechanical detection (MDT), vibration detection (MDT), and pressure pain thresholds (PPT) were measured before and three days after a single cupping treatment. Patients also kept a pain and medication diary (PaDi, MeDi) during the study. Results. Baseline characteristics were similar in the two groups. After cupping TG reported significantly less pain (PR: −17.9 mm VAS, 95%CI −29.2 to −6.6; PM: −19.7, 95%CI −32.2 to −7.2; PaDi: −1.5 points on NRS, 95%CI −2.5 to −0.4; all P < 0.05) and higher quality of life than WL (SF-36, Physical Functioning: 7.5, 95%CI 1.4 to 13.5; Bodily Pain: 14.9, 95%CI 4.4 to 25.4; Physical Component Score: 5.0, 95%CI 1.4 to 8.5; all P < 0.05). No significant effect was found for NDI, MDT, or VDT, but TG showed significantly higher PPT at pain-areas than WL (in lg(kPa); pain-maximum: 0.088, 95%CI 0.029 to 0.148, pain-adjacent: 0.118, 95%CI 0.038 to 0.199; both P < 0.01). Conclusion. A single application of traditional cupping might be an effective treatment for improving pain, quality of life, and hyperalgesia in CNP. 1. Introduction Patients with blank myogelosis, that is, hyperirri- table areas of skeletal muscle associated with small palpable nodules in taut bands of muscle fibres together with lowered microcirculation, were referred for dry cupping. to active trigger points, present in chronic non-specific neck pain patients but not in healthy controls [18]. However, it is still unknown if this process is restricted to the cervical area [16] or widespread [19]. Hyperalgesia in chronic non- specific neck pain also shows different patterns and seems to rely on different mechanisms than hyperalgesia in acute [19] and traumatic neck pain [15], respectively. Although there is only limited evidence for these treat- ments, conventional treatment options include the prescrip- tion of nonsteroidal anti-inflammatory drugs [20], physical therapy [21, 22] or exercise [23, 24]. According to the litera- ture [20, 25] and treatment guidelines [25] pharmacological therapy cannot be recommended, the same is true for man- ual therapy [26], or massages [21]. Dynamic and isometric exercises as part of physical therapy have also been proven to be only moderately effective in the long term [25]. Due to the limited treatment patients seek alternative treatment options, especially those patients with more intense pain [27, 28] and those who have not experienced improvements under conventional treatment [29]. Patients were included only if neck pain was clearly identified to be of mechanical origin and specific causes for their neck pain had been excluded in the medical history either by an orthopaedist or a neurologist. Specific causes included traumatic neck pain (e.g., WAD), inflammatory or malignant disease, congenital malformation of the spine, radicular symptoms such as radiating pain, paresis, prickling, or tingling, invasive treatments within the last 4 weeks, surgery to the spine within the last year, and corticosteroid or opioid treatment. Further exclusion criteria were pregnancy, serious acute or chronic organic diseases such as diabetes or cancer, mental disorders, and haemorrhagic tendency or anticoagulation treatment. Nonsteroidal pain medication and physiotherapy were allowed if the treatment regimen was not altered for four weeks before and continued during the study. This ensured that statistical evaluation of the effects of cupping treatment was not influenced by alterations in medications or physiotherapy during the study phase. Traditional cupping or wet cupping has been used in the treatment of pain and many other complaints for millennia [30]. A glass cup is utilized to create suction over a painful area after incisions are made to the skin. 1. Introduction [4], but also poor psychological health [1, 5], stress [6], low socioeconomic status [7], and smoking [8, 9]. Usually non- specific neck pain resolves within three to six months; but 14% of the patients will suffer from recurrent or persistent pain [10]. If neck pain persists for more than 3 months, it is considered chronic neck pain [11]. Neck pain, that is, pain between the occipital bone, the thoracic vertebra, and the extensions to the shoulder joint [1], is a major health-related socioeconomic problem and the lifetime prevalence is approximately 48.5% [2]. Neck pain can be caused by trauma, inflammatory diseases, or degeneration of the spine; however, most patients suffer from simple or non-specific neck pain, which is mainly caused by mechanical factors such as sprain and strains [3]. The aetiology of non-specific neck pain is not yet understood in detail, but different factors have been shown to contribute to the development and persistence of neck pain. They do not only include poor posture [3] and high physical load Besides the pain and the related impairment in daily ac- tivities, chronic neck pain is also associated with functional changes. For example dysfunctional microcirculation of the trapezius muscle [12, 13] has been reported as well as motor control disturbances of the neck musculature [14]. Me- chanical hyperalgesia, that is, increased response to painful mechanical stimulation has also been shown in chronic non- specific neck pain [15–17]; an effect which might be related Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 2 2 Medical Institutions (no. 09–3985). Between July 2009 and July 2010 50 patients aged 18 to 75 who suffered from neck pain for at least three months in a row with a minimum of 40 mm intensity on a 100 mm visual analogue scale (VAS) were included in the study. A specific inclusion criterion was based on the recommendations for traditional cupping [30, 39, 40]. Accordingly, patients show so-called plethora or overabundance. These terms refer to different signs and symptoms such as voluminous gelosis of the subskin, which indicates local blood congestion, swelling, and adhesions of the connective tissue in the neck region. A strong constitution, for example, high level of vitality, and high blood pressure, were further indicators for traditional cupping. 2.2. Outcome Measures 2.2.1. Pain. Pain at rest (PR) and maximal pain related to movement (PM, provoked pain by neck flexion, neck extension, lateral neck flexion, and neck rotation in either direction) [41] were recorded on a VAS graded from 0 (no pain at all) to 100 mm (worst pain imaginable). The minimal clinical important difference (MCID) for the VAS, a highly reliable instrument to measure pain intensity [42], is a reduction of 30%, which is the equivalent of a moderate pain reduction [43]. For PM the direction that elicited highest pain report was chosen for analysis. Baseline and postintervention pain scores were recorded at T1 and T2. Additionally patients kept a pain (PaDi) and medication diary (MeDi) from day 0 (7 days prior to T1) until T2, where they rated their pain three times daily on a 11-point numeric rating scale (NRS ranging from 0 = no pain to 10 = worst The aim of this pilot study was to test the efficacy of a single traditional cupping treatment in patients with chronic non-specific neck pain. Besides pain ratings we determined mechanical thresholds at pain-related and control areas to serve as more objective pain markers. We hypothesised that patients in the treatment group would report less pain at T2 compared to the waiting list control group. 1. Introduction By doing so, it is hypothesised that “congested” blood is sucked out of the skin thereby increasing blood and lymphatic circulation and relieving painful muscle tension [30, 31]. Within the last years the interest in traditional cupping has emerged and there is growing evidence that cupping might be effective in various pain conditions [32–37]. Michalsen et al. [37] for example, found that a single tra- ditional cupping treatment at the trapezius muscle was effec- tive in relieving the symptoms of the carpal tunnel syndrome as well as associated neck pain. L¨udtke et al. [36] investigated the effect of traditional cupping in Brachialgia parasthetica nocturna, that is, numb, tingling, and painful sensations in fingers or hands during the night. A single treatment signif- icantly reduced symptoms and the associated neck pain and no adverse events were observed. Farhadi et al. [34] found significantly reduced pain, functional disability, and pain medication in patients with low-back pain three months after traditional cupping compared to standard care. Cup- ping might further be effective in migraine and tension- type head-ache [32] and postherpetic pain [38]. However, despite growing evidence there is yet no RCT to investigate the effectiveness of traditional cupping in the treatment of chronic non-specific neck pain. All patients were recruited through advertisements in local newspapers and screened two times. First inclusion and exclusion criteria were checked in a standardised telephone interview, then the patients underwent a physical and neurological examination by the study physician at their first appointment. pain imaginable) and made notes of concurrent medication and treatments. VDT was quantified by a Rydel Seiffer tuning fork (64 Hz, 8/8 scale). It was placed over a bony prominence, for example, on the spinal process, the styloid process of ulna and the lateral malleolus and left there until the subject could not feel the vibration anymore. The arithmetic mean of three series was taken the individual vibration detection threshold [49]. 2.2.2. Questionnaires. Self-rated disability due to neck pain was assessed with the Neck Disability Index (NDI) [44], a 10-item questionnaire representing everyday activities. The MCID for the NDI is 10% improvement for uncomplicated neck pain [45]. The health-related quality of life was quantified by the German version of the Medical Outcomes Study Short Form-36 (SF-36) [46, 47]. The SF-36 provides a detailed health profile on the basis of eight health dimensions as well as sum scores for physical and mental health. Two versions were used in the study, the standard version (4- week time frame) for baseline assessment at T1 and the acute recall version (1-week time frame) at T2. The latter version was used because it was considered more sensitive to recent changes in health status [48]. PPT was measured by a pressure algometer (Algometer, SOMEDIC, Sweden) at pain-maximum, pain-adjacent, the thenar eminence, and the instep. It exerts forces up to 2000 kPa when used with a probe area of 1 cm2. The pressure pain threshold was measured in three ramps of increasing pressure intensities of ca. 50 kPa/s until the subject signalled the first sensation of pain in addition to the pressure sensation. The log-transformed arithmetic mean of these three series was taken the individual pressure pain threshold [49]. To evaluate the reliability of the sensory threshold meas- urements, the retest reliabilities were determined at the con- trol areas in the control group participants (WL, N = 23). Pearson’s correlation coefficients were r = 0.35 for MDT hand (P = 0.09), r = 0.66 for MDT foot (P = 0.001), r = 0.79 for PPT hand (P = 0.00001), r = 0.71 for PPT foot (P = 0.0001), r = 0.56 for VDT hand (P = 0.005), and r = 0.73 for VDT foot (P = 0.0001). The average correlation coefficients was r = 0.63 which indicates sufficient reliability. 2.2.3. General Health Outcome. pain imaginable) and made notes of concurrent medication and treatments. Within the SF-36 the General Health outcome was recorded on a 5-point Likert scale that ranged from “My health is much better than before treat- ment” to “My health is much worse than before treatment.” 2.2.4. Mechanical Sensory and Pain Thresholds. Sensory testing included determination of mechanical detection threshold (MDT), vibration detection threshold (VDT), and pressure pain threshold (PPT) and was conducted in the following areas: the site of maximal pain (pain-maximum), adjacent to the pain maximum (pain-adjacent), hand and foot on the right side. Pain-maximum and pain-adjacent were determined for each patient individually. First, the patient was given a diagram of the body on which to mark the most painful spot in the neck region. This spot, defined as pain-maximum, was verified by physical examination. The second spot, defined as adjacent to the painful area (pain-adjacent) was defined outside the painful area, that is, patients did not report pain in that area. Again physical examination was used to confirm the location. Both spots were marked in the pain diagram for precise replication of the measurements at T2. Thresholds were also determined at control areas, that is, right hand and right foot, in order to estimate reliability of measurements. All sensory measures were determined and calculated according to the standardised protocol for the quantitative sensory testing (QST) by Rolke et al. [49, 50], and MDT and PPT were logarithmised to reach normal distribution [49]. 2.2.5. Safety. All participants were asked to report any adverse events during the study period. The questionnaires relating to T2 also included an open question about relevant experiences and adverse events. 2.2.5. Safety. All participants were asked to report any adverse events during the study period. The questionnaires relating to T2 also included an open question about relevant experiences and adverse events. 2.2.6. Expectation. After randomisation all patients had to self-rate their expectations towards cupping therapy on a VAS ranging from 0 = “not effective at all” to 100 mm = “most effective.” 2.3. Intervention: Traditional Cupping Technique. Based on data from previous studies on traditional cupping [36, 37] and clinical experience, a single cupping treatment was considered sufficient. Cupping was performed by the study physician, who was trained in cupping and regularly performed cupping in a clinical setting. Patients were asked to lay topless on the massage couch. 2. Methods 2.1. Patients. The study protocol was approved by the in- stitutional review board of the University Duisburg-Essen 3 Evidence-Based Complementary and Alternative Medicine pain imaginable) and made notes of concurrent medication and treatments. pain imaginable) and made notes of concurrent medication and treatments. The study physician used the patients pain diagram (see Section 2.2.4) and physical examination to identify the areas of pain and the voluminous geloses of the subskin, which most commonly were found at the descending parts of the trapezius muscle. Mechanical detection threshold was measured with a set of von Frey filaments (Aesthesiometer, SOMEDIC, Sweden) that exert forces between 0.26 and 1080 mN. The threshold was determined by the method of limits, whereby the stimulus intensity is decreased until the patient can no longer perceive the touch and is then increased until the patient first perceives the touch again. Five series of descending and ascending stimulus intensities were made at pain-maximum, pain-adjacent, on the dorsa of the right hand and the right foot. The final threshold was the log-transformed geometric mean of these five series [49]. The cupping procedure involved the following steps: the skin was disinfected; superficial incisions were made with a disposable microlancet at the areas of pain and voluminous geloses; double-walled glass cups (2–6 glasses with diameters from 25 to 50 mm) were held inverted over an open flame to heat the air inside; the glass cup was placed on the incision. The air inside the cup cooled down and created a vacuum which sucked blood out through the incisions. The glasses were removed after 10 to 15 minutes, and the skin was Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine Medical examination T1 Baseline assessment Waiting list control group (no cupping) Postintervention assessment T2 Pain and medication diary Randomisation 0 7 10 Treatment group ( ) 1 × trad ( ) ay d itional cupping 1 × traditional cupping Figure 1: Study design. Randomisation 1 × traditional cupping Postintervention assessment Figure 1: Study design. however, since all drop outs were lost before T1 missing data could not be replaced by taking the last observation forward. disinfected and a plaster was applied. However, since bleed- ing generally stopped during treatment, this was only a pre- caution. Patients were asked not to take a bath or go swim- ming within the next 48 hours to prevent delays in wound healing. After some minutes of rest patients were free to leave. The pain diary (PaDi) was analysed by means of a repeated measurement ANCOVA. pain imaginable) and made notes of concurrent medication and treatments. Within the statistical model the group variable served as between-subject factor, the post intervention measures as dependent factors, and the average pain in the week before T1 as covariate. Medica- tion and concurrent treatments (MeDi) were continuously recorded in the diary and converted into relative amount of days under medication or treatment. 2.4. Study Design. After the telephone interview potential participants were invited to be assessed on whether they were eligible for the study. The study physician also informed them about the study details. Written informed consent was obtained and patients were then randomly assigned to either a treatment group or a waiting list control group by means of sequentially numbered, sealed opaque envelopes, prepared by the study coordinator, who was neither involved in treatment nor in measurement. Patients were handed out the pain and medication diary (PaDi, MeDi) and measurement and treatment appointments were scheduled. Figure 1 illustrates the study design. The General Health outcome was analysed by means of the Mann-Whitney U test. Because of the pilot character of the study the level of statistical significance was not adjusted. An alpha of 0.05 was chosen for all analyses. 3.3. Pain. Analysis of pain at rest (PR) shows a significant group difference at T2. TG reported 17.9 mm less pain on the 3. Results At baseline assessment (T1) study participants filled out the following questionnaires: pain at rest (PR), pain related to movement (PM), Neck Disability Index (NDI), and quality of life (SF-36). At last mechanical thresholds, that is, mechanical detection threshold (MDT), vibration detection threshold (VDT), and pressure pain threshold (PPT) were determined. At the end of T1 the treatment group received a single traditional cupping treatment whereas the waiting list control group received no treatment. Three days later participants returned for postintervention assessment (T2). They again filled out the questionnaires and underwent sensory testing. After they had completed the postinterven- tion assessment, the wait-list control group was offered the cupping treatment. 3.1. CONSORT Flowchart. After the first telephone screen- ing, 122 patients were invited for further evaluation. 50 of them fulfilled the study criteria and agreed to participate in the study. Three patients in the treatment group and two in the waiting list control group resigned from participation before T1, no data could be collected from these patients. Final statistical analyses were conducted on 22 patients in the treatment group and on 23 patients in the waiting list control group. Figure 2 shows a flowchart of patient recruitment. 3.2. Sample Characteristics. All baseline values were com- parable between the two groups, see Table 1. Two-thirds of participants in the study were female, the average age was 54.8 (TG) and 57.2 (WL). Study patients suffered for a very long time from neck pain; on average they reported 12.0 (TG) and 10.4 (WL) years of pain. The average pain intensity was rated 44.9 (TG) and 42.6 (WL). Expectation was comparable between the groups; therefore it was not included in further analysis. 2.5. Statistical Analyses. Treatment and waiting list con- trol group were compared using chi-square analysis for discrete data and independent t-tests for continuous data on demographics, pain history, and baseline variables. For each outcome measure except the pain diary the results of the intervention were compared by analyses of covariance (ANCOVA) taking the post treatment measurement (T2) as a dependent and group as a between-subject factor. Respective baseline value of the outcome (T1) served as a covariate. This approach was chosen according to Vickers and Altman [51]. The intention-to-treat principle was used in this study, Pre- and postintervention scores and estimated differ- ences are presented in Table 2 and described in detail below. 3.3. Pain. 3. Results Analysis of pain at rest (PR) shows a significant group difference at T2. TG reported 17.9 mm less pain on the Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 5 Table 1: Baseline demographic and clinical characteristics of trial groups. Sociodemographic and clinical characteristics TG (N = 22) mean ± SD WL (N = 23) mean ± SD P Age (years) 54.8 ± 9.6 57.2 ± 9.4 0.393 Sex (F/M) 18/7 16/9 0.544 BMI (kg/m2) 28.9 ± 5.6 27.1 ± 4.3 0.203 Pain at rest (PR) 44.9 ± 18.2 42.6 ± 17.8 0.810 Average neck pain at baseline (PaDi) 4.8 ± 1.1 4.6 ± 1.4 0.552 History of neck pain (years) 12.0 ± 10.3 10.4 ± 11.5 0.618 Expected effectiveness of cupping therapy (VAS from 0 = not effective at all to 100 = highly effective) 72.8 ± 18.9 68.3 ± 20.5 0.448 Table 1: Baseline demographic and clinical characteristics of trial groups. Table 2: Outcomes of subjective measures at T1 and T2. 3. Results T1 T2 Estimated difference at T2 ANCOVA TG (n = 22) (mean ± SD) WL (n = 23) (mean ± SD) TG (n = 22) (mean ± SD) WL (n = 23) (mean ± SD) diffTG versus WL∗ (95% CI) D f F P Pain at rest (PR) 44.9±18.2 42.6±17.8 28.5 ± 23.9 45.7 ± 16.4 −17.9 (−29.2 to −6.6) 44 10.2 0.003 Maximal pain related to movement (PM) 53.9±25.7 65.6±22.1 29.1 ± 20.9 53.8 ± 26.1 −19.7 (−32.2 to −7.2) 44 10.1 0.003 Neck Disability Index (NDI) 29.9 ± 11.8 31.1 ± 9.1 24.5 ± 13.5 29.0 ± 9.3 −3.6 (−8.7 to 1.6) 44 2.0 0.168 SF-36 Physical Functioning 74.5 ± 19.1 71.3 ± 20.7 80.0 ± 15.3 70.2 ± 19.2 7.5 (1.4 to 13.5) 44 6.2 0.017 SF-36 Role Physical 39.8 ± 37.5 39.1 ± 41.9 58.0 ± 41.8 51.1 ± 38.8 6.4 (−12.0 to 24.8) 44 0.5 0.483 SF-36 Bodily Pain 37.8 ± 9.3 39.7 ± 9.1 53.1 ± 22.9 39.3 ± 11.4 14.9 (4.4 to 25.4) 44 8.2 0.007 SF-36 General Health Perception 62.2 ± 14.2 64.0 ± 19.3 64.0 ± 14.8 61.3 ± 20.7 4.1 (−3.3 to 11.5) 44 1.3 0.268 SF-36 Vitality 59.5 ± 21.0 53.5 ± 19.6 61.4 ± 21.4 53.5 ± 23.8 2.1 (−5.1 to 9.2) 44 0.3 0.561 SF-36 Social Function 70.5 ± 25.7 69.6 ± 24.7 79.0 ± 26.6 73.9 ± 26.9 4.4 (−6.8 to 15.6) 44 0.6 0.434 SF-36 Role Emotional 81.8 ± 36.7 71.0 ± 39.3 81.8 ± 33.7 76.8 ± 39.5 −0.1 (−19.8 to 19.6) 44 0.0 0.991 SF-36 Mental Health 72.4 ± 15.9 68.2 ± 18.3 69.6 ± 21.4 68.5 ± 22.4 −3.4 (−10.7 to 4.0) 44 0.9 0.358 SF-36 Physical Component Score 37.8 ± 7.8 38.7 ± 8.6 43.3 ± 8.5 39.0 ± 7.4 5.0 (1.4 to 8.5) 44 7.8 0.008 SF-36 Mental Component Score 51.8 ± 10.8 48.7 ± 11.3 50.4 ± 11.7 49.8 ± 13.6 −2.1 (−7.1 to 3.0) 44 0.7 0.415 ∗Group differences and P values from an ANCOVA model with 2 groups, baseline values as covariate. Table 2: Outcomes of subjective measures at T1 and T2. 3. Results Enrollment Allocation Analysis Treatment Not meeting inclusion criteria (n = 72) Assessed for eligibility (n = 122) Allocated to treatment group (n = 25) Analysed (n = 22) Received allocated treatment (n = 23) Randomised (n = 50) Allocated to waiting list control group (n = 25) Received allocated treatment (n = 22) Analysed (n = 23) Resigning from the study before T1 (n = 3) Resigning from the study before T1 (n = 2) Figure 2: CONSORT flowchart of recruitment and study condi- tions. Enrollment Allocation Analysis Treatment Not meeting inclusion criteria (n = 72) Assessed for eligibility (n = 122) Allocated to treatment group (n = 25) Analysed (n = 22) Received allocated treatment (n = 23) Randomised (n = 50) Allocated to waiting list control group (n = 25) Received allocated treatment (n = 22) Analysed (n = 23) Resigning from the study before T1 (n = 3) Resigning from the study before T1 (n = 2) VAS than WL. There was also a significant group difference on maximal pain related to movement (PM). While both groups were comparable in their pain ratings at T1, TG reported 19.7 mm less movement-related pain than the WL at T2. VAS than WL. There was also a significant group difference on maximal pain related to movement (PM). While both groups were comparable in their pain ratings at T1, TG reported 19.7 mm less movement-related pain than the WL at T2. Pain diary (PaDi) shows a sudden decline in pain ratings in TG at day 2, that is, the day after cupping therapy whereas it remained relatively stable in WL (Figure 3). A repeated measures ANCOVA revealed a significant interaction time × group (F = 5.22, D f = 3/98, ε = 0.002, P = 0.005). Post hoc analyses confirmed a significant group difference at day 2 (Δ−1.5, 95%CI −2.5 to −0.4, P = 0.008) and single comparisons within TG also showed significant difference between baseline and day 2 (Δ−0.9, 95% CI −1.7 to −0.2, P = 0.014). The majority of the patients went without any concurrent treatment in the week before T1 (medication: 60.0%, physiotherapeutic treatment: 91.1%). Those who did, used medication in 27.8±22.2 and physiotherapy in 39.3±33.8 of the days. The use of medication and concurrent treatments during the study was not further analysed. Figure 2: CONSORT flowchart of recruitment and study condi- tions. 3. Results Figure 2: CONSORT flowchart of recruitment and study condi- tions. 6 Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine 6 0 2 4 6 8 1 2 3 4 Pain rating (NRS) TG WL ∗ ∗ ∗ ∗ ∗ ∗ Bas line e ay d Figure 3: Pain ratings (pain diary, NRS, mean ± SD) decreased in TG at the day after cupping. ∗P < 0.05. 0 2 4 6 8 1 2 3 4 Pain rating (NRS) TG WL ∗ ∗ ∗ ∗ ∗ ∗ Bas line e ay d However, two patients experienced more serious adverse events. As a result an ad hoc safety board was constituted to evaluate these adverse events and decide on further actions. The safety board was initiated by the principal investigator and consisted of the study physicians, the senior physicians of the Clinic for Complementary and Integrative Medicine, the head of the research group, an external statistician, and an external scientist, whose area of expertise is in safety issues and medical ethics. Two cases were presented and evaluated: one patient returned four days after treatment and reported worsened neck pain, a strong headache, and constant ear noises. The study physician examined the patient and diagnosed a cervical spine blockage. She was referred to an orthopedic for further diagnosis treatment. Later inquiries revealed that the symptoms had lasted for 2 to 3 weeks and improved subsequently. The neck pain, however, was neither better nor worse than before she participated in the study. Another patient complained of dizziness, nausea, and body misperception directly after treatment, so she had to lie down directly after treatment. Blood pressure and pulse measurement revealed normal circulatory function. The study physician diagnosed a transitory vagal reaction caused by the treatment and recommended her to rest until symptoms were resolved. After three hours lying and another hour sitting and walking the patient had mostly recovered. After examination the patient was sent home and a new appointment some days later was made. The patient then reported that the dizziness and nausea were fully resolved, but that the neck pain had worsened. The study physician offered her another treatment against the neck pain, which she refused. Later inquiries revealed that the pain had decreased within two weeks. The safety commission evaluated both incidents as adverse events, but not of a serious kind. 3. Results Further actions as a consequence of occurrence of the adverse events involved obligatory follow-up check of patients in WL within two days after treatment. No adverse events were reported for WL after treatment. Pain rating (NRS) Figure 3: Pain ratings (pain diary, NRS, mean ± SD) decreased in TG at the day after cupping. ∗P < 0.05. 3.4. Questionnaires. No significant differences at T2 were found for the Neck Disability Index (NDI). The same was true for the Mental Component Score (SF-36) and the following subscales of the SF-36: Role Physical, Vitality, Social Function, Role Emotional, and General Health per- ceptions. On the other hand, significant differences occurred in the subscales Physical Functioning, Bodily Pain, and the Physical Component Score. At T2 TG reported significantly higher values on these scales indicating higher quality of life. Analysis of the General Health Outcome (SF-36) revealed a significant group difference with a significant higher rank for the TG (Mann Whitney U test; mean rank TG: 18.8; WL: 27.0; U = 160.5; P = 0.019) indicating more positive ratings than WL. In detail 11 patients of 22 in TG rated their health at least somewhat better than before, only two did so in WL. The majority in WL rated their health about the same as before (18 of 23). Three patients in each groups even reported worse health (see also “safety issues”). 3.4. Questionnaires. No significant differences at T2 were found for the Neck Disability Index (NDI). The same was true for the Mental Component Score (SF-36) and the following subscales of the SF-36: Role Physical, Vitality, Social Function, Role Emotional, and General Health per- ceptions. On the other hand, significant differences occurred in the subscales Physical Functioning, Bodily Pain, and the Physical Component Score. At T2 TG reported significantly higher values on these scales indicating higher quality of life. Analysis of the General Health Outcome (SF-36) revealed a significant group difference with a significant higher rank for the TG (Mann Whitney U test; mean rank TG: 18.8; WL: 27.0; U = 160.5; P = 0.019) indicating more positive ratings than WL. In detail 11 patients of 22 in TG rated their health at least somewhat better than before, only two did so in WL. The majority in WL rated their health about the same as before (18 of 23). Three patients in each groups even reported worse health (see also “safety issues”). Evidence-Based Complementary and Alternative Medicine Since changes in the VAS and the NDI were also strongly correlated (r = 0.49, P = 0.001, N = 45), pain relief appears to be associated with reduced impairment. 4.2. Interpretation. In this study various pain measures such as pain at rest (PR), pain related to movement (PM), and pain diary (PaDi) data differed significantly between the TG and the WL after cupping. Thus, a single traditional cupping treatment appears to be effective in treating chronic non- specific neck pain. Since changes in the VAS and the NDI were also strongly correlated (r = 0.49, P = 0.001, N = 45), pain relief appears to be associated with reduced impairment. However, there were no significant differences in NDI at T2 and the estimated difference was fewer than 10 points of improvement, which is the minimum clinical important change (MCIC) for the NDI [45]. This might have been due to the already low NDI scores at the beginning or due to the short followup. Interestingly, the pain diary ratings indicate that cupping has immediate effects. That is, the effects of traditional cupping are present already on the day after cupping treatment. This conforms to clinical observations, in which traditional cupping often shows dramatic and immediate effects on pain and other complaints. 4.2. Interpretation. In this study various pain measures such as pain at rest (PR), pain related to movement (PM), and pain diary (PaDi) data differed significantly between the TG and the WL after cupping. Thus, a single traditional cupping treatment appears to be effective in treating chronic non- specific neck pain. Since changes in the VAS and the NDI were also strongly correlated (r = 0.49, P = 0.001, N = 45), pain relief appears to be associated with reduced impairment. However, there were no significant differences in NDI at T2 and the estimated difference was fewer than 10 points of improvement, which is the minimum clinical important change (MCIC) for the NDI [45]. This might have been due to the already low NDI scores at the beginning or due to the short followup. Interestingly, the pain diary ratings indicate that cupping has immediate effects. That is, the effects of traditional cupping are present already on the day after cupping treatment. This conforms to clinical observations, in which traditional cupping often shows dramatic and immediate effects on pain and other complaints. Evidence-Based Complementary and Alternative Medicine 7 Table 3: Mechanical detection and pain thresholds at T1 and T2 (mean ± SD). T1 T2 Estimated difference at T2 ANCOVA TG (n = 22) (mean ± SD) WL (n = 23) (mean ± SD) TG (n = 22) (mean ± SD) WL (n = 23) (mean ± SD) diffTG versus WL∗ (95% CI) D f F P MDT in log(mN) Pain- maximum 0.425 ± 0.427 0.443 ± 0.418 0.446 ± 0.508 0.411 ± 0.433 0.047 (−0.185 to 0.278) 44 0.686 Pain-sdjacent 0.290 ± 0.360 0.223 ± 0.374 0.382 ± 0.390 0.219 ± 0.477 0.124 (−0.094 to 0.341) 44 0.257 VDT in X/8 Pain-maximum 6.061 ± 1.542 5.986 ± 1.135 6.061 ± 1.398 5.956 ± 1.075 0.024 (−0.482 to 0.529) 44 0.447 Pain-adjacent 5.288 ± 1.527 5.601 ± 1.162 5.515 ± 1.186 5.580 ± 1.401 0.199 (−0.366 to 0.764) 44 0.217 PPT in log(kPa) Pain-maximum 2.349 ± 0.169 2.357 ± 0.192 2.381 ± 0.149 2.299 ± 0.192 0.088, (0.029 to 0.148) 44 0.005 Pain-adjacent 2.396 ± 0.203 2.418 ± 0.200 2.423 ± 0.195 2.321 ± 0.204 0.118 (0.038 to 0.199) 44 0.005 Baseline values were comparable between the groups. ∗Differences were estimated by an ANCOVA model with 2 groups and the respective baseline values as covariate. Table 3: Mechanical detection and pain thresholds at T1 and T2 (mean ± SD). .8 2 2 .6 2.4 2.2 2 T1 T2 Pressure in lg (kPa) Pain-adjacent ∗ .8 2 2 .6 2.4 2.2 2 T1 T2 Pressure in lg (kPa) Pain-maximum ∗ TG WL (a) .8 2 2 .6 2.4 2.2 2 T1 T2 Pressure in lg (kPa) Pain-adjacent ∗ (b) Figure 4: Course of pressure pain thresholds at pain-maximum and pain-adjacent (mean ± SD) ∗P < 0.05. .8 2 2 .6 2.4 2.2 2 T1 T2 Pressure in lg (kPa) Pain-maximum ∗ TG WL (a) T1 T2 (a) (b) Figure 4: Course of pressure pain thresholds at pain-maximum and pain-adjacent (mean ± SD) ∗P < 0.05. 4.2. Interpretation. In this study various pain measures such as pain at rest (PR), pain related to movement (PM), and pain diary (PaDi) data differed significantly between the TG and the WL after cupping. Thus, a single traditional cupping treatment appears to be effective in treating chronic non- specific neck pain. 4. Discussion 4.1. Principal Findings. To our knowledge, this is the first RCT where the effect of a single application of traditional cupping on chronic non-specific neck pain is investigated. Patients treated with cupping therapy showed significant improvements in their symptoms. Pain at rest (PR), maximal pain related to movement (PM), and bodily pain (SF- 36) were reduced after a single cupping treatment. Pain diary (PaDi) showed a significant decline in pain ratings already on the day after cupping. According to the quality of life questionnaires (SF-36), the cupping treatment also significantly decreased Bodily Pain and improved Physical Functioning as well as the Physical Component Score. 3.5. Mechanical Sensory and Pain Thresholds. MDT, VDT, and PPT for each group are listed in Table 3. Statistical analyses revealed no group differences for MDT or VDT, but for PPT. Significant differences in pressure pain thresholds were found at the pain-maximum and the pain-adjacent, but not at the control areas. Figure 4 displays the course of PPT at the pain-related areas. 3.6. Safety. Although most patients tolerated the treatment very well, adverse events were observed in some of the patients. One patient reported that the procedure itself was painful, other adverse events including slight reactions such as circulatory instability in the first minutes after treatment, tension headaches, a migraine attack, a reappearing tinnitus or wound healing itches. All of these adverse events were minor and transient. Cupping also showed an effect on one of the nonsubjec- tive parameters, the pressure-pain threshold (PPT), which is thought to reflect the functional status of (altered) pain perception. Pressure pain thresholds at pain-related areas increased or remained stable over time in the TG whereas patients of the WL control group became sensitised at those areas. Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine changes are impressive since the post intervention measure- ment was only four days after the treatment. However, the immediate pain relief and the changes on physical scales of the SF-36 suggest that traditional cupping might work on a very somatic level. Effects of cupping have also been found on pressure pain thresholds at pain-related areas. Although the differences in PPT were relatively small, they were found consistently, suggesting that cupping might exert its effects locally, probably at receptor level. p pp p However, there were no significant differences in NDI at T2 and the estimated difference was fewer than 10 points of improvement, which is the minimum clinical important change (MCIC) for the NDI [45]. This might have been due to the already low NDI scores at the beginning or due to the short followup. Interestingly, the pain diary ratings indicate that cupping has immediate effects. That is, the effects of traditional cupping are present already on the day after cupping treatment. This conforms to clinical observations, in which traditional cupping often shows dramatic and immediate effects on pain and other complaints. Different modes of actions might explain the effect of traditional cupping on chronic neck pain. They involve neural, haematological, immune, and psychological effects [34]. Stimulation of the skin causes several autonomous, hormonal, and immune reactions [52]; this also applies for injuries due to the incisions [53]. Blood vessels in the treated areas are dilated by release of vasodilators such as adenosine, noradrenaline, and histamine, which lead to increased blood circulation [54]. In the course of cupping treatment, blood and other interstitial fluids are drawn out from the skin by the vacuum. Traditional cupping is mainly used in patients Furthermore, Physical Functioning and the Physical Component Score (SF-36) changed significantly. These Evidence-Based Complementary and Alternative Medicine 8 with local blood congestion, swelling, and adhesions of the connective tissue in the neck region. It has been assumed that these congestions contain inflammatory extravasations [30, 35] and toxins. Cupping might therefore take the pressure offthe tissue and relieve the neck area from these toxic congestions, which also increases circulation and lymphatic flow. Since circulation has been shown to be dysfunctional in chronic neck pain patients [12], cupping might restore normal circulation. Increased circulation in turn improves oxygen supply and cell metabolism [30] reducing the amount of inflammatory or toxic substances. Evidence-Based Complementary and Alternative Medicine This might also explain the significant effects of cupping on pressure pain thresholds at pain-related areas. Muscle spasm, congestion, and restricted blood flow can cause ischemic pain [55]. Accumulated inflammatory substances in skin and tissue might further induce hypersensitivity to noxious stimuli [16, 56], which is reflected by lowered pressure pain thresholds [57]. Since traditional cupping is supposed to evacuate toxins and inflammatory agents from the affected area and to restore normal circulation, this might explain the local effects on pressure pain thresholds. study. However, baseline values were comparable between the groups. A further limitation is the rather mild baseline pain intensity. Pain intensities reported by the patients in this study were at the lower end of the inclusion criteria scale. Some patients even fell below the required pain intensity of 40 on the VAS. This can be regarded as a possible source of bias, since patients probably exaggerated their complaints during screening to ensure inclusion into the study. The ceiling effect due to the low baseline pain intensity, likely limited the possible absolute reduction in pain intensity. Furthermore, due to the same problem, the likelihood of aggravation was also high due to the natural course of disease. 4.4. Strengths of the Study. Despite the limitations we could observe a strong pain reduction (VAS) of approximately 32.8 ± 51.1% in the treatment group, compared to 24.6 ± 88.7% in the waiting list control group. This pain reduction is comparable to studies on dry cupping [59] or massage [60] but in contrast du these methods the effect in traditional cupping occurs almost immediately after treatment. The overall pain reduction is within the range of clinical signif- icance [43]. Moreover, treatment effects were also observed on pressure pain threshold, the concept of which is less transparent for the participant and therefore less open to presumption and hypothesising, which may make the results less biased than simple pain ratings. The blood volume loss together with the local vasodi- lation might further increase parasympathetic activity by somatosympathetic reflexes, which corresponds well with the observed self-reported relaxation. 5. Conclusion A single application of traditional cupping might be effective in the treatment of chronic non-specific neck pain. Further studies are necessary to confirm these results and to evaluate the effectiveness of cupping compared to standard treat- ments. Studies investigating the effects of repeated traditional cupping interventions in different intervals and long-term observations are needed as well. Although measurements of sensory thresholds give possible hints on the physiology of pain processing, further investigations aiming at the mechanisms of action are necessary, too. However, the results of this first study and the patients’ experiences with cupping therapy support the assumption that cupping might be a safe and effective treatment for chronic non-specific neck pain. 4.3. Limitations of the Study. The interpretation of the results might be limited due to choice of the passive control group. We are aware of the fact that unspecific effects such as expectation, conditioning, or environmental effects may have contributed to the observed overall effect size [35]. However, to date there are no suitable sham devices [58], even though there is an urgent need for a suitable sham procedure. Sham cupping by means of adhesives to keep the glass in place have been tried, but in our experience even cupping na¨ıve subjects are likely to discover the sham intervention, even more so in traditional cupping than in dry cupping. Another serious problem is the impracticability of experimental blinding the assessor due to superficial wounds and visible cupping marks. On the other hand, traditional cupping is applied commonly in clinical CAM settings and has been proven to be helpful in alleviating several pain conditions [34, 36, 37], and patients request this treatment. Therefore there is a need for clinical trials on the topic, evaluating the efficacy and safety of these procedures. In conclusion, instead of ignoring the fact that there is a patient request for this reasonably invasive procedure and that there is limited data on efficacy and safety available, we decided to run an RCT with the best possible methodological approach, even though we are aware of its limitations. Conflict of Interests All authors disclose any commercial association that might create a conflict of interest in connection with the submitted paper. There is especially no competing financial interest for any of the authors. Evidence-Based Complementary and Alternative Medicine Despite the invasiveness of traditional cupping the treatment group felt very relaxed after cupping treatment, on average they rated relaxation at 62.2 ± 20.1 mm on a 100 mm VAS from 0 = “not relaxed at all” to 100 mm = “very relaxed.” In the worst case the reflex might cause a vasovagal syncope, as observed in one patient. References Gross, T. Kay, M. Hondras et al., “Manual therapy for mechanical neck disorders: a systematic review,” Manual Therapy, vol. 7, no. 3, pp. 131–149, 2002. [23] T. M. Kay, A. Gross, C. Goldsmith, P. L. Santaguida, J. Hoving, and G. Bronfort, “Exercises for mechanical neck disorders,” Cochrane Database of Systematic Reviews, no. 3, Article ID CD004250, 2005. [8] S. Hogg-Johnson, G. van der Velde, L. J. Carroll et al., “The burden and determinants of neck pain in the general pop- ulation. 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Confounding adjustment performance of ordinal analysis methods in stroke studies
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PLOS ONE RESEARCH ARTICLE Thomas P. ZonneveldID1, Annette Aigner2, Rolf H. H. Groenwold3,4, Ale Algra5,6, Paul J. Nederkoorn1, Ulrike Grittner7,8, Nyika D. Kruyt9, Bob SiegerinkID3,8* 1 Department of Neurology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands, 2 Institute of Public Health, Charite´-Universita¨tsmedizin Berlin, Berlin, Germany, 3 Department of Clinical Epidemiology, Leiden University Medical Centre, Leiden, The Netherlands, 4 Department of Biomedical Data Sciences, Leiden University Medical Centre, Leiden, The Netherlands, 5 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands, 6 Department of Neurology and Neurosurgery, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands, 7 Berlin Institute of Health, Charite´-Universita¨tsmedizin Berlin, Berlin, Germany, 8 Center for Stroke research Berlin, Charite´-Universita¨tsmedizin Berlin, Berlin, Germany, 9 Department of Neurology and Clinical Neuropsychology, Leiden University Medical Center, Leiden, The Netherlands a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * bob.siegerink@charite.de OPEN ACCESS In stroke studies, ordinal logistic regression (OLR) is often used to analyze outcome on the modified Rankin Scale (mRS), whereas the non-parametric Mann-Whitney measure of superiority (MWS) has also been suggested. It is unclear how these perform comparatively when confounding adjustment is warranted. Aims Our aim is to quantify the performance of OLR and MWS in different confounding variable settings. Editor: Aristeidis H. Katsanos, University of Ioannina School of Medicine, GREECE Received: October 28, 2019 Accepted: March 28, 2020 Published: April 16, 2020 Methods We set up a simulation study with three different scenarios; (1) dichotomous confounding variables, (2) continuous confounding variables, and (3) confounding variable settings mim- icking a study on functional outcome after stroke. We compared adjusted ordinal logistic regression (aOLR) and stratified Mann-Whitney measure of superiority (sMWS), and also used propensity scores to stratify the MWS (psMWS). For comparability, OLR estimates were transformed to a MWS. We report bias, the percentage of runs that produced a point estimate deviating by more than 0.05 points (point estimate variation), and the coverage probability. Copyright: © 2020 Zonneveld et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. * bob.siegerink@charite.de Background OPEN ACCESS Citation: Zonneveld TP, Aigner A, Groenwold RHH, Algra A, Nederkoorn PJ, Grittner U, et al. (2020) Confounding adjustment performance of ordinal analysis methods in stroke studies. PLoS ONE 15 (4): e0231670. https://doi.org/10.1371/journal. pone.0231670 Confounding adjustment performance of ordinal analysis methods in stroke studies Thomas P. ZonneveldID1, Annette Aigner2, Rolf H. H. Groenwold3,4, Ale Algra5,6, Paul J. Nederkoorn1, Ulrike Grittner7,8, Nyika D. Kruyt9, Bob SiegerinkID3,8* Thomas P. ZonneveldID1, Annette Aigner2, Rolf H. H. Groenwold3,4, Ale Algra5,6, Paul J. Nederkoorn1, Ulrike Grittner7,8, Nyika D. Kruyt9, Bob SiegerinkID3,8* Citation Citation Zonneveld, T. P., Aigner, A., Groenwold, R. H. H., Algra, A., Nederkoorn, P. J., Grittner, U., … Siegerink, B. (2020). Confounding adjustment performance of ordinal analysis methods in stroke studies. Plos One, 15(4). doi:10.1371/journal.pone.0231670 Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3182735 Version: Publisher's Version License: Creative Commons CC BY 4.0 license Downloaded from: https://hdl.handle.net/1887/3182735 License: Note: To cite this publication please use the final published version (if applicable). PLOS ONE Introduction The ordinal modified Rankin Scale (mRS) measures functional outcome after stroke on a 7-step scale from 0 (no symptoms) to 6 (death), and is the primary outcome measure in most stroke trials. [1] To analyze the differences in mRS between treatment arms, pivotal stroke trials primarily use the ordinal logistic regression (OLR) method. [2–4] The OLR produces a single effect size estimate (a common odds ratio) based on the odds ratios for each cut-point across the mRS, and this estimate can be interpreted as the odds ratio of ending up one cate- gory higher on the scale. Because OLR is based on several assumptions, such as a linear and proportional effect of the independent variables on the outcome variable, [5] the Mann-Whit- ney measure of superiority (MWS) was recently proposed as a more robust analysis method of an ordinal outcome scale. [6, 7] In contrast to regression methods, the MWS is a non- parametric rank-based test based on proversions, which are one to one comparisons of out- come between observations. In short, each observation in one group (A) is compared to each observation in the other group (B), and the following three complementary probabilities (Ps) are derived: P(A>B), P(A = B), and P(B>A). The MWS for A is then given by the formula P (A>B) + 0.5P(A = B). As a result, the MWS ranges from 0 to 1, with 0.5 as the value of no dif- ference between groups A and B. Importantly, OLR and MWS differ fundamentally in their confounding adjustment tech- nique. In regression methods such as the OLR, independent variables can be added to the equation. However, these variables are also subject to aforementioned assumptions. As a non- parametric method, the MWS uses stratification for confounding adjustment: the basic concept is that the proversions are performed only within the defined strata. For example, when adjust- ing for sex, proversions are only made between males from group A versus males from group B, and females from group A versus females from group B. Stratification is however linked to estimation problems. Most notably, residual confounding and instability through empty cells might occur, especially when adjusting for multiple confounding variables. [8] A possible solu- tion to overcome these issues is to form strata based on percentiles of propensity scores, which estimate the probability of being exposed based on measured confounding variables. Results Funding: The author(s) received no specific funding for this work. In scenario 1, there was no bias in both sMWS and aOLR, with similar point estimate varia- tion and coverage probabilities. In scenario 2, sMWS resulted in more bias (0.04 versus 0.00), and higher point estimate variation (41.6% versus 3.3%), whereas coverage Competing interests: The authors have declared that no competing interests exist. 1 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies probabilities were similar. In scenario 3, there was no bias in both methods, point estimate variation was higher in the sMWS (6.7%) versus aOLR (1.1%), and coverage probabilities were 0.98 (sMWS) versus 0.95 (aOLR). With psMWS, bias remained 0.00, with less point estimate variation (1.5%) and a coverage probability of 0.95. Conclusions The bias of both adjustment methods was similar in our stroke simulation scenario, and the higher point estimate variation in the MWS improved with propensity score based stratifica- tion. The stratified MWS is a valid alternative for adjusted OLR only when the ratio of number of strata versus number of observations is relatively low, but propensity score based stratifi- cation extends the application range of the MWS. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 Scenarios We generated data in three distinct scenarios, differing from each other only in their con- founding variable settings (Table 1). In scenario 1, we modeled five dichotomous confounding variables all with a prevalence of 0.5 and all with a regression coefficient of ln (1.5) (which is equivalent to an odds ratio of 1.5) in relation to the outcome. In scenario 2, we modeled five continuous confounding variables, with a standard normal distribution and regression coeffi- cient of ln (1.5) in relation to the outcome. In scenario 3, we modeled five varying confounding variables, with distributions and regression coefficients reflecting known important character- istics (sex, age, stroke severity, previous stroke, systolic blood pressure) associated with func- tional outcome after stroke. [10, 11] In our main simulations we generated 1000 observations, which we changed to 250 and 4000 in sensitivity analyses. Each scenario was run 1000 times. All simulations were performed in Stata/IC 15.1 for Windows (32 bit), with full code provided in the appendix. Data generation process First, we generated a seven-step ordinal outcome variable, based on the presence or value of the confounding variables and their assumed relationship with the outcome (as specified in the respective scenario). Second, we constructed a dichotomous exposure variable also based on the confounding variables present, yet conditionally independent of the outcome. Impor- tantly, we did not model a direct relationship between the exposure variable and the ordinal outcome variable, nor did we model a correlation between any of the confounding variables. See appendix 1 for a detailed description of our data generation process and appendix 2 for the full Stata code used. Introduction [9] How these different confounding adjustment techniques of OLR and MWS perform comparatively has not been investigated previously. Our study aims to explore to what extent MWS is a viable option in stroke research when confounding adjustment is necessary. There- fore, our objective was to quantify the bias/variance trade-off of these methods in simulation models with varying confounding conditions, focusing on conditions typically present in a stroke patient cohort. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 2 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies https://doi.org/10.1371/journal.pone.0231670.t001 Abbreviations: RC = regression coefficient; NIHSS = National Institutes of Health Stroke Scale; SBP = systolic blood pressure; SD = standard deviation; IQR = interquartile range; ln = natural logarithm.  = per unit increase. Scenario 2: Continuous confounding variables In the scenario with five continuous confounding variables (resulting in 1024 possible strata for sMWS), aOLR outperformed sMWS; bias was 0.04 in the sMWS versus 0.00 in the aOLR, and point estimate variation was 41.6% in the sMWS versus 3.3% in the aOLR. The coverage probability was 96% for both methods. With psMWS, bias was 0.02, point estimate variation was 8.1%, and coverage probability was 88%. See Fig 2 for the boxplots (including the results for 1 to 4 continuous confounding variables). Scenario 1: Dichotomous confounding variables In the scenario with five dichotomous confounding variables (resulting in 32 possible strata for sMWS), sMWS and aOLR performed similar; bias was 0.00 in both methods, and point esti- mate variation (percentage of runs that produced a point estimate deviating more than 0.05 MWS from the true effect) was 2.1% in the sMWS versus 1.8% in the aOLR. The coverage prob- ability was 96% in the sMWS versus 95% in the aOLR. Propensity score based strata adjustment in the MWS (psMWS) resulted in a bias of 0.01, a point estimate variation of 2.1%, and cover- age probability of 93%. See Fig 1 for the boxplots (including the results for 1 to 4 dichotomous confounding variables). Comparison of analysis methods For each run, we performed a crude OLR and MWS analysis, and an adjusted analysis for both methods; regression adjustment in OLR (aOLR) and stratified adjustment in MWS (sMWS). Ordinal and continuous confounding variables were stratified based on quartiles; this resulted in up to 32 (2^5) possible strata in scenario 1, up to 1024 (4^5) possible strata in scenario 2, and up to 256 (24424) possible strata in scenario 3. We calculated a propensity score per observation based on all confounding variables present in the respective scenario. This score Table 1. Confounding variable settings per scenario. Scenario Confounding variables Type Distribution Prevalence RCs (βj) 1 x1, x2, x3, x4, x5 Dichotomous Binary 50% ln(1.50) 2 x1, x2, x3, x4, x5 Continuous Normal Mean = 0 (SD = 1) ln(1.50) 3 x1; female sex Dichotomous Binary 50% ln(1.10) x2; age Continuous Normal Mean = 69 (SD = 10) ln(1.01) x3; NIHSS Ordinal Right-skewed Median = 9 [IQR = 5–14] ln(1.05) x4; previous stroke Dichotomous Binary 50% ln(1.10) x5; SBP Continuous Normal Mean = 160 (SD = 15) ln(1.01) Abbreviations: RC = regression coefficient; NIHSS = National Institutes of Health Stroke Scale; SBP = systolic blood pressure; SD = standard deviation; Table 1. Confounding variable settings per scenario. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 3 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies was subsequently divided into quartiles for the propensity based stratified MWS (psMWS). For comparison purposes, we converted the odds ratios (ORs) generated by the OLR to Mann- Whitney measures of superiority, with the following approximation formula: MWS = (OR/ (OR-1)2) x ((OR-1)-ln(OR)). [6] Outcome parameters The validity of each method is assessed by the extent of the bias, which we defined as the differ- ence between the mean of the observed point estimates and the simulated, true effect. In order to quantify the variation in (point) estimates of each method, we report the percentage of runs that produced a point estimate deviating more than 0.05 MWS from the true effect (i.e. an estimate lower than 0.45 or higher than 0.55, roughly equivalent to an OR lower than 0.74 or higher than 1.35 using the approximation formula stated above). Finally, we also report the coverage probability, defined as the proportion of 95%- confidence intervals encompassing the true effect. With our 1000 runs, calculated coverage probabilities within the range of 93.6%–96.4% are compatible with a true coverage of 95%. Of note, as the number of prover- sions decreases when the number of strata increases, there were no or only very little prover- sions to construct the MWS estimate in some runs. As this results in extreme estimates and impossibility to construct a valid confidence interval, we discarded runs that resulted in less than 11 proversions. We created boxplots of the five analysis methods’ (OLR, MWS, aOLR, sMWS, psMWS) point estimates, displaying the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers not shown). Scenario 3: Varying confounding variables In the scenario with five varying confounding variables (resulting in 256 possible strata for sMWS), sMWS and aOLR performed similar; bias was 0.00 in both methods, and point esti- mate variation was 6.7% in the sMWS versus 1.1% in the aOLR. The coverage probability was 98% for sMWS and 95% in the aOLR. With psMWS, bias was 0.00, point estimate variation PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 4 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies Fig 1. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 1. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). Fig 1. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 1. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). https://doi.org/10.1371/journal.pone.0231670.g001 https://doi.org/10.1371/journal.pone.0231670.g001 was 1.5%, and coverage probability was 95%. See Fig 3 for the boxplots (including the results for 1 to 4 varying confounding variables). PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 Varying sample size (Table 2) Sensitivity analyses with 250 observations in scenario 1 resulted in similar bias (sMWS 0.00, aOLR 0.00, psMWS 0.01) and coverage probabilities (sMWS 96%, aOLR 95%, psMWS 96%) as the main analyses, but with higher point estimate variation (sMWS 30.5%, aOLR 25.0%, psMWS 23.7%). In scenario 2, bias was also similar to the main analyses (sMWS 0.05, aOLR 0.00, psMWS 0.02), but point estimate variation increased particularly in the sMWS (91.0% versus 32.2% with aOLR, and 34.5% with psMWS). Coverage probabilities were 100% (sMWS) versus 95% (aOLR), and 93% in the psMWS. In scenario 3, bias was similar to the main analy- ses (sMWS 0.01, aOLR 0.00, psMWS 0.00), but point estimate variation increased particularly in the sMWS (58.3% versus 22.5% with aOLR, and 20.4% with psMWS). Coverage probabili- ties were 98% (sMWS) versus 94% (aOLR), and 96% in the psMWS. Sensitivity analyses with 4000 observations in scenario 1 resulted in similar bias (sMWS 0.00, aOLR 0.00, psMWS 0.01) and coverage probabilities (sMWS 95%, aOLR 95%, psMWS PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 5 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies Fig 2. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 2. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). Fig 2. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 2. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). https://doi.org/10.1371/journal.pone.0231670.g002 86%), but, as expected, with lower point estimate variation (all methods 0.0%). Varying sample size (Table 2) In scenario 2, bias was also similar to the main analyses (sMWS 0.03, aOLR 0.00, psMWS 0.02), and point esti- mate variation lowered proportionally (sMWS 14.7%, aOLR 0.0%, psMWS 0.1%). However, the coverage probability was only 49% with sMWS versus 95% with aOLR, and 71% with psMWS. In scenario 3, bias was 0.00 and point estimate variation was 0.0% in all analysis methods. Cov- erage probabilities were 95% (sMWS) versus 94% (aOLR), and 92% in the sMWS. Discussion In our final simulation scenario with confounding variable settings based on stroke cohorts (scenario 3), we found that both stratified MWS (sMWS) and adjusted OLR (aOLR) produced unbiased point estimates. The variation in point estimates was higher for sMWS, but this was fixed with propensity score based stratification in the MWS (psMWS). Interestingly, when modeling fewer observations sMWS performed worse than aOLR, but psMWS produced simi- lar results to aOLR. When modeling a larger number of observations, differences disappeared and all methods produced unbiased and precise point estimates. In the scenario with dichoto- mous confounding variables (scenario 1), both methods performed similar in terms of bias 6 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies Fig 3. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 3. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). https://doi.org/10.1371/journal.pone.0231670.g003 Fig 3. Bias of Mann-Whitney measure of superiority (MWS), ordinal logistic regression (OLR), stratified Mann-Whitney measure of superiority (sMWS), adjusted ordinal logistic regression (aOLR), and propensity score based stratified Mann-Whitney measure of superiority (psMWS) in scenario 3. The psMWS was not performed in the scenario with one confounding variable. Runs (N): 1000. The x-axis shows the number of confounding variables modeled. The y-axis shows the bias, with estimates from the OLR analyses converted to a MWS. Boxplots display the lower adjacent value, 25th percentile, median, 75th percentile, and upper adjacent value (extreme outliers are not displayed). https://doi.org/10.1371/journal.pone.0231670.g003 Table 2. Varying sample sizes (sensitivity analyses). Results shown for scenarios with five confounding variables. Abbreviations: sMWS = stratified Mann-Whitney measure of superiority; aOLR = adjusted ordinal logistic regression; psMWS = propensity score based stratified Mann-Whitney measure of superiority; PEV = point estimate variation (percentage of runs in which the difference between point estimate and true effect is more than 0.05 MWS); CP = coverage probability. y p y; j g g ; p p p y Mann-Whitney measure of superiority; PEV = point estimate variation (percentage of runs in which the difference between point estimate and true effect is more than 0.05 MWS); CP = coverage probability. itney measure of superiority; aOLR = adjusted ordinal logistic regression; psMWS = propensity score based stratified https://doi.org/10.1371/journal.pone.0231670.t002 int estimate variation (percentage of runs in which the difference between point estimate and true effect is more than https://doi.org/10.1371/journal.pone.0231670.t002 Discussion 250 1000 4000 Scenario Outcome sMWS aOLR psMWS sMWS aOLR psMWS sMWS aOLR psMWS 1 Bias 0.00 0.00 0.01 0.00 0.00 0.01 0.00 0.00 0.01 PEV (%) 30.5 25.0 23.7 2.1 1.8 2.1 0.0 0.0 0.0 CP probability 0.96 0.95 0.96 0.96 0.95 0.93 0.95 0.95 0.86 2 Bias 0.05 0.00 0.02 0.04 0.00 0.02 0.03 0.00 0.02 PEV (%) 91.0 32.2 34.5 41.6 3.3 8.1 14.7 0.0 0.1 CP probability 1.00 0.95 0.93 0.96 0.96 0.88 0.49 0.95 0.71 3 Bias 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PEV (%) 58.3 22.5 20.4 6.7 1.1 1.5 0.0 0.0 0.0 CP probability 0.98 0.94 0.96 0.98 0.95 0.95 0.95 0.94 0.92 g sample sizes (sensitivity analyses). Results shown for scenarios with five confounding variables. Table 2. Varying sample sizes (sensitivity analyses). Results shown for scenarios with five confounding variables. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 7 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies and point estimate variation. In the scenario with continuous confounding variables (scenario 2), sMWS resulted in more bias and higher point estimate variation than aOLR, as can be expected from any stratification based methodology. [12] Although psMWS resulted in improved results compared to sMWS, performance of aOLR remained superior in this scenario. To our knowledge, this is the first study directly comparing the performance of confounding adjustment of a parametric model (OLR) with a non-parametric test (MWS) regarding bias and precision of resulting effect estimates. Although it is well known that stratification tech- niques are generally less effective, [13] we still compared these methods head-to-head as it reflects the choice that stroke researchers have to make in scientific practice. Our comparison was focused on quantifying the differences and provides researchers with more detailed charac- teristics of these analyses methods. Intuitively, the MWS seems primarily suited for situations when no adjustment for confounding is needed, such as in primary analyses of interventional studies. However, our simulations showed that sMWS also performs comparably to aOLR in a range of confounding variable settings. As expected, increasing the number of continuous con- founding variables (and thus strata) renders the sMWS more biased, which can only partly be corrected by psMWS. As in any simulation study, conclusions stated above pertain to the modeled scenarios and might not translate to other settings with different confounding settings. Discussion Furthermore, our simulations do not address other issues relevant when deciding which analysis technique should be applied. These issues include residual confounding, measurement error and misclas- sification, model misspecification, and missing data patterns. Although important, we believe these issues are in some sense secondary to the more basic question that we addressed in our simulations. Another limitation is that we modeled proportional effects of our confounding variables on the outcome; further research should focus on exploring the comparative perfor- mance of MWS and OLR when the proportional odds assumption is violated. [14] Another limitation is inseparably linked with the nature of the MWS; as it performs proversions between two groups only, it can only be used when studying a binary exposure variable. This might not be a problem in most intervention studies, but dichotomization of a non-dichoto- mous exposure invariably leads to a loss of information. Of note, we chose to exclude varia- tions on both analysis methods, such as the proportional odds model or the permutation approach excluding tied proversions. [7, 15] These variations were left out as we expected a similar performance as the methods from which they were derived, and also because they are used infrequently in clinical practice. We firmly believe that the choice on how the exposure is modeled should be based on subject matter knowledge in combination with a weighing of potential drawbacks of analysis techniques. Therefore, we are not able to provide a general statement whether the benefits of the assumption free MWS approach outweigh drawbacks that come from the required categorization of both the exposure and the confounding variables. Other research fields than stroke might have different constellations of known confounding factors, which renders it difficult to extrapolate our results to other fields. Yet, as we provide the used Stata code, readers could modify the provided code in the appendix to generate results more relevant to their specific setting. In conclusion, the confounding variable settings in our stroke simulation scenario resulted in an unbiased performance of both methods, and the higher point estimate variation in the stratified MWS was corrected with propensity score based stratification. Continuous and ordi- nal confounding variables strained the performance of stratified MWS, and this led to unac- ceptable problems when fitting a large number of strata over a small number of observations. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 Author Contributions Conceptualization: Thomas P. Zonneveld, Rolf H. H. Groenwold, Ale Algra, Ulrike Grittner, Bob Siegerink. Conceptualization: Thomas P. Zonneveld, Rolf H. H. Groenwold, Ale Algra, Ulrike Grittner, Bob Siegerink. Data curation: Thomas P. Zonneveld, Bob Siegerink. Data curation: Thomas P. Zonneveld, Bob Siegerink. Formal analysis: Thomas P. Zonneveld, Bob Siegerink. Formal analysis: Thomas P. Zonneveld, Bob Siegerink. Funding acquisition: Paul J. Nederkoorn, Bob Siegerink. Investigation: Thomas P. Zonneveld, Bob Siegerink. Methodology: Thomas P. Zonneveld, Annette Aigner, Rolf H. H. Groenwold, Ale Algra, Ulrike Grittner, Bob Siegerink. Project administration: Bob Siegerink. Project administration: Bob Siegerink. Resources: Paul J. Nederkoorn, Nyika D. Kruyt, Bob Siegerink. Resources: Paul J. Nederkoorn, Nyika D. Kruyt, Bob Siegerink. Software: Bob Siegerink. Software: Bob Siegerink. Supervision: Paul J. Nederkoorn, Ulrike Grittner, Nyika D. Kruyt, Bob Siegerink. Validation: Annette Aigner, Bob Siegerink. Validation: Annette Aigner, Bob Siegerink. Visualization: Bob Siegerink. Visualization: Bob Siegerink. Writing – original draft: Thomas P. Zonneveld. Writing – review & editing: Annette Aigner, Ale Algra, Paul J. Nederkoorn, Nyika D. Kruyt, Bob Siegerink. Discussion In future stroke research, the stratified MWS is a valid analysis method only when adjustment is needed for a limited number of confounding variables, and when sufficient observations are available to prevent model instability due to empty cells. If it is not possible to keep this ratio PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 8 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies of number of strata versus number of observations relatively low, OLR is the superior analysis method. Propensity score based stratification improves the confounding adjustment perfor- mance of MWS, and this should be weighed against the specific limitations of any regression method. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 Supporting information S1 Appendix. (DOCX) References 1. van Swieten JC, Koudstaal PJ, Visser MC, Schouten HJ, van Gijn J. Interobserver agreement for the assessment of handicap in stroke patients. Stroke. 1988; 19(5):604–7. Epub 1988/05/01. https://doi. org/10.1161/01.str.19.5.604 PMID: 3363593. 2. Berkhemer OA, Fransen PS, Beumer D, van den Berg LA, Lingsma HF, Yoo AJ, et al. A randomized trial of intraarterial treatment for acute ischemic stroke. The New England journal of medicine. 2015; 372(1):11–20. Epub 2014/12/18. https://doi.org/10.1056/NEJMoa1411587 PMID: 25517348. 3. Goyal M, Demchuk AM, Menon BK, Eesa M, Rempel JL, Thornton J, et al. Randomized assessment of rapid endovascular treatment of ischemic stroke. The New England journal of medicine. 2015; 372 (11):1019–30. Epub 2015/02/12. https://doi.org/10.1056/NEJMoa1414905 PMID: 25671798. 4. Jovin TG, Chamorro A, Cobo E, de Miquel MA, Molina CA, Rovira A, et al. Thrombectomy within 8 hours after symptom onset in ischemic stroke. The New England journal of medicine. 2015; 372 (24):2296–306. Epub 2015/04/18. https://doi.org/10.1056/NEJMoa1503780 PMID: 25882510. 5. Brant R. Assessing Proportionality in the Proportional Odds Model for Ordinal Logistic Regression. Bio- metrics. 1990; 46:1171–8. PMID: 2085632 6. Rahlfs VW, Zimmermann H, Lees KR. Effect size measures and their relationships in stroke studies. Stroke. 2014; 45(2):627–33. https://doi.org/10.1161/STROKEAHA.113.003151 PMID: 24370754 PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 9 / 10 PLOS ONE Confounding adjustment performance of ordinal analysis methods in stroke studies 7. Howard G, Waller JL, Voeks JH, Howard VJ, Jauch EC, Lees KR, et al. A simple, assumption-free, and clinically interpretable approach for analysis of modified Rankin outcomes. Stroke. 2012; 43(3):664–9. https://doi.org/10.1161/STROKEAHA.111.632935 PMID: 22343650 8. Groenwold RH, Klungel OD, Altman DG, van der Graaf Y, Hoes AW, Moons KGM. Adjustment for con- tinuous confounders: an example of how to prevent residual confounding. (1488–2329 (Electronic)). 9. Austin PC. An Introduction to Propensity Score Methods for Reducing the Effects of Confounding in Observational Studies. (1532–7906 (Electronic)). 10. Westendorp WF, Vermeij JD, Zock E, Hooijenga IJ, Kruyt ND, Bosboom HJ, et al. The Preventive Anti- biotics in Stroke Study (PASS): a pragmatic randomised open-label masked endpoint clinical trial. Lan- cet (London, England). 2015; 385(9977):1519–26. Epub 2015/01/24. https://doi.org/10.1016/s0140- 6736(14)62456-9 PMID: 25612858. 11. Gensicke H, Strbian D, Zinkstok SM, Scheitz JF, Bill O, Hametner C, et al. Intravenous Thrombolysis in Patients Dependent on the Daily Help of Others Before Stroke. Stroke. 2016; 47(2):450–6. Epub 2016/ 01/23. https://doi.org/10.1161/STROKEAHA.115.011674 PMID: 26797662. 12. Cochran WG. The effectiveness of adjustment by subclassification in removing bias in observational studies. Biometrics. 1968; 24(2):295–313. Epub 1968/06/01. PMID: 5683871. PLOS ONE | https://doi.org/10.1371/journal.pone.0231670 April 16, 2020 References 13. Senn S, Graf E, Caputo A. Stratification for the propensity score compared with linear regression tech- niques to assess the effect of treatment or exposure. Stat Med. 2007; 26(30):5529–44. Epub 2007/12/ 07. https://doi.org/10.1002/sim.3133 PMID: 18058851. 14. Bender R, Grouven U. Using binary logistic regression models for ordinal data with non-proportional odds. (0895–4356 (Print)). 15. Williams R. Generalized Ordered Logit/Partial Proportional Odds Models for Ordinal Dependent Vari- ables. The Stata Journal. 2006; 6(1):58–82. https://doi.org/10.1177/1536867x0600600104 10 / 10
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Simulation and measurement of optimized microwave reflectivity for carbon nanotube absorber by controlling electromagnetic factors
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Simulation and measurement of optimized microwave reflectivity for carbon nanotube absorber by controlling electromagnetic factors Received: 27 April 2016 Accepted: 18 October 2016 Published: xx xx xxxx Danfeng Zhang1, Zhifeng Hao1,2, Yannan Qian1, Yinxin Huang1, Bizeng1, Zhenda Yang1 & W Qibai1 Heat-treatments may change the defect and surface organic groups of carbon nanotubes (CNTs), and lead to significant changes in the microwave electromagnetic parameter of CNTs. In this paper, the effect of heat-treatment time and temperature on the complex dielectric constant and permeability as well as the microwave reflectivity of CNTs was investigated. The experimental results indicated that the microwave absorption property of CNTs arises mainly from the high permittivity and consequent dielectric loss. Moreover, the heat-treatment resulted in increased dielectric constant of CNTs and significant improvement of the microwave absorption at frequency values of 2–18 GHz. The microwave reflectivity of CNT composites with a coating thickness of 3 mm was simulated by using the electromagnetic parameters. The absorption peak of CNTs treated at 700 °C had an amplitude of R = −48 dB, which occurred at 9 GHz. Below −10 dB, the composites treated at 900 °C had a bandwidth of 7 GHz. The position of the absorption peak concurred with the measured results. The results indicated that the microwave-absorption properties can be modified by adjusting heat-treatment temperature and time. In recent years, carbon-based nano-materials, such as carbon nanotubes (CNTs), carbon nanoparticles, and car- bon onion have been considered as electromagnetic shielding and absorption materials with wide bandwidth, high absorption, tunable electromagnetic properties, and high reliability1–9. A good absorption material must effectively absorb and attenuate the incident electromagnetic wave. The design of electromagnetic absorption materials and structure is based on the transmission theory of electromagnetic waves in a medium. In addition, large complex permittivity and permeability of the materials are required for effective absorption, and impedance matching of the materials should be considered. Careful selection and preparation of an impedance matching layer with excellent complex permittivity are essential for dielectric loss-absorbing materials. The frequency char- acteristics of absorbing materials are determined by the thickness of the coating, the correlation between the concentration and frequency of the absorber, and weight proportion of the materials. CNTs, as a representative one-dimensional nanomaterial, have extraordinary electromagnetic wave-absorption properties and, hence, are considered as the promising excellent absorbent materials that exhibit good compatibility and possess excellent light quality and broad absorbing band width. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Guangdong University of Technology, Guangzhou, 510006, China. 2Foshan University, Foshan, 528000, China. Correspondence and requests for materials should be addressed to D.Z. (email: zhangdanfenggdut@126.com) or Z.H. (email: zfhao@fosu.edu.cn) Received: 27 April 2016 Accepted: 18 October 2016 Published: xx xx xxxx Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 Simulation and measurement of optimized microwave reflectivity for carbon nanotube absorber by controlling electromagnetic factors Abundant dangling bonds in the surface of CNTs could enhance the superficial polarization and the dielectric loss. Moreover, the high specific surface area could result in the mul- tiple scattering. These factors play an important role in the CNTs’ absorption properties, such as the high absorp- tion and wide-frequency band. CNTs have a series of comprehensive performances such as thermal and electrical conductivity, high strength, excellent corrosion resistance, thermal shock resistance, and high-temperature resist- ance and, hence, act as a high-temperature microwave absorber8. The low density of CNTs is favorable for obtain- ing light-weight composites. Therefore, CNTs have become one of the most important research topics in the new generation of “thin, light, wide, strong” absorbing materials.h g g g g The permittivity and permeability behaviors of composites fabricated from multi-walled CNTs with mag- netic impurity Ni and wax had been studied at frequencies of 3–18 GHz9. The 1.5-mm-thick composites 1Guangdong University of Technology, Guangzhou, 510006, China. 2Foshan University, Foshan, 528000, China. Correspondence and requests for materials should be addressed to D.Z. (email: zhangdanfenggdut@126.com) or Z.H. (email: zfhao@fosu.edu.cn) Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 1 www.nature.com/scientificreports/ exhibited excellent microwave absorption of the reflection loss (−20 dB). Kim10 fabricated hybrids of oxidized multi-walled CNTs and silver powder in a polyurethane matrix for electromagnetic interference shielding. Qi et al.11 synthesized bamboo-like multi-walled CNTs (BCNTs), which exhibited a reflection loss of −13.32 dB at 14.33 GHz, and a frequency bandwidth of >3 GHz was as lower than the reflection loss of −10 dB. Liu et al.12 found that the microwave absorption can be optimized at a given concentration and the absorption peak shifts with CNT content. Hou et al.13 suggested that a bilayer structure could increase the electromagnetic absorp- tion of the CNT composite. Zhai et al.14 investigated microwave-absorbing materials are composed of hydrogen- ated butadiene-acrylonitrile rubber (HNBR), carbon fiber (CF), carbon black (CB) and BCNTs. The reflection losses of −49.3 dB, −13.1 dB and −7.1 dB occurred at HNBR/BCNTs, HNBR/CF and HNBR/CB weight ratios of 100/10, 100/15 and 100/30, respectively. Despite excellent absorption properties, the mechanical properties of HNBR/BCNTs composite materials were also improved15. p p Heat treatments will change the defects and surface organic groups, such as the hydroxyl and carboxyl of the CNTs. The effect of CNT content on the microwave dielectric performance of composite materials has been extensively investigated12, 14, 16–18. Experimental p Calculating the reflectivity of absorbing wave coating by using the transmission line the- ory. Electromagnetic waves incident from free space into the medium interface may partially reflect on the surface and partially enter the interior of layer or multilayer absorbent materials of a certain thickness with the metallic substrates. Attenuation of electromagnetic waves or the electromagnetic-wave absorption of materials results from multiple repeated reflections between the material surface and the metal surface in the back. The pas- sage of electromagnetic waves through the coating surface and N layer dielectric to the metallic base plate is con- sidered as a multi-node lossy transmission line segment of a terminal short-circuit terminal. The characteristic impedance of the electromagnetic wave between each internode is considered, rather than the times of reflection and refraction interlayer and the degree of absorption. The total characteristic impedance of the entire coating is obtained via the recurrence relation, where the entire coating is considered as a monolayer during calculation of the reflectivity (absorption). This process is referred to as the transmission line method, which is used to calculate the reflectivity of the microwave-absorption coatings. l y p g Here, the coating on the metallic substrate is considered during vertical incidence of the electromagnetic wave. Using the transmission line method, the reflectivity R (absorption, dB) resulting from incidence of an elec- tromagnetic wave on an N layer coating can be determined as follows: = − + R Z Z Z Z 20 lg (1) n a n a (1) where Za is the intrinsic impedance of air, and Zn is the input impedance of the interface between the N layer material and the air, and the reflectivity unit is decibels (dB). Eq. (1) can be normalized as follows: where Za is the intrinsic impedance of air, and Zn is the input impedance of the interface between the N layer material and the air, and the reflectivity unit is decibels (dB). Eq. (1) can be normalized as follows: = − + R Z Z 20 lg 1 1 (2) n n (2) where Zn is the input impedance of the normalized N layer material. The recursion relation of each layer imped- ance is given as: where Zn is the input impedance of the normalized N layer material. Simulation and measurement of optimized microwave reflectivity for carbon nanotube absorber by controlling electromagnetic factors However, the dependence of the absorption intensity and frequency on the heat-treatment condition of CNTs has scarcely been quantified. Therefore, in this work, CNTs are heat-treated at different temperatures and times, and the corresponding pre- and post-treatment microwave dielectric properties are compared. The relevant electromagnetic parameters of the CNTs are also measured. Furthermore, the reflec- tivity of the CNT absorber coating is simulated based on the transmission line theory of an electromagnetic wave in a medium, and this reflectivity is compared with the experimental results. Experimental reduced, and the absorption entered the medium of electromagnetic should be enhanced. Therefore, the optimal value of the electromagnetic parameter is confirmed via simulation, and the optimized reflectivity (absorption) of the absorber coating is subsequently obtained. reduced, and the absorption entered the medium of electromagnetic should be enhanced. Therefore, the optimal value of the electromagnetic parameter is confirmed via simulation, and the optimized reflectivity (absorption) of the absorber coating is subsequently obtained. Preparation and measurement of nanotubes coating samples. A certain amount of CNTs was weighed, and a mixture of sulfuric acid and nitric acid was prepared in a volume ratio of 1:2. The mixed acid and CNTs were homogeneously mixed in a drying flask, heated to 155 °C, and refluxed for 5 h. The mixture was then filtered (i.e., to filter the mixed acid), washed with deionized water, and further filtered until a pH of 7 was obtained for the solution. After low-temperature drying under protective atmosphere, the CNTs were heat-treated at 700 °C and 900 °C, respectively, for 1, 2, 3, and 4 h. Figure 1 shows the scanning electron micro- scope (SEM) and transmission electron microscope (TEM) photos of carbon tubes treated with the mixed acid and then heat-treated at 700 °C. As the photos shown, the ports of the carbon tubes are broken and open. These carbon tubes with the pipe diameter of 20–40-nm and the lengths exceeding 1 μm have straight bar and arc morphologies.hfih p g The heat-treated CNTs and paraffin were mixed in a weight ratio of 85:15. These mixtures were then placed in the two stick rubber mixing machine, dispersed for 20 min, and pressed into a mold. The resulting circular composites, with inner diameter, outer diameter and length of 3.0 mm, 7.0 mm and 2–3 mm, respectively, meet the requirements of the coaxial measurement. An AV3618 vector network analyzer can be used to determine the electromagnetic parameters of the measured samples at frequencies ranging from 2 to 18 GHz. The electromag- netic parameters of paraffin (ε′: ∼2.2 and μ′: ∼0.6) are significantly smaller than those of the CNTs. Therefore, the electromagnetic parameters of CNTs are taken as the electromagnetic parameters of the composites. Equation (6) is obtained by substituting the electromagnetic parameters of the heat-treated carbon tube/paraffin composite coating into equation (2). Here, the thickness of the composite coating is set to 2 mm to calculate the microwave reflectivity. Experimental The recursion relation of each layer imped- ance is given as: η γ η γ = + + − − Z Z th d Z th d ( ) ( ) (3) n n n n n n n n n 1 1 η µ ε = / (4) n rn rn γ π µ ε = j f c 2 / (5) n rn rn (3) (4) (5) where c: velocity of light, f: frequency of the electromagnetic wave, μm and εm: complex permeability and permit- tivity of the N layer material, respectively, dn: thickness of the N layer material, and th: hyperbolic tangent func- tion. The substrate is metallic (Z0 = 0) and, hence, the overall impedance (from the first to the N layer coating) can be determined from the recursion formula. In addition, the reflectivity R(N) is calculated based on the obtained characteristic impedance of the surface coating (N layer).l where c: velocity of light, f: frequency of the electromagnetic wave, μm and εm: complex permeability and permit- tivity of the N layer material, respectively, dn: thickness of the N layer material, and th: hyperbolic tangent func- tion. The substrate is metallic (Z0 = 0) and, hence, the overall impedance (from the first to the N layer coating) can be determined from the recursion formula. In addition, the reflectivity R(N) is calculated based on the obtained characteristic impedance of the surface coating (N layer).l g y When the reflectivity of the absorber coating is calculated via the transmission line method, the characteris- tic impedance of the coating is decided by the complex permittivity ε (or εr) and permeability μ (or μr), which is used to represent the electromagnetic property of the absorber. The permittivity and permeability under an alternating magnetic field are expressed as ε = ε′ + jε″ and μ = μ′ + jμ″ (in plural form). Considering the effect of the medium on electromagnetic-wave absorption, ε″ and μ″ > ε′ and μ′ is preferable. Impedance matching of the materials is also considered, i.e., the reflection of electromagnetic wave on the incidence interface should be Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Morphology of the CNT samples heat-treated at 700 °C: (a) SEM image and (b) TEM image. Figure 1. Morphology of the CNT samples heat-treated at 700 °C: (a) SEM image and (b) TEM image. Experimental In equation (6), R is the reflectivity (dB), µr is the relative permeability, εr is the relative dielectric con- stant, d is the coating thickness (mm), c is the light velocity of free space, and f is frequency. ε µ ε µ =    −    + µ ε π µ ε π R h j d h j d 20log tan 1 tan 1 (6) f c r r f c r r 10 2 2 r r r r (6) The measured electromagnetic parameters are imported into Matlab, and R is simulated via the transmission line method for different coating thicknesses. The effect of the heat-treatment temperature and coating thickness on R was also determined. The 180 × 180 mm standard absorption sample plates (see Fig. 2), with a 2-mm-thick coating, are prepared from CNTs and silicone resin composites. The practical reflectivity of the composites is measured via the Arch method (see Fig. 3). Results and Discussion Relative complex permittivity and permeability of the paraffin-CNTs composites. The real part (ε′) of the complex dielectric constant of the CNT composites is measured at microwave frequencies of 2–18 GHz. Figure 4 shows the relationship between the real part of complex dielectric constant and frequency for CNTs heat- treated at temperatures of 700 °C and 900 °C, respectively, for 1, 2, 3 and 4 h. As shown in Fig. 4, the maximum value of real parts (i.e., ε′ = 21) for heat-treated CNTs occurs in the low-frequency band (2–8 GHz).f In the high-frequency band (9–18 GHz), the heat treatment has only a modest effect on ε′, as evidenced by smaller changes in its value compared to those occurring in the low-frequency band. The experimental results indicate that ε′ may be affected by the heat-treatment temperature and time. Excessively high temperatures (e.g., 900 °C) should reduce the density of defects and groups, leading to a reduction in the number of electric dipoles and, in turn, the dielectric constant of CNTs. Furthermore, excessively long or short heat-treatment time will lead Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 3 ntificreports/ Figure 2. The coating samples were painted onto the 180 mm × 180 mm standard aluminium plate. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. The coating samples were painted onto the 180 mm × 180 mm standard aluminium plate. Figure 3. Equipment used to evaluate the practical reflectivity of absorbent materials, via the Arch method. Figure 3. Equipment used to evaluate the practical reflectivity of absorbent materials, via the Arch method. Figure 4. Real part (ε′) of the complex dielectric constant of CNT composites heat-treated for 1, 2, 3, and 4 h at temperatures of 700 °C and 900 °C; ε′ of the untreated composites is denoted as 0 h. Figure 4. Real part (ε′) of the complex dielectric constant of CNT composites heat-treated for 1, 2, 3, and 4 h at temperatures of 700 °C and 900 °C; ε′ of the untreated composites is denoted as 0 h. to a decrease in the dielectric constant. Short times are inadequate for complete optimization of the structure of the CNTs, whereas excessive times will lead to a decrease in the number of CNTs and electric dipoles. This will to a decrease in the dielectric constant. o a decrease in the dielectric constant. Short times are inadequate for complete optimization of the structure o he CNTs, whereas excessive times will lead to a decrease in the number of CNTs and electric dipoles. This wil Results and Discussion ε″ increases with increasing heat-treatment time, reaching a maximum value (i.e., 17) at heat-treating time of 3 h, and decreasing thereafter. This indicates that sufficient heat-treatment time is required for structural optimization of CNTs, and insufficient time will lead to an unstable ε″. However, excessive heat-treatment time may reduce the number of CNTs, leading to a reduction in the number of electric dipoles. The change in ε″ is consistent with the change in ε′. Heat treatments may modify the surface structure of CNTs, reduce the defect density, transform the structure, and increase the dielectric constant. The complex dielectric constant obtained via heat treating at 700 °C is larger than that obtained at 900 °C. The shift of the complex dielectric constant to the low-frequency band is condu- cive for absorption at low frequency. Excessive heat-treatment temperatures may erode carbon tubes, lead to a reduction of electric dipoles, and result in a decrease in the dielectric constant. Therefore, the complex dielectric constant and frequency of CNTs can be tailored by varying the heat-treatment temperature and time. Figures 6 and 7 show the frequency-dependence of the real (μ′) and imaginary (μ″) parts, respectively, of the permeability of the untreated and (three of the aforementioned) heat-treated CNTs. As Fig. 6 shows, the original untreated CNTs have a μ′ of ∼1.1. The high-temperature treatment has only a slight effect on μ′, which is almost constant at low frequency. At high frequencies, μ′ of the treated CNTs reaches a maximum value of 1.4. Fig. 7 displays the imaginary part (i.e., μ″) of the complex permeability constant for both the treated and untreated composites. As shown in Fig. 7, μ″ of the treated CNTs exhibits the same behavior as the μ″ of the untreated CNTs. This similarity results possibly from the fact that the heat-treatment induced change in the number of electric dipoles affects mainly the dielectric constant rather than the permeability. Reflection loss of CNT composites: simulation and measurement. The correlation between the electromagnetic parameter and reflectivity of materials indicates that the absorption reflectivity of these mate- rials will increase with increasing real component of the complex dielectric constant. Figure 8 shows the simu- lated reflectivity of the heat-treated and the untreated composites; these composites consist of 3-mm-thick CNTs, which account for 15 wt.% of the composite. Figure 8(a) shows the reflectivity of CNT composites treated at 700 °C. Results and Discussion Short times are inadequate for complete optimization of the structure of the CNTs, whereas excessive times will lead to a decrease in the number of CNTs and electric dipoles. This will Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 4 www.nature.com/scientificreports/ Figure 5. Imaginary part (ε″) of the complex dielectric constant of CNT composites heat-treated for 1, 2, 3, and 4 h at temperatures of 700 °C and 900 °C; ε″ of the untreated composites is denoted as 0 h. Figure 5. Imaginary part (ε″) of the complex dielectric constant of CNT composites heat-treated for 1, 2, 3, and 4 h at temperatures of 700 °C and 900 °C; ε″ of the untreated composites is denoted as 0 h. lead to a decrease in the dielectric constant of the CNTs. The maximum ε′ is obtained at a heat-treatment time of 3 h. As CNTs are non-magnetic materials, their absorption originates either from polarization or multiple scat- tering due to the large specific surface area. Compared with single-walled CNTs (SWCNTs), multi-walled CNTs (MWCNTs) have higher defect densities, and therefore higher permittivity, and their microwave-absorption abil- ity arises mainly from dielectric loss. Previous studies have indicated that the defects and impurities in CNTs are influenced by the polarization of the composite materials19, 20. The electromagnetic-wave absorption of CNTs arises mainly from electric dipole steering and interfacial polarization21, 22 because the increasing density of defects and groups leads to the increasing density of electric dipoles. Under an external electromagnetic field, the defects and dangling bonds in the CNTs would become the polarization center, and the interfacial polarization of the CNTs as well as the dielectric relaxation and dielectric loss increase. Figure 5 shows the frequency dependence of the imaginary part (ε″) of the complex dielectric constant of the CNT composites heat-treated for 1–4 h at 700 °C and 900 °C. As shown in Fig. 5, ε″ of the heat-treated CNTs exhibits a higher frequency dependence than the ε″ of the untreated CNTs. The maximum ε″ of the untreated CNTs (i.e., ε″ = 5.5) occurs at ∼9 GNz. However, a maximum ε″ of 17 (i.e., ε″ = 17) is obtained for the heat-treated CNTs. The maximum ε″, associated with each heat-treatment time, occurs in the low-frequency band. The peak in the dielectric loss indicates that the highest absorption occurs at 7–9 GHz. www.nature.com/scientificreports/ www.nature.com/scientificreports/ tificreports/ Figure 6. Real part (μ′) of the complex permeability of CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ′ of the untreated composites is denoted as 0 h. Figure 7. Imaginary part (μ″) of the complex permeability of the CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ″ of the untreated composites is denoted as 0 h. Figure 6. Real part (μ′) of the complex permeability of CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ′ of the untreated composites is denoted as 0 h. Figure 6. Real part (μ′) of the complex permeability of CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ′ of the untreated composites is denoted as 0 h. Figure 7. Imaginary part (μ″) of the complex permeability of the CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ″ of the untreated composites is denoted as 0 h. Figure 7. Imaginary part (μ″) of the complex permeability of the CNT composites heat-treated for 2, 3, and 4 h at temperatures of 700 °C and 900 °C; μ″ of the untreated composites is denoted as 0 h. Figure 8. Simulated reflectivity of CNT composites heat-treated for 3 and 4 h at temperatures of 700 °C and 900 °C. The simulated reflectivity of the untreated composites is denoted as 0 h. Figure 8. Simulated reflectivity of CNT composites heat-treated for 3 and 4 h at temperatures of 700 °C and 900 °C. The simulated reflectivity of the untreated composites is denoted as 0 h. heat treatment may reduce the internal defects of the CNTs, making the structure more perfect and imparting the intrinsic characteristics (e.g., high dielectric loss) of the CNTs. For the time considered here, the dielectric con- stant of the carbon tubes increases. However, excessive heat-treatment times would erode the number of carbon tubes and lead to a reduction of electric dipoles, the dielectric constant and, consequently, the reflectivity absorp- tion peak. The observed increase in the absorption peak is, as in the case of previous studies16–18, attributed to an increase in the dielectric losses. When the heat-treatment temperature is increased to 900 °C (see Fig. Results and Discussion As the figure shows, the microwave-absorbing reflectivity of the untreated composites occurs at ∼7 GHz, and the maximum absorption-peak reflectivity is −25 dB. Heat treating at the temperature (700 °C) may yield the improved absorption of the CNTs. For example, a peak value of −48 dB and a bandwidth of ∼5 GHz below −10 dB are obtained for the 3 h heat-treated CNT composites. These experimental results are consistent with those obtained, at frequencies of 7–9 GHz, for the imaginary part of the complex dielectric constant. When the heat-treatment time is increased to 4 h, the reflectance absorption peak shifts to low frequency, towards the peak corresponding to the untreated CNTs, and has an amplitude of ∼−37 dB (6.5 GHz). The high temperature of the Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 5 www.nature.com/scientificreports/ 8(b)), the simulated reflectivity absorption peak of the 3 h-treated composites decreases to −36 dB (at ∼10 GHz). However, a bandwidth of ∼7 GHz, which is expected to increase, occurs below −10 dB. These results indicate that the real and imaginary part of the complex dielectric constant may be modified, and the microwave absorption effect of heat treatment may reduce the internal defects of the CNTs, making the structure more perfect and imparting the intrinsic characteristics (e.g., high dielectric loss) of the CNTs. For the time considered here, the dielectric con- stant of the carbon tubes increases. However, excessive heat-treatment times would erode the number of carbon tubes and lead to a reduction of electric dipoles, the dielectric constant and, consequently, the reflectivity absorp- tion peak. The observed increase in the absorption peak is, as in the case of previous studies16–18, attributed to an increase in the dielectric losses. When the heat-treatment temperature is increased to 900 °C (see Fig. 8(b)), the simulated reflectivity absorption peak of the 3 h-treated composites decreases to −36 dB (at ∼10 GHz). However, a bandwidth of ∼7 GHz, which is expected to increase, occurs below −10 dB. These results indicate that the real and imaginary part of the complex dielectric constant may be modified, and the microwave absorption effect of Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 6 ntificreports/ Figure 9. The experimental reflectivity curve of 3-mm-thick CNT composite coatings with a CNT content of 15 wt.%. The composites were heat-treated for 3 h at temperatures of 700 °C and 900 °C. www.nature.com/scientificreports/ Figure 9. The experimental reflectivity curve of 3-mm-thick CNT composite coatings with a CNT content of 15 wt.%. The composites were heat-treated for 3 h at temperatures of 700 °C and 900 °C. CNTs further tailored by controlling the heat-treatment temperature and time of the CNTs. This could ensure fulfillment of requirements for various frequency band and bandwidth.l CNTs further tailored by controlling the heat-treatment temperature and time of the CNTs. This could ensure fulfillment of requirements for various frequency band and bandwidth.l i Figure 9 shows the measured reflectivity curve of a 3-mm-thick composite coating with a CNT content of 15 wt.%; this curve is obtained via the Arch method. As shows in Fig. 9, the absorption peak of the compos- ites treated at 700 °C has a minimum value of −15.2 dB (8 GHz). www.nature.com/scientificreports/ The corresponding peak of the composite heat-treated at 900 °C has a minimum value of ∼−14.2 dB (11 GHz). This trend is relatively consistent with the simulated results. The theoretical and experimental studies on CNT composites reveal that the interface prop- erties of CNTs are influenced by the heat-treatment process. These properties will ultimately influence the elec- tromagnetic factors and microwave-absorption characteristics. The effect of interfacial polarization was also demonstrated by comparing the microwave-absorption characteristics of CNT/polymer and decorated CNT/ polymer composites23–25. These observations indicate that strong interfacial polarization is essential for high microwave absorption. References pp g p 3. Park, S. H., Theilmann, P. T., Asbeck, P. M. & Bandaru, P. R. Enhanced electromagnetic interference shielding through the use o functionalized carbon-nanotube-reactive polymer composites. IEEE Trans. Nanotechnol. 9, 464–469 (2010).i p y p ( ) 4. Qin, F. & Brosseau, C. A review and analysis of microwave absorption in polymer composites filled with carbonaceous particles. J Appl. Phys. 111, 061301–061325 (2012). pp y 5. 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Controllable and large-scale synthesis of carbon nanofibers, bamboo-Like nanotubes, and chains of nanospheres over Fe/SnO2 and their microwave-absorption properties. J Phys. Chem. Conclusionhf The effect of heat-treatment temperature and time on the electromagnetic parameters and microwave absorption of CNTs was investigated. The experimental results indicate that heat-treatments can increase the dielectric con- stant of CNTs and significantly improve their microwave absorption at frequency values of 2–18 GHz. In addition, the microwave reflectivity of the CNT composites, with a coating thickness of 3 mm, are simulated by using the electromagnetic parameters. The absorption peak of CNTs treated at 700 °C has an amplitude of R = −48 dB, which occurs at 9 GHz. Below −10 dB, the composites treated at 900 °C have a bandwidth of 7 GHz. The position of the absorption peak concurs with the measured results. The results indicate that the microwave-absorption properties can be modified by adjusting the heat-treatment temperature and time. 1. Wu, J. & Kong, L. High microwave permittivity of multiwalled carbon nanotube composites. Appl. Phys. Lett. 84, 4956–4958 (2004). 2. Kuzhir, P. P., Paddubskaya, A. G., Maksimenko, S. A., Kuznetsov, V. L. & Moseenkov, S. et al. Carbon onion composites for EMC applications. IEEE Trans. Electromagn. Compat. 54, 6–16 (2012).h Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 Acknowledgements g This work is supported by the Science and Technology Program of Guangdong Province of China (Nos. 2014B010106005 and 2013B051000077), and the Science and Technology Program of Guangzhou City of China (No. 201508030018). g This work is supported by the Science and Technology Program of Guangdong Province of China (Nos. 2014B010106005 and 2013B051000077), and the Science and Technology Program of Guangzhou City of China (No. 201508030018). 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Microwave absorption of single-walled carbon nanotubes/soluble cross-linked polyurethane composites. J. Phys. Chem. C 111, 13696–13700 (2007). 13. Hou, C. L., Li, T. H., Zhao, T. K., Zhang, W. J. & Cheng, Y. L. Electromagnetic wave absorbing properties of carbon nanotubes doped rare metal/pure carbon nanotubes double-layer polymer composites. Mater. Design 33, 413–418 (2012).f p y p y p g , ( ) 14. Zhai, Y. H., Zhang, Y. & Ren, W. T. Electromagnetic characteristic and microwave absorbing performance of different carbon-b hydrogenated acrylonitrile- butadiene rubber composites. Mater. Chem. Phys. 133, 176–181 (2012). Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 7 Additional Informationh Competing Interests: The authors declare that they have no competing interests. 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. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 7: 479 | DOI:10.1038/s41598-017-00372-9 8
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Netrin-1 Confines Rhombic Lip-Derived Neurons to the CNS
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:35982199 Citation Citation Yung, Andrea R., Noah R. Druckenbrod, Jean-François Cloutier, Zhuhao Wu, Marc Tessier- Lavigne, and Lisa V. Goodrich. 2018. “Netrin-1 Confines Rhombic Lip-Derived Neurons to the CNS.” Cell reports 22 (7): 1666-1680. doi:10.1016/j.celrep.2018.01.068. http://dx.doi.org/10.1016/ j.celrep.2018.01.068. Published Version doi:10.1016/j.celrep.2018.01.068 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). *Correspondence: lisa_goodrich@hms.harvard.edu. 4Lead Contact 5Present address: Department of Biology, Stanford University, Stanford, CA 94306, USA SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and seven figures and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.01.068. AUTHOR CONTRIBUTIONS A.R.Y. and L.V.G. designed the research, analyzed the results, and wrote the manuscript. N.R.D. discovered the phenotype and A.R.Y. performed all the experiments. J.-F.C. provided the Wnt1Cre; DCC−/−; Neo1fl/fl, and Neo1−/− tissue. Z.W. and M.T.-L. provided the DCC+/−; Neo1Gt/+ mice and tissue. J.-F.C., Z.W., and M.T.-L. also shaped the course of the study and provided input on the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Netrin-1 Confines Rhombic Lip-Derived Neurons to the CNS Andrea R. Yung1, Noah R. Druckenbrod1, Jean-François Cloutier2, Zhuhao Wu3, Marc Tessier-Lavigne2,5, and Lisa V. Goodrich1,4,* 1Department of Neurobiology, Harvard Medical School, Boston, MA, USA 2Department of Neurology & Neurosurgery Montreal Neurological Institute McGill University 2Department of Neurology & Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada Author Manuscript 3Laboratory of Brain Development & Repair, The Rockefeller University, New York, NY 10065, USA HHS Public Access Author Manuscript Published in final edited form as: Published in final edited form as: Cell Rep. 2018 February 13; 22(7): 1666–1680. do Cell Rep. 2018 February 13; 22(7): 1666–1680. doi:10.1016/j.celrep.2018.01.068. SUMMARY During brainstem development, newborn neurons originating from the rhombic lip embark on exceptionally long migrations to generate nuclei important for audition, movement, and respiration. Along the way, this highly motile population passes several cranial nerves yet remains confined to the CNS. We found that Ntn1 accumulates beneath the pial surface separating the CNS from the PNS, with gaps at nerve entry sites. In mice null for Ntn1 or its receptor DCC, hindbrain neurons enter cranial nerves and migrate into the periphery. CNS neurons also escape when Ntn1 is selectively lost from the sub-pial region (SPR), and conversely, expression of Ntn1 throughout the mutant hindbrain can prevent their departure. These findings identify a permissive role for Ntn1 in maintaining the CNS-PNS boundary. We propose that Ntn1 confines rhombic lip-derived neurons by providing a preferred substrate for tangentially migrating neurons in the SPR, preventing their entry into nerve roots. Author Manuscript In Brief In Brief Author Manuscript Yung et al. Page 2 Page 2 Yung et al. show that Ntn1 prevents pontine neurons from migrating into the periphery along cranial nerves by providing a preferred substrate in the sub-pial region. These findings introduce a local, permissive role for Ntn1 in the maintenance of the CNS-PNS boundary in the developing mouse hindbrain. Author Manuscript Author Manuscript Yung et al. show that Ntn1 prevents pontine neurons from migrating into the periphery along cranial nerves by providing a preferred substrate in the sub-pial region. These findings introduce a local, permissive role for Ntn1 in the maintenance of the CNS-PNS boundary in the developing mouse hindbrain. Cell Rep. Author manuscript; available in PMC 2018 March 30. INTRODUCTION A basic organizing principle of the nervous system is the segregation of the peripheral nervous system (PNS) and CNS, which are anatomically and functionally distinct yet linked by nerves. This is particularly apparent in the vertebrate brainstem, which houses ten cranial nerves as well as a constellation of nuclei that govern functions critical to life, from motor coordination to auditory processing (Farago et al., 2006; Wang et al., 2005). Many of these nuclei are composed of neurons originating from the rhombic lip, a transient strip of proliferating neuroepithelium lining the fourth ventricle during development (Ray and Dymecki, 2009). The formation of hindbrain nuclei, therefore, depends on the successful tangential migration of newborn neurons from the rhombic lip to their final destinations. This route is unusually long and complex, especially since the surface of the hindbrain is broken by multiple cranial nerve roots that the rhombic lip derivatives must ignore. Although several guidance cues play critical roles sculpting the trajectory of tangentially migrating neurons in vivo (reviewed in Kratochwil et al., 2017), nothing is known about the molecular mechanisms that confine these neurons to the CNS, despite opportunities to deviate into the periphery. Author Manuscript Author Manuscript Pontine neurons (PNs) traverse one of the longest migratory routes in the hindbrain, ultimately settling at the midline to supply excitatory mossy fiber input to the cerebellum (Kratochwil et al., 2017). PNs originate from the rhombic lip in rhombomeres (r)6–r8 from embryonic day (E)12.5 to E16.5. They extend long leading processes (Ono and Kawamura, 1990; Yee et al., 1999) as they migrate beneath the pial surface, maneuvering between the trigeminal (Vth), facial (VIIth), and vestibulocochlear (VIIIth) nerve roots and arriving at the Cell Rep. Author manuscript; available in PMC 2018 March 30. Page 3 Page 3 Yung et al. Yung et al. ventral midline of r3–r4 several days later (Nichols and Bruce, 2006) (Figure 1A). This navigation depends on the activity of several guidance cues, including Slits, which are secreted by the facial motor nucleus to prevent premature ventral migration (Geisen et al., 2008), and meninges-derived chemokine SDF-1, which keeps PNs from migrating into the neuroepithelium (Zhu et al., 2009). What prevents PNs from escaping in the opposite direction, into the periphery, is unknown. Author Manuscript Auth Author Manuscript One of the first guidance cues implicated in PN migration is the classic chemoattractant Netrin-1 (Ntn1). RESULTS Author Manuscript INTRODUCTION PNs are highly sensitive to Ntn1 and can migrate toward a source of Ntn1 over unusually long distances in vitro (Yee et al., 1999); in vivo, the pontine nuclei are missing in mice severely hypomorphic for Ntn1 (Serafini et al., 1996; Yee et al., 1999). These data were originally interpreted to indicate that a floor-plate-derived gradient of Ntn1 guides PNs to the midline during the final leg of their migration (Zelina et al., 2014), much as Ntn1 was proposed to guide commissural axon growth in the developing spinal cord (Kennedy et al., 2006; Serafini et al., 1996). However, Ntn1 is also expressed in the ventricular zone (Kennedy et al., 1994; Serafini et al., 1996), and this source is required for proper commissure formation (Charron et al., 2003). The protein itself is deposited in the sub-pial region (SPR) adjacent to the basement membrane (BM) surrounding the neural tube (Kennedy et al., 2006; MacLennan et al., 1997; Varadarajan et al., 2017). Recent genetic studies have underscored the importance of SPR-localized Ntn1 for commissural axon guidance (Dominici et al., 2017; Varadarajan et al., 2017; Yamauchi et al., 2017), consistent with documented functions for Ntn1 in the BM of other tissues (Liu et al., 2004; Srinivasan et al., 2003; Yebra et al., 2003). This suggests that additional roles for locally produced Ntn1 in the developing nervous system likely remain to be found, particularly in cases such as PN migration, where neurons migrate along the pial surface rather than through the neuroepithelium. Indeed, the complete range of Ntn1’s effects on PN migration is still unclear, due both to the hypomorphic nature of the original allele and to the lack of information about the ultimate fate of PNs. Author Manuscript Here, we show that Ntn1 functions as a permissive cue to confine PNs to the CNS. We propose that Ntn1 in the sub-pial region provides a preferred corridor for migrating rhombic lip-derived neurons, allowing them to distinguish the appropriate migratory substrate and avoid opportunities to migrate instead into the periphery. These findings introduce another local function for Ntn1 and establish an additional molecular explanation for how CNS-PNS segregation is achieved in the brainstem, a hub for CNS-PNS interactions that are vital for life. Cell Rep. Author manuscript; available in PMC 2018 March 30. Ntn1 Protein Is Enriched in the SPR in the Developing Hindbrain Migrating PNs follow a stereotyped pathway to the ventral midline that can be divided into three phases: (1) a short ventral migration marking the departure from the rhombic lip; (2) a relatively straight rostral migration between and past the vestibulocochlear (VIIIth), facial (VIIth), and trigeminal (Vth) nerves, respectively; and (3) a final ventral turn before resting at the midline (Figure 1A) (Geisen et al., 2008; Nichols and Bruce, 2006). The entire migration takes place in the space beneath the pia, which we call the sub-pial region (SPR). Cell Rep. Author manuscript; available in PMC 2018 March 30. Page 4 Yung et al. While the molecules that define many aspects of this complex migratory route have been identified, the cues that instruct PNs to stay in the SPR and avoid cranial nerve roots remain unknown. During the peak of migration at E15.5, PNs traverse hundreds of microns as they move past the cranial nerves. Throughout this journey, PNs express the Ntn1 receptor Deleted in Colorectal Carcinoma (DCC) and are exposed to Ntn1 produced in the floor plate (FP) and ventricular zone (Yee et al., 1999). When Ntn1 levels are reduced, rare spinal cord interneuron axons can be found in dorsal root ganglia (Laumonnerie et al., 2014), hinting at a role in setting the CNS-PNS boundary. We therefore hypothesized that Ntn1 not only guides PNs to the midline but also keeps them contained within the CNS. Author Manuscript Author Author Manuscript Since Ntn1 is a potent secreted cue, we examined the localization of Ntn1 relative to its sources in the hindbrain at the onset of PN migration at E13.5 (de Diego et al., 2002; Yee et al., 1999). Immunostaining revealed that Ntn1 protein is widely distributed (Figures 1B and 1B’; n = 2 animals), accumulating in the FP, on commissural axons, and in the SPR in the vicinity of the laminin-positive pial BM, as described previously (Dominici et al., 2017; Kennedy et al., 2006; MacLennan et al., 1997; Varadarajan et al., 2017). Notably, Ntn1 is absent from nerve roots, where cranial nerves project into or out of the CNS via gaps in the pial BM at stereotyped locations (Figures 1C and 1C’; n = 2 animals). Thus, Ntn1 protein is present where PNs migrate, but not at sites they avoid. Ntn1 Protein Is Enriched in the SPR in the Developing Hindbrain Author Manuscript To determine whether migrating PNs might encounter and respond to Ntn1 in the SPR, we stained for PN markers Pax6 and DCC at E13.5, when PNs have begun exiting the rhombic lip, and at E15.5, when they are passing by cranial nerves. At E13.5, Pax6+/DCC+ cells cluster beneath the pial BM (Figures 1D–1E’; n = 4 animals) and maintain this position as they navigate near the facial (VII) and vestibulocochlear (VIII) nerves at E15.5 (Figure 1F). Since Ntn1 is enriched in the SPR, migrating DCC+ PNs likely encounter Ntn1 from early on. Given the conspicuous absence of Ntn1 at nerve roots—which migrating PNs avoid— this pattern of distribution suggests that Ntn1 may contribute to the confinement of tangentially migrating neurons in the hindbrain by providing an attractive substrate. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Loss of Ntn1 Causes PNs to Exit the Hindbrain and Enter the Periphery Earlier analyses of hypomorphic Ntn1 animals (Ntn1trap/trap) suggested that Ntn1 mediates the final ventral migration to the midline, as PNs complete most of the first and second phases of their migration (Zelina et al., 2014). However, phenotypic analyses of Ntn1 null animals (Ntn1−/−) showed that residual Ntn1 in hypomorphs masks the full extent of its role in guidance (Bin et al., 2015; Yung et al., 2015). We posited that complete loss of Ntn1 might reveal additional functions earlier in migration, particularly since PNs are exposed to Ntn1 far before their final ventral turn. Author Manuscript To visualize PN distribution, we collected transverse sections of embryonic Ntn1−/− heads spanning the anterior extramural stream (AES) through which PNs travel. In E15.5 control animals (n = 4), the AES is identifiable as a dense stripe of Pax6+ nuclei and DCC+ processes traveling beneath the pial surface (Figures 2B–2D), giving rise to the pontine nuclei at the midline, which first appear at late E14.5 (Nichols and Bruce, 2006). In contrast, in Ntn1−/− animals (n = 6), the AES was missing, and there were ectopic streams of Pax6+ and DCC+ neurons immediately ventral to the stereotyped location of the AES, as if the PNs Page 5 Yung et al. Yung et al. had been diverted into the periphery (Figures 2B’ and 2C’). Ectopic Pax6+ nuclei and a few DCC+ processes were rarely found in the trigeminal (Vth) ganglion (Figure 2D’). However, the facial and vestibulocochlear nerves contained similar numbers of ectopic nuclei (Figure 2E; n = 6 nerves each). We focused on the vestibulocochlear (VIIIth) nerve as a site of exit, as it is the first nerve root that migrating PNs pass and because the cochlea is an enclosed, easily recognizable landmark. These findings suggest that, in addition to mediating the final ventral turn to the midline, Ntn1 acts earlier to prevent PNs from migrating along cranial nerves and into the periphery. Author Manuscript Auth Author Manuscript To confirm the origin and identity of these neurons, we genetically labeled PNs by providing tamoxifen to E13.5 Atoh1CreERT2;Ai14 mice crossed onto the Ntn1 null background. tdTomato+ cells also expressed both Pax6 and DCC (Figures 2G–2H’’’; n = 3 animals), demonstrating that the ectopic neurons in the cochlea derive from the rhombic lip. Loss of Ntn1 Causes PNs to Exit the Hindbrain and Enter the Periphery Likewise, in Ntn1 mutants, Pax6+ neurons first reach the cochlea at E13.5 and increase in number steadily up until E15.5 (Figure 2F; n = 6 cochleae per time point), matching the timing of PN production and migration. These neurons accumulate mostly in the base and middle turns of the cochlea, which lie closest to the hindbrain. Taken together, these data demonstrate that the Pax6+ neurons invading Ntn1−/− cochleae are bona fide PNs. Author Manuscript Despite their ectopic location, the misrouted PNs survived and differentiated within the cochlea. At E18.5, 103.5 ± 42.6 (mean ± SD) Pax6+ PNs were present in the cochlea, but instead of integrating into the spiral ganglion, the PNs, which express low levels of Gata3, formed a rind around the Gata3-high spiral ganglion neurons (SGNs; Figures 3A–3C’; n = 3 Ntn1−/−). The gross organization of the spiral ganglion was remarkably normal, as visualized by crossing a MafBGFP allele (Moriguchi et al., 2006) into the Ntn1−/− background. As in control embryos, GFP+ SGNs extended orderly bundles of radial fibers toward the hair cells in mutants (Figures 3D and 3F; n = 3 Ntn1−/−), beneath an overlying swath of processes from GFP−/Tuj+ PNs (Figures 3E and 3G). Since Ntn1 mutants die at birth, we could not examine the fate of ectopic PNs in adults. Nonetheless, these data show that PNs thrive in the cochlea but stay segregated from the SGNs. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. DCC Is Also Required for PN Confinement Since PNs express DCC and fail to reach the midline in DCC−/− animals (Fazeli et al., 1997; Yee et al., 1999), we hypothesized that Ntn1-DCC signaling underlies their confinement. Consistent with this idea, DCC−/− animals contain ectopic neurons in the cochlea that were assumed to be displaced SGNs (Kim et al., 2016). We found not only that ectopic neurons in DCC−/− cochlea express Pax6, indicative of PN identity instead, but also that Pax6+ neurons are also present elsewhere in the periphery, such as in the VIIth and VIIIth nerves, demonstrating that DCC enables Ntn1-mediated confinement. However, there were not as many neurons in DCC−/− cochleae as in Ntn1 mutants (Figure 4A; n ≥ 6 cochlea per genotype), hinting that another receptor also contributes. Author Manuscript Ntn1 signals through many receptors, including Unc5 family members, Down syndrome cell adhesion molecule (DSCAM), integrins, and the DCC ortholog Neogenin (reviewed in Lai Wing Sun et al., 2011). We predicted that Neogenin might influence Ntn1-mediated confinement, since DCC and Neogenin collaborate to mediate midline crossing in commissural neurons (Xu et al., 2014). As in previous reports (Brugeaud et al., 2014; Fitzgerald et al., 2007; van den Heuvel et al., 2013), we observed low levels of Neogenin throughout the E15.5 hindbrain (Figures 4B and 4B’; n = 2 Ntn1+/−), with stronger expression in the surrounding mesenchyme and SGNs. We did not detect Neogenin in migrating PNs and found no obvious qualitative differences in the size or location of the pontine nuclei or the trajectory of the AES in either Neo1 hypomorphs (Neo1Gt/Gt; Bae et al., 2009; Leighton et al., 2001) (Figures 4D–4G; n = 3 Neo1Gt/Gt) or null animals (Kam et al., 2016) (Figures S3A and S3B; n = 3 Neo1−/−). Ectopic PNs in the cochleae also did not express Neogenin (Figures 4C and 4C’; n = 3 Ntn1−/−), suggesting that Neogenin is not required for PN migration or confinement. Nonetheless, increasing numbers of Pax6+ neurons accumulated in the cochlea, as more copies of Neo1 were lost in the DCC null background (Figure 4A). Thus, Neogenin affects Ntn1-mediated PN confinement when DCC is absent, but not when DCC is present. Author Manuscript Author Manuscript Multiple Populations of Neurons Escape the CNS in Ntn1 Mutants In addition to PNs, many other rhombic lip derivatives migrate through the SPR, including neurons of the cochlear nucleus, inferior olive, and external cuneate nucleus (Machold and Fishell, 2005; Nichols and Bruce, 2006; Wang et al., 2005). As these neurons also respond to Ntn1 (Alcántara et al., 2000; Bloch-Gallego et al., 1999; Causeret et al., 2002; Howell et al., 2007; Riyadh et al., 2014; Marcos et al., 2009), we wondered if Ntn1’s role in confinement extends to other rhombic lip derivatives. Since Ntn1 is expressed in the neural tube as early as E9.5 (Kennedy et al., 1994; Serafini et al., 1994; Yee et al., 1999), we examined the trajectory of some of the earliest born rhombic lip-derived neurons in the CNS by injecting tamoxifen at E9.5 into Atoh1CreERT2;Ai14 mice crossed to the Ntn1 null background. As expected, many commissural neurons were labeled, shown by the presence of tdTomato+ processes crossing the midline at E11.5 (Figures S1A’–S1A’’’; n = 2 controls). In Ntn1−/− animals, tdTomato+ neurons resided in more dorsal locations, and the commissure failed to form. Additionally, many labeled cell bodies and processes were found outside the hindbrain Author Manuscript Yung et al. Yung et al. Page 6 Page 6 near and along cranial nerve roots (Figures S1B’–S1B’’’; n = 3 mutants). As in older animals, the tdTomato+ cells escaped through the Vth, VIIth, and VIIIth nerves, raising the possibility that later born neurons such as PNs depend on these existing ectopic tracts to exit the CNS. However, we found that, within the VIIIth nerve, PNs were not physically associated with any other ectopic DCC+ axons and were consistently present independent of other ectopic projections, such as those from the earlier born neurons in the ventral cochlear nucleus (Figure S2). These observations argue that the departure of PNs is not secondary to earlier phenotypes. Thus, multiple populations of neurons exit the CNS in the absence of Ntn1, indicating that Ntn1 plays a general role in establishing the CNS-PNS boundary. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Neogenin Functions Non-Cell-Autonomously in PN Migration A redundant function for Neogenin within PNs would provide the simplest explanation for why DCC−/−; Neo1Gt/Gt double mutants, but not DCC−/− or Neo1Gt/Gt single mutants, more closely mimic the phenotype in Ntn1−/− animals. To test whether PNs upregulate Neogenin in the absence of DCC, we crossed the Neo1Gt/+ allele into the DCC−/− background. This allowed us to assay β-galactosidase activity as a proxy for Neo1 expression, which is more Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Yung et al. Page 7 sensitive than immunostaining. As expected, β-galactosidase reaction product was present in SGNs and the surrounding mesenchyme at E15.5, but not in the AES in either control or mutants (Figures 4H–4I’; n = 3 control, 4 DCC−/−). Thus, Neogenin is unlikely to compensate for DCC in migrating PNs. Author Manuscript To be sure that early or low levels of Neogenin in rhombic lip-derived neurons do not explain the stronger phenotype in DCC−/−; Neo1Gt/Gt double mutants, we used Wnt1Cre and a floxed Neo1 allele (Kam et al., 2016) to remove Neogenin from Wnt1+ rhombic lip precursors in a DCC null background. Deletion of Neo1 from early PNs did not enhance the DCC phenotype (Figures S3C and S3D; n = 3 conditional mutants), making it highly unlikely that Neogenin and DCC function redundantly in PNs. Additionally, though more PNs migrated all the way into the cochlea in DCC−/−; Neo1Gt/Gt double mutants than in DCC−/− single mutants, similar numbers entered the nerve roots in E15.5 animals of both genotypes. Ntn1, therefore, appears to act largely through DCC to prevent PNs from crossing the CNS-PNS boundary but may influence their subsequent behavior through Neogenin expressed in other tissues. Author Manuscript Ntn1−/− Mutants Retain Boundary Cap Cells at Nerve Roots Ntn1−/− Mutants Retain Boundary Cap Cells at Nerve Roots Our results show that, in addition to its canonical role as a chemoattractant, Ntn1 contributes to CNS-PNS segregation, raising the question of how Ntn1 mediates this function. Many cellular structures contribute to the compartmentalization of the CNS. Neural crest-derived boundary cap cells (BCCs), for example, reside at all spinal nerve roots, and loss of BCCs or the cues they secrete results in the ectopic migration of motor neurons into the ventral root (Bron et al., 2007; Garrett et al., 2016; Mauti et al., 2007; Vermeren et al., 2003). The role of BCCs in the hindbrain is less well understood, though they reside at the trigeminal and facial nerve roots in mice (Garrett et al., 2016) and chicks (Niederländer and Lumsden, 1996). Loss of Ntn1 could alter the position of BCCs at cranial nerve roots, in turn permitting the departure of CNS neurons along nerves. Author Manuscript To test this possibility, we used RNAscope to detect Egr2, one of the only markers for BCCs (Vermeren et al., 2003), and counterstained for laminin to assess the distribution of BCCs at the Vth, VIIth, and VIIIth nerves. At E11.5, when DCC+ processes have already entered the periphery (Figure S1), Egr2+ BCCs were observed at nerve entry and exit sites in both controls and mutants (Figures 5A–5B’’; n = 3 animals per genotype). This result is not unexpected, given that, based on the number of ectopic CNS neurons in the periphery, the Ntn1−/− hindbrain phenotype is much more severe than what was previously described in animals lacking BCCs (Vermeren et al., 2003). These data show that Ntn1 maintains the CNS-PNS divide through mechanisms distinct from those of BCCs. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Ectopic Neurons Exit the CNS Independent of Defects in BM Organization In addition to BCCs, an effective CNS-PNS border depends on BM integrity, which is maintained, in part, by radial glial endfeet lining the pial surface. Deletion of BM components or detachment of radial glial endfeet from the pial surface causes BM rupture, defects in neuronal migration, extrusion of cortical neurons into the subarachnoid space, and ectopic migration of spinal cord motor neurons into the ventral root (Beggs et al., 2003; Cell Rep. Author manuscript; available in PMC 2018 March 30. Page 8 Page 8 Yung et al. Yung et al. Halfter et al., 2002; Lee and Song, 2013; Moore et al., 2002; Nakagawa et al., 2015; Satz et al., 2010). Since Netrins affect BM integrity in some tissues (Abraira et al., 2008; Liu et al., 2004; Srinivasan et al., 2003; Yebra et al., 2003; Ziel et al., 2009), we wondered whether loss of Ntn1 might cause defects in the pial BM or in the organization of the radial glial endfeet, thereby enabling PN exodus. Halfter et al., 2002; Lee and Song, 2013; Moore et al., 2002; Nakagawa et al., 2015; Satz et al., 2010). Since Netrins affect BM integrity in some tissues (Abraira et al., 2008; Liu et al., 2004; Srinivasan et al., 2003; Yebra et al., 2003; Ziel et al., 2009), we wondered whether loss of Ntn1 might cause defects in the pial BM or in the organization of the radial glial endfeet, thereby enabling PN exodus. Author Manuscript Auth Author Manuscript To assess BM integrity prior to the earliest signs of the phenotype, we performed transmission electron microscopy (TEM) of E10.5 control and Ntn1−/− animals. At this age, the BM looks like a thin, diffuse rope surrounding the hindbrain, and we were able to follow the BM from the ventral edge of the VIIIth nerve root to the midline. The appearance of the BM was highly variable, altering in thickness, smoothness, and curvature, with no discernable pattern in WT and null animals (Figures 6C and 6D; n = 3 WT, 4 Ntn1−/−). In rare cases, we observed what appeared to be ectopic processes reaching into the periphery, yet the surrounding BM still did not look diminished in a way that would, a priori, enable neurons to exit. Author Manuscript Several days later, the BM in Ntn1−/− mutants still looked intact overall, as assessed by laminin staining. Ectopic Neurons Exit the CNS Independent of Defects in BM Organization However, small ectopic breaks were consistently observed near the AES and the vestibulocochlear nerve (Figures 5E–5F’’; n = 3 Ntn1−/−), where ectopic DCC+ processes protrude, resulting in a significant decrease in the area covered by laminin adjacent to the AES (Figure 5G; n = 4 control, 5 Ntn1−/− ears). These breaks appeared independent of impaired radial glia architecture, whose RC2/Nestin+ endfeet remained attached to the pial surface in E15.5 mutants, as in controls (Figures 5H and 5I; n = 2 control, 3 Ntn1−/−). Moreover, at the sites of BM breaks, the radial glial endfeet projected further without showing obvious changes in their morphology or organization (Figure 5I’). Since BM integrity is normal at E10.5, with no apparent changes in radial glia organization at E15.5, it is unlikely that Ntn1 is required for BM integrity per se, consistent with the fact that Ntn1 has no effects on laminin assembly in vitro (Schneiders et al., 2007). Altogether, the lack of defects in key cell types that contribute to CNS integrity indicate that Ntn1 in the SPR acts directly on migrating neurons to keep them in the CNS. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. SPR-Localized Ntn1 Produced by Hindbrain Progenitors Is Required for Confinement Our results contrast with those from previous studies that reported no phenotypes in the spiral ganglion of Ntn1 hypomorphs (Howell et al., 2007; Kim et al., 2016). These differences could be attributed to the hypomorphic nature of the Ntn1trap/trap mice, which show a weaker phenotype: many PNs get close to their final destination (Figures S4D and S4D’), and there are significantly fewer Pax6+ cells in E15.5 Ntn1trap/trap cochleae (Figures S4B–S4C’; n = 6 cochleae). The number of ectopic neurons remained unchanged at E18.5 (Figure S4E; n = 6 cochleae), further indicating that, unlike null animals, the phenotype does not worsen as later born PNs migrate out. Despite this difference, the pontine nuclei are absent in Ntn1trap/trap animals, indicating that confinement and guidance are differentially affected by the loss of Ntn1, possibly due to differences in the availability or localization of Ntn1 in vivo. Author Manuscript In the developing hindbrain, Ntn1 is present both in the FP and in the SPR (Figure 1) (Dominici et al., 2017; MacLennan et al., 1997). We wondered whether the role in Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Page 9 Yung et al. Page 9 confinement might be attributed specifically to Ntn1 in the SPR, which is primarily supplied by progenitors in the ventricular zone (Dominici et al., 2017; Varadarajan et al., 2017). Using NestinCre/+ (Zimmerman et al., 1994) and a floxed allele of Ntn1, we significantly reduced Ntn1 in the SPR of NestinCre/+; Ntn1fl/− (Nestin conditional knockout [cKO]) animals (Figures 6A–6D; n = 2 controls and 3 Nestin cKO). Despite the presence of residual Ntn1 protein (Figures 6C and 6D) and transcript (Figures 6E and 6F) at the FP, PNs migrated ectopically into the ear (Figures 6G and 6H; n = 2 control and 3 Nestin cKO), partially phenocopying Ntn1−/− animals and fully phenocopying the hypomorphs (Figure 6I; n = 6 ears per genotype), which showed a similar distribution of Ntn1 protein, i.e., a severe decrease in the SPR (Figure 6D) with residual Ntn1 present at the midline (Figures 6C and S4; n ≥ 3 animals per genotype). These results support two conclusions. First, Ntn1 derived from the ventricular zone—which provides most of the Ntn1 in the SPR—ensures the compartmentalization of the CNS and PNS. SPR-Localized Ntn1 Produced by Hindbrain Progenitors Is Required for Confinement Second, residual Ntn1 in hypomorphs and from the FP of Nestin cKO animals is sufficient to reduce the departure of CNS neurons into the periphery, but not to guide them reliably to the midline, as the pontine nuclei are missing in the hypomorph (Serafini et al., 1996; Yee et al., 1999). Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Overexpression of Ntn1 throughout the CNS Rescues CNS-PNS Boundary Integrity Our results raise the possibility that Ntn1 serves dual functions in the developing hindbrain, both securing the CNS-PNS boundary and attracting PNs to the ventral midline. In this model, the low levels of Ntn1 that persist in Ntn1 hypomorphs may be sufficient to establish a partially functional boundary, but not to mediate guidance to the midline, thereby explaining phenotypic differences between the hypomorphic and null mutants. Thus, Ntn1 could act instructively in a gradient to direct PNs to the midline and permissively in the SPR to keep them in the CNS. Author Manuscript To disambiguate these two possible functions, we disrupted Ntn1’s role as a guidance cue by altering its pattern of distribution using a Cre-dependent Ntn1 conditional expressor (Ntn1CE/+), which produces a myc-tagged chick Ntn1 protein (cNtn1) with the same biological activity as mouse Ntn1 (mNtn1; Serafini et al., 1994). E11.5 NestinCre/+; Ntn1CE/+ animals showed widespread expression of cNtn1-myc throughout the hindbrain, overlaid on top of endogenous mNtn1 protein in the FP and SPR (Figures 7A–7B’; see also Figure S5; n = 4 NestinCre/+; Ntn1CE/+). Despite this clear change in Ntn1 protein distribution, PN migration appeared qualitatively normal: PNs reached the midline (Figures 7E and 7F), and no ectopic neurons were observed in the periphery (data not shown). To determine whether the lack of a phenotype reflected a dominant role for endogenous mNtn1, we crossed the NestinCre/+; Ntn1CE/+ animals onto the Ntn1−/− background. Both the cNtn1 transcript (Figure S6; n = 3 NestinCre/+; Ntn1CE/+; Ntn1−/−) and protein (Figures 7C–7D’; n = 2 NestinCre/+; Ntn1CE/+; Ntn1−/−) were present throughout the hindbrain, with cNtn1-myc enriched in the SPR but reduced at the FP. Thus, we significantly altered Ntn1 localization, thereby distorting any directional information that might be encoded in a gradient while maintaining a rich source of Ntn1 in the SPR. Author Manuscript Despite the drastic change in Ntn1 distribution, PN migration was surprisingly normal in all NestinCre/+; Ntn1CE/+; Ntn1−/− embryos (n = 4), as evidenced by the presence of both a well- Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Page 10 defined AES and PNs at the midline where the pontine nuclei are normally found (Figure 7G). Although some PNs reached the midline in all animals, the extent of rescue varied, even between the two sides of the animal. Overexpression of Ntn1 throughout the CNS Rescues CNS-PNS Boundary Integrity We observed a complete rescue in 4 out of 8 cases (two per embryo), as defined by a qualitatively normal AES and no PNs detected in the periphery (Figures 7H and 7I). In the other cases, the AES was misshapen and sometimes accompanied by clusters of Pax6+ neurons in the proximal segment of the vestibulocochlear nerve or sparse Pax6+ neurons in other cranial nerves (Figure 7J). Although PNs appear to be resistant to major disruptions in the pattern of Ntn1 expression, these occasional errors may reflect some requirement for the WT pattern of Ntn1 expression. Alternatively, the degree of rescue may be sensitive to slight variations in the timing or efficiency of NestinCre- mediated recombination. Importantly, none of the embryos contained Pax6+ neurons in the cochlea. Thus, broad expression of Ntn1 is sufficient to restrict PNs from migrating into the periphery, consistent with the model that Ntn1 acts locally to provide a preferred substrate for neuronal migration, thereby keeping neurons confined to the CNS. Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. DISCUSSION In the developing hindbrain, rhombic lip-derived neurons migrate long distances to form brainstem nuclei amidst a crowded network of nerves linking the CNS and PNS. We show here that SPR-localized Ntn1 maintains the CNS-PNS divide by preventing these highly motile neurons from straying into cranial nerves and entering the periphery. Our findings point to a model in which Ntn1 in the SPR acts as a preferred substrate for migrating neurons, thereby keeping them away from nerve roots devoid of Ntn1. Like flags marking a hiking trail, Ntn1 facilitates the successful migration of rhombic lip-derived neurons by establishing a preferred corridor for growth. Without this corridor, the neurons wander off trail, losing track of and failing to reach their destination. Author Manuscript In support of the idea that Ntn1 acts in the SPR to keep migrating neurons on track, migrating PNs express the Ntn1 receptor DCC and respond to Ntn1 in vitro (Yee et al., 1999). Ntn1 protein is also notably enriched in the SPR but absent at cranial nerve roots, which rhombic lip derivatives avoid. Thus, DCC+ PNs may prefer the Ntn1-rich environment surrounding the nerve roots so much that they reliably migrate around them, with the Ntn1-negative gap discouraging their entry. In agreement with this interpretation, the amount of Ntn1 in the SPR correlates with the strength of the confinement phenotype. For instance, using NestinCre to selectively reduce Ntn1 in the SPR but not the FP caused many PNs to enter the periphery. More strikingly, no ectopic PNs were observed in the cochlea when NestinCre was used to restore cNtn1-myc only to the SPR in the null background, where Ntn1 is never produced by the FP and the broad ectopic distribution of Ntn1 throughout the hindbrain obscures any positional information normally encoded by localized Ntn1. Thus, the pattern of Ntn1 expression does not seem to matter for the confinement of migrating neurons, as long as Ntn1 protein accumulates in the SPR. Author Manuscript Although a direct role for Ntn1 seems most likely, indirect effects might also contribute to the overall phenotype. For example, the departure of PNs could be facilitated by the presence of errant axons from earlier born neurons that breached the CNS-PNS border. However, such a mechanism is unlikely to account for the entire phenotype, as both neuronal Yung et al. Page 11 Yung et al. DISCUSSION Page 11 cell bodies and processes have already entered the periphery at E11.5 (arrows in Figure S1B’’), the earliest point when we can detect a phenotype. Likewise, PNs appear segregated from other DCC+ ectopic axons in the VIIIth nerve, and these ectopias can arise independently (Figure S2). Thus, the departure of neurons does not seem to depend, a priori, on the presence of a pre-existing ectopic axon tract. It is, of course, possible that PNs occasionally migrate along earlier born ectopic processes as they escape the CNS, similar to the fasciculation of PN leading processes within the normal AES (Ono and Kawamura, 1990) and of later born axons that follow pioneer axons toward their targets. However, this would not rule out or diminish the role of Ntn1 in the confinement of rhombic lip-derived neurons overall. Author Manuscript Author Manuscript In another scenario, SPR-localized Ntn1 could promote or maintain a physically sound CNS-PNS boundary, in addition to affecting neuronal migration. While we cannot rule out subtle changes, the overall organization of the CNS-PNS boundary appeared intact in Ntn1−/− animals. BCCs were present at nerve roots, and there were no obvious changes in the integrity of the BM surrounding the hindbrain, consistent with the fact that Ntn1 has no effect on BM assembly in vitro (Schneiders et al., 2007). These data suggest that Ntn1 acts directly on PNs to corral them within the SPR, thereby preventing them from leaving the CNS altogether. Our findings add to a growing body of evidence supporting a permissive role for Ntn1 (Dominici et al., 2017; Varadarajan et al., 2017; Yamauchi et al., 2017), and they expand the repertoire of Ntn1 functions in the developing nervous system. Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Distinct Functions for Ntn1 in Confinement along the Rostrocaudal Axis Compared to other aspects of neural development, little is known about the initiation or maintenance of the CNS-PNS boundary, which selectively permits the passage of neural processes— but not cell bodies—into peripheral nerves. Studies in the spinal cord have highlighted the importance of BCCs (Vermeren et al., 2003) and chemorepellents such as Ntn5 (Garrett et al., 2016), Sema3B, Sema3G, and Sema6A (Bron et al., 2007; Mauti et al., 2007) in retaining motor neuron cell bodies inside the CNS, even as they extend their axons out to the periphery. In stark contrast, confinement phenotypes have not been reported in the hindbrain, although BCCs express similar repellents at cranial nerve roots. For example, we found no evidence of ectopic Pax6+ neurons in the cochlea of Ntn5−/− mice (data not shown), despite the presence of ectopic motor neurons in the spinal cord (Garrett et al., 2016). This discrepancy underscores two points. First, the molecular mechanisms that define the CNS-PNS boundary in the vertebrate brainstem remain unknown; and second, the hindbrain and the spinal cord may have evolved unique ways of maintaining the CNS-PNS boundary. Author Manuscript Author Manuscript Indeed, our work illustrates that the same molecule may have distinct functions in hindbrain versus spinal cord confinement. In the spinal cord, Ntn1 plays a relatively limited role, preventing the axons of a single population of neurons from straying into the periphery. Notably, the cell bodies do not follow in Ntn1 mutants (Laumonnerie et al., 2014). Thus, in this context, the misrouting of CNS axons into the PNS is much like other axon guidance phenotypes. In contrast, in the hindbrain, Ntn1 signaling appears to play an integral role in defining the CNS-PNS boundary, as evidenced by both the sheer number of neurons exiting Yung et al. Page 12 Page 12 the CNS and, most importantly, the departure of cell bodies, which an effective CNS-PNS boundary absolutely forbids. These differences in Ntn1 function may reflect the distinct developmental demands of the two brain regions: whereas migration is limited in the spinal cord, there is extensive migration of multiple populations of neurons over long distances and past multiple nerve roots in the hindbrain. As such, having a centrally derived cue play a weightier role in confinement may offer the greater fidelity and robustness needed for rhombic-lip derivatives to complete their migratory routes successfully. Distinct Functions for Ntn1 in Confinement along the Rostrocaudal Axis It remains to be seen whether another centrally derived cue might play a more prominent role in confinement in the spinal cord, particularly since most motor neurons stay within the CNS, even when all BCCs are ablated (Vermeren et al., 2003). Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Finding Unity in Ntn1’s Diverse Functions: a Role for Locally Produced Ntn1 in Neural Development Author Manuscript In addition to being the archetype of diffusible guidance cues, much of Ntn1’s prominence can be attributed to its versatility. Beyond its role in axon guidance, cell migration, and confinement, Ntn1 modulates angiogenesis and tissue morphogenesis, cell adhesion, synapse formation, and cell survival in cancer (reviewed in Cirulli and Yebra, 2007; Lai Wing Sun et al., 2011). Historically, a large emphasis has been placed on the division between long- and short-range functions, which are categorized based on where Ntn1 acts relative to the source of its expression. For instance, textbook models of Ntn1 as a long-range attractant depict commissural axons navigating along an increasing gradient of FP-derived Ntn1 in the spinal cord. The situation in vivo, however, is more complicated. Although Ntn1 is distributed in a gradient in the spinal cord (Kennedy et al., 2006) and can act over a distance in vitro (Kennedy et al., 1994; Yee et al., 1999), it was purified as a heparin-binding protein (Serafini et al., 1994) and found to interact with BM components, including type IV collagen and heparin sulfate proteoglycans (Geisbrecht et al., 2003; Geisen et al., 2008; Kappler et al., 2000). This had raised the possibility that it might function at both short- and long-range (Serafini et al., 1994; Kennedy et al., 1994), and a local role in short-range guidance was soon demonstrated at the optic nerve head (Deiner et al., 1997). Short-range functions have also been demonstrated during tissue morphogenesis, such as BM breakdown in the inner ear (Nishitani et al., 2017; Salminen et al., 2000) or adhesion between two cell layers in the mammary gland (Srinivasan et al., 2003). Author Manuscript Our findings add to a growing body of work that suggest that many of Ntn1’s other functions in the nervous system may be grounded in local signaling, a shared mechanism that may provide a foundation for its diverse roles. Membrane-tethered versions of Ntn, for example, can rescue guidance defects in the Drosophila nerve cord and visual system that were previously ascribed to soluble Ntn (Brankatschk and Dickson, 2006; Timofeev et al., 2012). More recently, several groups have demonstrated that commissural guidance depends on ventricular- zone-derived Ntn1 accumulating in the SPR and along the commissural axons (Dominici et al., 2017; Varadarajan et al., 2017; Yamauchi et al., 2017), expanding on related observations (Charron et al., 2003; Kennedy et al., 2006). Cell-Autonomous and Non-Cell-Autonomous Functions for Multiple Ntn1 Receptors in Confinement Author Manuscript Author Manuscript Our findings provide additional evidence for Ntn1’s multifunctionality, which likely depends on its diverse repertoire of receptors. PNs express receptors mediating both attraction, such as DCC, and repulsion, such as Unc5B/C. However, Ntn1-mediated confinement does not seem to depend on repulsion, since PNs remain within the CNS in Unc5b and Unc5c mutants (Di Meglio et al., 2013; Kim and Ackerman, 2011). Moreover, both HoxB4+ (Unc5B-low) and HoxB4− (Unc5B-high) PNs (Di Meglio et al., 2013) escape into the periphery in Ntn1 hypomorphs (Figure S7), indicating that differential responsiveness to Ntn1 cannot account for the partial phenotype. Thus, Unc5B/Cs appear to influence only the later stages of Ntn1-mediated PN migration, comparable to the way Unc5A/Cs position commissural neuron cell bodies in the spinal cord but are not required for confinement of their axons (Laumonnerie et al., 2014). Author Manuscript In contrast, as an obligate receptor expressed on commissural axons and PNs, DCC mediates confinement in both the spinal cord and hindbrain. We also discovered a surprising role for Neogenin in trans, as Neo1 is neither expressed nor required in migrating PNs, though it is present at low levels throughout the neuroepithelium and at higher levels on cranial nerves and in the surrounding mesenchyme. These data suggest that complete containment depends both on Ntn1-DCC signaling within PNs and also on Ntn1-receptor interactions in the environment. Since Ntn1-Neogenin interactions mediate adhesion in the developing mammary gland (Srinivasan et al., 2003), similar interactions in the BM around the VIIIth nerve could prevent movement into the nerve root, providing an additional safeguard for CNS-PNS segregation. However, any effects of Ntn1 signaling on the structural integrity of the CNS-PNS border are likely to be subtle, as the BM did not appear strikingly different in Ntn1−/− mutants by electron microscopy (EM) or immunostaining. Moreover, PNs stay confined to the CNS in ISPD mutants (data not shown), which have fragmented BMs (Wright et al., 2012). Hence, disrupting boundaries alone is not sufficient to induce the departure of CNS neurons, indicating that Ntn1 plays an active signaling role across multiple cell types in confining migrating neurons to the CNS. Author Manuscript Page 13 Page 13 Yung et al. Cell Rep. Author manuscript; available in PMC 2018 March 30. EXPERIMENTAL PROCEDURES Further details and an outline of resources used in this work can be found in the Supplemental Experimental Procedures. Finding Unity in Ntn1’s Diverse Functions: a Role for Locally Produced Ntn1 in Neural Development We have similarly revealed a role for SPR-localized Ntn1 in cellular confinement, providing an alternative explanation for both the reduced number of PNs in Ntn1trap/trap mice, which was thought to reflect Ntn1’s tropic and trophic roles (Yee et al., 1999), and the presence of ectopic neurons in DCC−/− cochleae, which was attributed to a mis-positioning of SGNs (Kim et al., 2016). Thus, across multiple species and in multiple regions of the nervous system, Ntn1 appears to act locally to mediate its purported long-range functions. Author Manuscript Given the clear importance of Ntn1 for nervous system wiring, the possibility that Ntn1 may not act as a long-range instructive cue for PNs raises the question of where the directional information comes from. One idea is that a gradient of Ntn1 activity is achieved through interactions with other cues in the environment. Indeed, every confirmed Ntn1 receptor also interacts with additional ligands (Ahmed et al., 2011; Karaulanov et al., 2009; Rajagopalan et al., 2004; Yamagishi et al., 2011), raising the possibility that Ntn1 is a crucial collaborator for many guidance pathways, perhaps mediating short-range interactions that are necessary Yung et al. Page 14 Page 14 Yung et al. for axons to grow reliably toward other ligands. This could occur either directly, i.e., by binding to the same receptors, or indirectly, i.e., by attaching migrating neurons to the BM, where they may be steered by other cues such as Slits. This may explain Ntn1’s ability to augment the effect of other guidance molecules synergistically (reviewed in Morales and Kania, 2017). Thus, even 20 years after its discovery, Ntn1 continues to inform new models for how the complex networks of the nervous system are constructed reliably and accurately using relatively few guidance cues. Author Manuscript Statistical Analysis All statistical comparisons were done using Prism software (GraphPad, La Jolla, CA, USA) and presented as mean ± SD. Statistical significance was determined by a Student’s t test when comparing between two groups. If more than two groups were being considered, a one-way ANOVA was performed with Tukey’s multiple comparisons test. In cases of the latter, the multiplicity adjusted p values were included in the figures, and the p value of the ANOVA was reported in the figure legends. Sample size for all experiments was determined empirically based on standards in the field. Specific details for each experiment are included in the text (n values and their meanings) or in the figure legends (statistical tests used). Author Manuscript Animal Models Author Manuscript The following mouse strains were used and genotyped as described previously: Ntn1fl/fl, Ntn1+/− (Yung et al., 2015), Ntn1CE/+ (Nishitani et al., 2017), DCC+/−; Neo1Gt/+ (Fazeli et al., 1997; Leighton et al., 2001; Xu et al., 2014), Neo1−/− and Neo1fl/fl (Kam et al., 2016), Atoh1CreERT2 (Machold and Fishell, 2005), Six3Cre (Furuta et al., 2000), NestinCre (Tronche et al., 1999), MafBGFP (Moriguchi et al., 2006), and Ai14 Cre-dependent tdTomato (Madisen et al., 2010). Mice were maintained on a C57BL/6 background. Noon on the day of the plug was considered E0.5. Tamoxifen (Sigma-Aldrich) injections were carried out at 20 mg/mL in sunflower oil at 1 mg/10 g of body weight. 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UNC-6 (netrin) orients the invasive membrane of the anchor cell in C. elegans. Nat. Cell Biol. 2009; 11:183–189. [PubMed: 19098902] Zimmerman L, Parr B, Lendahl U, Cunningham M, McKay R, Gavin B, Mann J, Vassileva G, McMahon A. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron. 1994; 12:11–24. [PubMed: 8292356] Author Manuscript Author Manuscript Author Manuscript Yung et al. Page 20 Highlights • Ntn1 is enriched in the sub-pial region (SPR) but absent at cranial nerve roots • Pontine neurons exit the CNS along nerves when SPR-localized Ntn1 is removed • DCC and Neogenin act in distinct cell types to confine pontine neurons to the CNS • Restoring Ntn1 to the SPR rescues the CNS-PNS boundary and pontine neuron migration Author Manuscript Author Manuscript Author Manuscript Author Manuscript Yung et al. Page 21 Figure 1. Ntn1 Protein Is Enriched in the SPR in the Developing Hindbrain (A) Schematic depicting the three phases of PN migration (green) across multiple rhombomere segments (shaded in gray) and a view of the AES in an E15.5 transverse section. Thick dashed line indicates plane of section. D, dorsal; A, anterior; P, posterior; V, ventral. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Figure 1. Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. AES, anterior extramural stream; fp, floor plate; hb, hindbrain; 4th vent., fourth ventricle; VII, facial nerve; VIII, vestibulocochlear nerve. nerve roots (yellow arrowheads). Low (D) and high (E–E’) magnification images show ventrally migrating pontine neurons in the SPR, even as they avoid cranial nerve roots later in their migration (F; E15.5). Pontine neurons express Pax6 (red, D and E) and DCC (green, E–F). Cell Rep. Author manuscript; available in PMC 2018 March 30. References Ntn1 Protein Is Enriched in the SPR in the Developing Hindbrain (A) Schematic depicting the three phases of PN migration (green) across multiple rhombomere segments (shaded in gray) and a view of the AES in an E15.5 transverse section. Thick dashed line indicates plane of section. D, dorsal; A, anterior; P, posterior; V, ventral. Author Manuscript (B–F) Immunostains of transverse embryonic head sections. At E13.5, low-power (B and B’) and high-power (C and C’) images show strong Ntn1 staining at the FP, on crossing commissural axons at the midline, and in the sub-pial region (SPR) adjacent to the laminin- positive pial basement membrane (magenta). Curiously, Ntn1 appears to be absent from nerve roots (yellow arrowheads). Low (D) and high (E–E’) magnification images show ventrally migrating pontine neurons in the SPR, even as they avoid cranial nerve roots later in their migration (F; E15.5). Pontine neurons express Pax6 (red, D and E) and DCC (green, E–F). AES, anterior extramural stream; fp, floor plate; hb, hindbrain; 4th vent., fourth ventricle; VII, facial nerve; VIII, vestibulocochlear nerve. Page 22 Yung et al. Yung et al. Page 22 nerve roots (yellow arrowheads). Low (D) and high (E–E’) magnification images show ventrally migrating pontine neurons in the SPR, even as they avoid cranial nerve roots later in their migration (F; E15.5). Pontine neurons express Pax6 (red, D and E) and DCC (green, E–F). Author Manuscript AES, anterior extramural stream; fp, floor plate; hb, hindbrain; 4th vent., fourth ventricle; VII, facial nerve; VIII, vestibulocochlear nerve. Author Manuscript Author Manuscript Author Manuscript Figure 2. PNs Exit the CNS along Cranial Nerves in the Absence of Ntn1 (A) Schematic of the PN migratory route (anterior extramural stream, green) across multiple rhombomeres (numbered, shaded in gray) and relative to cranial nerve roots. (B–D’) E15.5 transverse head sections immunostained for DCC and Pax6 to label migrating PNs (yellow arrowheads), which normally travel rostrally beneath the pial surface toward the midline, shown at three rostro-caudal levels (B, C, and D), as indicated by the dashed lines in (A) A mix of WT and Ntn1+/−tissues are shown as controls In Ntn1−/−animals the AES ng et al. Page 23 Yung et al. Yung et al. Page 23 Author Manuscript Figure 2. Cell Rep. Author manuscript; available in PMC 2018 March 30. (G–H’’’) Low- (G and H) and high-power (H’–H’’’) images of fate-mapped PNs in the cochlea which were labeled with tdTomato (G, H, H’), DCC (G, H, H’’), and Pax6 (H’’) in Atoh1CreERT2/+; Ai14; Ntn1+/− (G) and Ntn1−/− (H–H’’’) embryos exposed to tamoxifen at E13.5. A merged image is shown in (H’’’). References PNs Exit the CNS along Cranial Nerves in the Absence of Ntn1 (A) Schematic of the PN migratory route (anterior extramural stream, green) across multiple rhombomeres (numbered, shaded in gray) and relative to cranial nerve roots. Author Manuscript Author Manuscript Figure 2. PNs Exit the CNS along Cranial Nerves in the Absence of Ntn1 Figure 2. PNs Exit the CNS along Cranial Nerves in the Absence of Ntn1 (A) Schematic of the PN migratory route (anterior extramural stream, green) across multiple rhombomeres (numbered, shaded in gray) and relative to cranial nerve roots. (B–D’) E15.5 transverse head sections immunostained for DCC and Pax6 to label migrating PNs (yellow arrowheads), which normally travel rostrally beneath the pial surface toward the midline, shown at three rostro-caudal levels (B, C, and D), as indicated by the dashed lines in (A). A mix of WT and Ntn1+/− tissues are shown as controls. In Ntn1−/− animals, the AES is missing. PNs, instead, diverge into the VIIIth (B’) and VIIth (C’) nerves. Rare ectopic processes are present in the Vth nerve (D’, white arrowheads). (B–D’) E15.5 transverse head sections immunostained for DCC and Pax6 to label migrating PNs (yellow arrowheads), which normally travel rostrally beneath the pial surface toward the midline, shown at three rostro-caudal levels (B, C, and D), as indicated by the dashed lines in (A). A mix of WT and Ntn1+/− tissues are shown as controls. In Ntn1−/− animals, the AES is missing. PNs, instead, diverge into the VIIIth (B’) and VIIth (C’) nerves. Rare ectopic processes are present in the Vth nerve (D’, white arrowheads). Author Manuscript (E and F) Quantification of the number of Pax6+ neurons in the VIIth and VIIIth nerves (E) (mean ± SD, Student’s t test) and in the base and middle turns of control and Ntn1−/− cochlear sections over time (F) (mean ± SD). (E and F) Quantification of the number of Pax6+ neurons in the VIIth and VIIIth nerves (E) (mean ± SD, Student’s t test) and in the base and middle turns of control and Ntn1−/− cochlear sections over time (F) (mean ± SD). Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Yung et al. Dotted lines indicate the cochlea; roman numerals indicate cranial nerves. hb, hindbrain. Asterisks indicate cochlear duct. All scale bars indicate 200 µm unless otherwise noted. Dotted lines indicate the cochlea; roman numerals indicate cranial nerves. hb, hindbrain. Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. radial fibers (G; white arrowheads). Merged images shown in (E) and (G). sg, spiral ganglion. References Page 24 (G–H’’’) Low- (G and H) and high-power (H’–H’’’) images of fate-mapped PNs in the cochlea which were labeled with tdTomato (G, H, H’), DCC (G, H, H’’), and Pax6 (H’’) in Atoh1CreERT2/+; Ai14; Ntn1+/− (G) and Ntn1−/− (H–H’’’) embryos exposed to tamoxifen at E13.5. A merged image is shown in (H’’’). Dotted lines indicate the cochlea; roman numerals indicate cranial nerves. hb, hindbrain. Asterisks indicate cochlear duct. All scale bars indicate 200 µm unless otherwise noted. See also Figures S1 and S2. Author Manuscript Author Manuscript Author Manuscript Yung et al. Figure 3. Ectopic Neurons Do Not Integrate into the Spiral Ganglion (A–C’) E18.5 transverse sections of the base of the cochlea immunostained for Pax6 (A and A’) and Gata3 (B and B’). Only mutant cochleae contain Pax6+ neurons (yellow arrowheads), which form a rind around SGNs that express higher levels of Gata3 (C’). Page 25 Author Manuscript Author Manuscript Author Manuscript Figure 3. Ectopic Neurons Do Not Integrate into the Spiral Ganglion g p g p g (A–C’) E18.5 transverse sections of the base of the cochlea immunostained for Pax6 (A and A’) and Gata3 (B and B’). Only mutant cochleae contain Pax6+ neurons (yellow arrowheads), which form a rind around SGNs that express higher levels of Gata3 (C’). (D–G) Whole-mount immunostains of E18.5 cochleae from control and Ntn1−/− embryos also harboring a MafBGFP allele, which is expressed in SGNs. GFP+ SGN processes (green, D–G) form bundles extending radially to hair cells in both Ntn1+/− controls (D and E) and mutant mice (F and G). In addition, Ntn1−/− cochleae contain Tuj-positive PNs (red, E and G) that extend MafBGFP -negative GFP-processes longitudinally over the SGNs and their Author Manuscript Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Yung et al. Page 26 Page 26 radial fibers (G; white arrowheads). Merged images shown in (E) and (G). sg, spiral ganglion. Author Manuscript Author Manuscript Author Manuscript Figure 4. DCC−/−; Neo1Gt/Gt Double Mutants Phenocopy Ntn1−/− Mutants, but Neogenin Acts Non-Cell-Autonomously (A) The number of Pax6+ PNs found in the base and middle turns of the cochlea in E18.5 DCC and Neo1 gene trap (Neo1Gt) single and double mutants (mean ± SD, p < 0.0001, F = 14.33; DF = 28; one-way ANOVA, Tukey’s multiple comparisons test). Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. References (B–C’) Immunostained E15.5 transverse sections show that Neogenin is expressed strongly in MafB+ SGNs and in the surrounding mesenchyme in control (B) and Ntn1−/− (C) tissue. ung et al. Page 27 Yung et al. Page 27 Author Manuscript Author Manuscript Author Manuscript Figure 4. DCC−/−; Neo1Gt/Gt Double Mutants Phenocopy Ntn1−/− Mutants, but Neogenin Acts Non-Cell-Autonomously (A) The number of Pax6+ PNs found in the base and middle turns of the cochlea in E18.5 DCC and Neo1 gene trap (Neo1Gt) single and double mutants (mean ± SD, p < 0.0001, F = 14.33; DF = 28; one-way ANOVA, Tukey’s multiple comparisons test). (B–C’) Immunostained E15.5 transverse sections show that Neogenin is expressed strongly in MafB+ SGNs and in the surrounding mesenchyme in control (B) and Ntn1−/− (C) tissue. High-power images of the boxed areas show that MafB+ SGNs normally express Neogenin (B’), but ectopic MafB- PNs in the ear do not (C’, yellow arrowheads). Figure 4. DCC−/−; Neo1Gt/Gt Double Mutants Phenocopy Ntn1−/− Mutants, but Neogenin Acts Non Cell Autonomously re 4. DCC−/−; Neo1Gt/Gt Double Mutants Phenocopy Ntn1−/− Mutants, but Neogenin Acts Cell-Autonomously y (A) The number of Pax6+ PNs found in the base and middle turns of the cochlea in E18.5 DCC and Neo1 gene trap (Neo1Gt) single and double mutants (mean ± SD, p < 0.0001, F = 14.33; DF = 28; one-way ANOVA, Tukey’s multiple comparisons test). Author Manuscript (B–C’) Immunostained E15.5 transverse sections show that Neogenin is expressed strongly in MafB+ SGNs and in the surrounding mesenchyme in control (B) and Ntn1−/− (C) tissue. High-power images of the boxed areas show that MafB+ SGNs normally express Neogenin (B’), but ectopic MafB- PNs in the ear do not (C’, yellow arrowheads). Page 28 Yung et al. Yung et al. (D–G) Ventral (D and E) and sagittal (F and G) views of heterozygous (D and F) and homozygous (E and G) Neo1 E15.5 brains immunostained for Pax6. Rostral is up in (D) and (E) and to the right in (F) and (G). Author Manuscript (H–I’) X-gal reactions (blue) in eosin-stained tissue from E15.5 control and DCC−/− embryos carrying the Neo1Gt allele, which drives expression of β-galactosidase in Neogenin + cells. aes, anterior extramural stream; BP, basilar pons; cb, cerebellum; hb, hindbrain; SO, superior olive. References No signal is detected in the AES (black arrowheads) in DCC+/−; Neo1Gt/+ (H, H’) or DCC−/−; Neo1Gt/+ (I and I’) animals, shown at low (H and I) and high (H’ and I’) magnification (H–I’) X-gal reactions (blue) in eosin-stained tissue from E15.5 control and DCC−/− embryos carrying the Neo1Gt allele, which drives expression of β-galactosidase in Neogenin + cells. No signal is detected in the AES (black arrowheads) in DCC+/−; Neo1Gt/+ (H, H’) or DCC−/−; Neo1Gt/+ (I and I’) animals, shown at low (H and I) and high (H’ and I’) magnification aes, anterior extramural stream; BP, basilar pons; cb, cerebellum; hb, hindbrain; SO, superior olive. See also Figures S3 and S7. Author Manuscript Author Manuscript Author Manuscript Yung et al. Page 29 igure 5. Neurons Exit the CNS Independent of Defects in BCCs, Radial Glial Endfeet, and the asement Membrane A–B’’) High-power images of E11.5 transverse head sections near the VIIIth nerve root how that Egr2+ BCCs (blue) are present at gaps in laminin (magenta) in both WT (A–A’’) nd Ntn1−/− (B–B’’) animals. In Ntn1−/− mutants, ectopic DCC+ processes exit the CNS espite the presence of BCCs (hollow yellow arrowheads). C and D) TEM images of the basement membrane (BM, hollow black arrowheads) urrounding the WT (C) and Ntn1−/− (D) hindbrain. E G) Immunostains of transverse sections from WT (E) and Ntn1−/−(F) E15 5 embryos Author Manuscript Author Manuscript Figure 5. Neurons Exit the CNS Independent of Defects in BCCs, Radial Glial Endfeet, and the Basement Membrane (A–B’’) High-power images of E11.5 transverse head sections near the VIIIth nerve root show that Egr2+ BCCs (blue) are present at gaps in laminin (magenta) in both WT (A–A’’) and Ntn1−/− (B–B’’) animals. In Ntn1−/− mutants, ectopic DCC+ processes exit the CNS despite the presence of BCCs (hollow yellow arrowheads). (C and D) TEM images of the basement membrane (BM, hollow black arrowheads) surrounding the WT (C) and Ntn1−/− (D) hindbrain. (E–G) Immunostains of transverse sections from WT (E) and Ntn1−/− (F) E15.5 embryos. Figure 5. Neurons Exit the CNS Independent of Defects in BCCs, Radial Glial Endfeet, and the Basement Membrane eurons Exit the CNS Independent of Defects in BCCs, Radial Glial Endfeet, and the Membrane Author Manuscript (A–B’’) High-power images of E11.5 transverse head sections near the VIIIth nerve root show that Egr2+ BCCs (blue) are present at gaps in laminin (magenta) in both WT (A–A’’) and Ntn1−/− (B–B’’) animals. Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. References In Ntn1−/− mutants, ectopic DCC+ processes exit the CNS despite the presence of BCCs (hollow yellow arrowheads). (A–B’’) High-power images of E11.5 transverse head sections near the VIIIth nerve root show that Egr2+ BCCs (blue) are present at gaps in laminin (magenta) in both WT (A–A’’) and Ntn1−/− (B–B’’) animals. In Ntn1−/− mutants, ectopic DCC+ processes exit the CNS despite the presence of BCCs (hollow yellow arrowheads). (C and D) TEM images of the basement membrane (BM, hollow black arrowheads) surrounding the WT (C) and Ntn1−/− (D) hindbrain. (E–G) Immunostains of transverse sections from WT (E) and Ntn1−/− (F) E15.5 embryos. Low- (E and F) and high-power (E’–F’’) images of laminin (blue) and DCC (green) show an Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Yung et al. Page 30 ectopic break in the BM (yellow arrowheads, F’ and F’’) in Ntn1 mutants, quantified in (G) (mean ± SD, Student’s t test). Author Manuscript (H–I’) Stains on the same WT (H) and mutant (I and I’) sections for RC2, a radial glia marker, show that the radial glia endfeet (red) remain attached to the laminin-positive BM (blue) in the mutant, even extending together with PN processes through breaks in the laminin (yellow arrowhead), shown also in a single-channel image for RC2 in (I’). hb, hindbrain; VIII, vestibulocochlear nerve; WT, wild-type. (H–I’) Stains on the same WT (H) and mutant (I and I’) sections for RC2, a radial glia marker, show that the radial glia endfeet (red) remain attached to the laminin-positive BM (blue) in the mutant, even extending together with PN processes through breaks in the laminin (yellow arrowhead), shown also in a single-channel image for RC2 in (I’). hb, hindbrain; VIII, vestibulocochlear nerve; WT, wild-type. Author Manuscript Author Manuscript Author Manuscript Page 31 Yung et al. Figure 6. Ntn1 in the SPR, but Not the FP, Is Required for PN Confinement (A–D) Immunostaining for Ntn1 shows depletion from the SPR of E11.5 Nestin cKO animals (B and B’) compared to controls (A and A’), with maintained expression in the FP (arrowheads). Ntn1 intensity was measured at the FP or in the SPR (white circles in A’), quantified in (C) and (D), respectively (mean ± SD). For (C), F = 12.06; DF = 19; p = 0.0004; for (D), F = 14.73; DF= 16; p = 0.0002; one-way ANOVA with Tukey’s multiple comparisons test. Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. fp, floor plate; hb, hindbrain; 4th, fourth ventricle. Asterisks indicate cochlear duct. All scale bars indicate 200 µm. See also Figure S4. References (E and F) In situ hybridization for Ntn1 further illustrates that relative to NestinCre/+; Author Manuscript Author Manuscript Author Manuscript Figure 6. Ntn1 in the SPR, but Not the FP, Is Required for PN Confinement (A D) Immunostaining for Ntn1 shows depletion from the SPR of E11.5 Nestin cKO animals (B and B’) compared to controls (A and A’), with maintained expression in the FP (arrowheads). Ntn1 intensity was measured at the FP or in the SPR (white circles in A’), quantified in (C) and (D), respectively (mean ± SD). For (C), F = 12.06; DF = 19; p = 0.0004; for (D), F = 14.73; DF= 16; p = 0.0002; one-way ANOVA with Tukey’s multiple comparisons test. (E and F) In situ hybridization for Ntn1 further illustrates that relative to NestinCre/+; Ntn1fl/+ animals (E), Ntn1 is selectively reduced in the ventricular zone of E15.5 Nestin cKO embryos (F). Author Manuscript (G–I) DCC (green) and Pax6 (red) immunostains on E15.5 transverse head sections. Nestin cKO animals (H) retain the AES (yellow arrowheads), but it is smaller and deformed compared to controls (G), and there are many Pax6+ nuclei in the cochlea, quantified in (I). Depleting Ntn1 from the SPR is sufficient to partly recapitulate the null phenotype and fully phenocopy the hypomorph (I; mean ± SD) (F = 7.542; DF = 15; p = 0.0054; one-way ANOVA with Tukey’s multiple comparisons test). Refer to Figure S4 for raw data for the gene trap allele. Dotted lines indicate the outline of the cochlea. Cell Rep. Author manuscript; available in PMC 2018 March 30. Yung et al. Yung et al. Page 32 fp, floor plate; hb, hindbrain; 4th, fourth ventricle. Asterisks indicate cochlear duct. All scale bars indicate 200 µm. fp, floor plate; hb, hindbrain; 4th, fourth ventricle. Asterisks indicate cochlear duct. All scale bars indicate 200 µm. See also Figure S4. Author Manuscript See also Figure S4. See also Figure S4. Author Manuscript Author Manuscript Yung et al. Yung et al. Page 33 Figure 7. Broadly Expressing cNtn1 in the Hindbrain Can Rescue Confinement Defects in Ntn1−/− Animals (A–D’) Transverse sections through E11.5 conditional expressor tissue immunostained for mNtn1 (A–D) and cNtn1-myc (A’–D’). cNtn1-myc is broadly distributed throughout the hindbrain (A’), overlaid on top of endogenous Ntn1 protein (A). Whereas mNtn1 is enriche at the FP, cNtn1 is relatively reduced, but both are enriched in the SPR (B and B’). Cell Rep. Author manuscript; available in PMC 2018 March 30. Cell Rep. Author manuscript; available in PMC 2018 March 30. References Low- power images (C, C’) show that a similar distribution of cNtn1 persists in the null background, where despite the absence of mNtn1 (C), cNtn1 is present throughout the hindbrain, with less in the floor plate (C’). High-power images (D, D’) show that without Author Manuscript Author Manuscript Author Manuscript Figure 7. Broadly Expressing cNtn1 in the Hindbrain Can Rescue Confinement Defects in Ntn1−/− Animals Author Manuscript (A–D’) Transverse sections through E11.5 conditional expressor tissue immunostained for mNtn1 (A–D) and cNtn1-myc (A’–D’). cNtn1-myc is broadly distributed throughout the hindbrain (A’), overlaid on top of endogenous Ntn1 protein (A). Whereas mNtn1 is enriched at the FP, cNtn1 is relatively reduced, but both are enriched in the SPR (B and B’). Low- power images (C, C’) show that a similar distribution of cNtn1 persists in the null background, where despite the absence of mNtn1 (C), cNtn1 is present throughout the hindbrain, with less in the floor plate (C’). High-power images (D, D’) show that without Page 34 Yung et al. Yung et al. mNtn1 (D), cNtn1 is the only Ntn1 enriched in the SPR (D’). In all cases, note the absence of any Ntn1 near nerve entry roots (yellow arrowheads). Author Manuscript Author Manuscript (E–J) E15.5 transverse sections immunostained for Pax6 (red) and DCC (green). Single- channel images of Pax6 (E–G) show PNs accumulating at the midline of all conditional expressors, in both Ntn1+/− (F) and Ntn1−/− (G) backgrounds. Conditional expression of cNtn1-myc rescued confinement in some Ntn1−/− animals (I), as shown by a qualitatively normal AES (white arrowheads). In others, we observed a partial rescue in the form of a misshapen AES and a cluster of ectopic neurons in the nerve (J). Control sections at the midline (E) and AES (H) are provided for comparison. hb, hindbrain; 4th, fourth ventricle. Roman numerals indicate cranial nerves. Asterisks indicate cochlear duct. All scale bars indicate 100 µm. See also Figures S5 and S6. Author Manuscript Author Manuscript Author Manuscript
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Estimation of Head Accelerations in Crashes Using Neural Networks and Sensors Embedded in the Protective Helmet
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    Citation: Bracali, A.; Baldanzini, N. Estimation of Head Accelerations in Crashes Using Neural Networks and Sensors Embedded in the Protective Helmet. Sensors 2022, 22, 5592. https://doi.org/10.3390/s22155592 Academic Editor: Carlo Ricciardi Received: 30 May 2022 Accepted: 22 July 2022 Published: 26 July 2022 Citation: Bracali, A.; Baldanzini, N. Estimation of Head Accelerations in Crashes Using Neural Networks and Sensors Embedded in the Protective Helmet. Sensors 2022, 22, 5592. https://doi.org/10.3390/s22155592 sensors sensors sensors Andrea Bracali * and Niccolò Baldanzini Andrea Bracali * and Niccolò Baldanzini Department of Industrial Engineering, University of Florence, 50139 Firenze, Italy; niccolo.baldanzini@unifi.it * Correspondence: andrea.bracali@unifi.it; Tel.: +39-3479-284-137 Abstract: Traumatic Brain Injuries (TBIs) are one of the most frequent and severe outcomes of a Powered Two-Wheeler (PTW) crash. Early diagnosis and treatment can greatly reduce permanent consequences. Despite the fact that devices to track head kinematics have been developed for sports applications, they all have limitations, which hamper their use in everyday road applications. In this study, a new technical solution based on accelerometers integrated in a motorcycle helmet is presented, and the related methodology to estimate linear and rotational acceleration of the head with deep Artificial Neural Networks (dANNs) is developed. A finite element model of helmet coupled with a Hybrid III head model was used to generate data needed for the neural network training. Input data to the dANN model were time signals of (virtual) accelerometers placed on the inner surface of the helmet shell, while the output data were the components of linear and rotational head accelerations. The network was capable of estimating, with good accuracy, time patterns of the acceleration components in all impact conditions that require medical treatment. The correlation between the reference and estimated values was high for all parameters and for both linear and rotational acceleration, with coefficients of determination (R2) ranging from 0.91 to 0.97. Keywords: traumatic brain injuries (TBIs); linear acceleration; rotational acceleration; safety; helmet sensors; neural networks 1. Introduction The possibility of estimating the linear and rotational accelerations of the head during a crash becomes key to predicting TBIs, but the estimation process is extremely difficult in real-world conditions (e.g., impacts between football players, motorcyclists’ road crashes, skiers’ falls). Since a helmet is the most common solution to mitigate TBIs, several technical solutions used the helmet as part of the measuring system. Systems incorporating microelectromechanical system (MEMS) inertial sensors into helmets were developed and employed, with the Head Impact Telemetry System (HITS) [15] being one of the earliest and most widely used [11,16–21]. The HIT system is composed of six single-axis accelerometers oriented normal to the skull, and it is specifically designed to measure head accelerations by elastically coupling the accelerometers in contact with the head, isolating them from the helmet shell. The linear acceleration is estimated with an optimization method, while the rotational acceleration is computed assuming a pivot point located about 10 cm below the head Centre of Gravity (CoG). A development of HIT with a new sensor layout resulted in the 6DOF (Degrees of Freedom) HIT measurement device [22], providing both the linear and the rotational accelerations and iteratively solving the optimization problem [12,20,23]. y g The most recent helmeted device was the gForce Tracker (GFT) [24], characterized by a triaxial accelerometer and a triaxial gyroscope embedded inside a casing attached to the helmet. This technology provides the maximum values of the resultant linear acceleration and rotational velocity obtained from a power fit regression. Other devices require a rigid connection to the head such as mount-guards, earplugs and bands. These devices provide more accurate data at the expense of comfort and user-friendliness, and they may cause an acceptability problem to end users such as motorcyclists or bikers. Although the indirect identification of the head kinematics by using helmet dynamics was extensively deepened in previous research, all measurement devices fitted to the helmet and capable of estimating kinematic parameters as a function of time require sensors to be in contact with the head to overcome the difficulties due to the relative movement between head and helmet. The helmet rotation in relation to the head is primarily affected by the coupling of different head and helmet sizes as well as by a combination of padding compression, chin strap tension, and friction between the helmet and the head. 1. Introduction Despite attempts to minimize the incidence and severity of head injuries with im- proved protective equipment, closed-head impacts represent the highest percentage of Traumatic Brain Injuries (TBIs) diagnosed each year among the civil population in the United States (US) [1]. In 2017, 61,000 TBI-related deaths occurred in the United States, and motor vehicle crash was the second most relevant category after suicide [2]. In addition, TBIs, regardless of severity level, can lead to difficulties in performing daily activities, such as gait impairment [3]. Worldwide, Vulnerable Road Users (VRUs) account for more than half of all global deaths in road crashes (the events related to two- and three-wheeled vehi- cles represent 26% of all deaths) [4]. Therefore, models capable of properly estimating TBI are needed to perform real-time estimation of the injuries and thus to improve protective devices. Received: 30 May 2022 Accepted: 22 July 2022 Published: 26 July 2022 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. Currently, TBI risk assessment is made using criteria coupling a biomechanical metric and an injury risk function. There are two types of biomechanical metrics: based on kinematic parameters of the head or on the brain tissue deformation during the impact. Most of the existing injury criteria are based on the head kinematics since measurements, either on a dummy or a volunteer, are easier than measuring brain tissue response. An overview of these metrics was provided by Gabler et al. [5]. The latest findings on the key role played by rotational acceleration on brain injuries led the United Nations Economic Commission for Europe to revise the ECE 22.05 helmet homologation standard [6]. The new regulation, ECE 22.06, took effect on January 2021 and introduced new tests for homologation also based on the rotational acceleration [7]. Several studies [8–14] were conducted to improve the knowledge of TBI and to develop a method or an injury criterion Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sensors Sensors 2022, 22, 5592. https://doi.org/10.3390/s22155592 Sensors 2022, 22, 5592 2 of 16 2 of 16 to estimate injuries based on kinematic parameters. 1. Introduction This dependency produces different rotational velocities and rotational acceleration sustained by the helmet and head during an impact. Additionally, the presence of the foam component between the helmet outer shell and the head significantly reduces its linear acceleration compared to the helmet one. Manoogian et al. [25] demonstrated that the helmet peak linear acceleration is approximately 10 times the head peak acceleration. More recently, Joodaki et al. [26] found that the peak linear acceleration of the helmet was 2/5 times greater than the head one, while the helmet peak angular velocity was greater or smaller than the head one according to the impact conditions; in some tests, the helmet rotated more than 30 deg relative to the head. g The technical solution investigated in this paper is based on a novel sensing system characterized by a new layout of the accelerometers, which are placed on the inner side of the helmet shell. In this configuration, the signal processing for the determination of the head kinematics cannot rely on simplifying assumptions, as typical of previous studies. The high number of parameters influencing head kinematics increases the complexity level and suggests the use of deep learning techniques for the estimation task. At present, there are a limited number of applications of deep Artificial Neural Net- works (dANNs) for identifying the impact load history of structures [27–29]. dANNs are computing models used for information processing that only need data for supervised learning. They are often used to identify and model a complex functional relationship or pattern between input and output data with a black-box approach. ANNs can detect com- plex non-linear relationships between variables through their training phase. Nowadays, several software offer packages to develop ANNs in an easy and friendly way with multiple training algorithms [30]. However, ANNs also have some disadvantages: performances achieved are highly reliant on the quantity and quality of data given as input and output to train the networks; collecting the necessary amount of data to train the network can be Sensors 2022, 22, 5592 3 of 16 3 of 16 costly and time-consuming [31]. Staszewski [28] and Ghajari [27] used an dANN model to identify the impact load acting on a composite plate; specifically, in Ghajari [27], the effects of signal features, network architectures and sensor placements on the performance of the dANN model were analyzed. 1. Introduction Most recently, Zhou [29] proposed a novel impact load identi- fication method of non-linear structures by using deep Recurrent Neural Networks (RNNs), verifying this method in three non-linear cases: damped Duffing oscillator, non-linear three-degree-of-freedom system and non-linear composite plate. Finally, deep learning neural networks techniques were recently implemented to recognize human activities from wearable sensors [32,33]. To date, there is no application for the identification of a body (e.g., head) acceleration with sensors embedded in a second body (e.g., helmet), which exhibits a relative movement to the first one. This paper contributes to the methodological development of a device for the real-time estimation of TBIs. Specifically, it proposes a novel method to estimate, as time functions, the components of head linear and rotational accelerations from signals provided by twelve single-axis accelerometers embedded in the outer shell of a motorcycle helmet. The sensors are organized in orthogonally oriented pairs at six different locations, with their sensing axis tangential to the helmet, as described in [34]. The system’s non-linearity at the helmet– head interface is modeled within the estimation process using deep learning ANNs. They were trained using data collected from finite element simulations, which is a time-saving and cost-effective technique compared to carrying out the same number of experimental tests. 2. Materials and Methods Head impact scenarios were defined considering previous research on head impacts. A wide and representative set of impact conditions was defined and reproduced with a finite element model to generate the necessary datasets. The Artificial Neural Network architecture was defined to optimize the estimation of head kinematics. The procedure implemented to develop and validate the technology is described at the end of this section. 2.1. Head Impact Scenarios A head impact scenario can be defined by two different variables: relative position between the helmeted head and the ground and the impact speed vector. Considering a reference scenario (Figure 1a) where the helmeted head was oriented such that the transverse plane of the head was parallel to the ground, the relative position in a generic scenario was defined by sequentially rotating the ground around the X-axis and Y-axis, while the helmeted head position was unchanged. Different ranges were considered for these two parameters: the angle β around the X-axis was varied between −100◦and 100◦, but the angle γ around the Y-axis was varied between −125◦and 115◦. g γ The impact speed vector was defined through its tangential and normal components to the ground and the orientation of the tangential component in the ground plane. Vehicle impact speed varies between 20 and 88 km/h in urban accidents [35]. Different studies reproducing typical oblique impacts in motorcycle crashes through experimental impact tests [36] or FE analyses [27,37] observed that the angular kinematics of the head remained quite constant at high tangential speeds. Cernicchi et al. [38] simulated motorcycle head impacts using two different Vn magnitudes: 2.20 and 5.66 m/s and a tangential velocity ranging from 0 to 60 m/s. They observed that at constant Vn, the angular acceleration peak did not vary above a specific Vt threshold (V∗ t ). However, as Vn increased, the value of V∗ t seemed also to increase (for Vn = 2.20 m/s, V∗ t = 4.21 m/s, while for Vn = 5.66 m/s, V∗ t = 8.57 m/s). Based on previous research, the magnitude of the impact speed ranged between 8 and 78 km/h to reproduce real-world head impacts in urban crashes. This magnitude was obtained by combining the normal speed component Vn, which ranged between 2 and 12 m/s, and the tangential speed component Vt, which varied between 3 and 18 m/s. A range of 360◦was considered for the orientation of the tangential component in the ground plane (angle θ). 4 of 16 Sensors 2022, 22, 5592 (b) X y Z (b) X y Z X Y (a) Figure 1. (a) Start of the first head-impact scenario; (b) scatter plot of the shell mesh nodes (blue points) and the impact points (black points) for the entire training dataset. X Y (a) (b) (a) Y X X y Figure 1. 2.2. Finite Element Head and Helmet Model Head impact scenarios were simulated using a Hybrid III (HIII) Head finite element model (https://biocorellc.com/finite-element-models/) (accessed on 21 July 2022) dis- tributed by the Biomechanics and Research, LLC (Biocore) [40], which was coupled with an AGV X3000 full-face helmet provided by the Dainese company. The original HIII model included both the head and neck, but only the head model was used in this study, in accordance with the ECE 22.06 standard [7]. The HIII head was previously validated by the University of Virginia Center for Applied Biomechanics [40], simulating the NHTSA Head Drop Certification Test. The present head includes three main parts: skin rubber layer, rigid skull and head mount. Both the head skin and mount used hexahedral solid elements, but the quadrilateral shell elements were used to mesh the rigid skull. The helmet model consists of an outer shell, an energy-absorbing liner, a chin pad and a chin strap. Further information about the helmet finite element model used in this paper can be found in [34]. This model was updated, including the accelerometers of the measurement device. Further information about the procedure adopted to select the accelerometer locations can be found in [34]. 2.1. Head Impact Scenarios (a) Start of the first head-impact scenario; (b) scatter plot of the shell mesh nodes (blue points) and the impact points (black points) for the entire training dataset. The latter five parameters (rotational angles around X-axis and Y-axis, normal and tangential impact speed components and tangential speed orientation) were combined using the Latin Hypercube Sampling (LHS) method [39] to define three different datasets of, respectively, 2000, 200 and 300 simulations. The purpose of the three datasets will be clarified later in Section 3. The impact points on the helmet outer shell for the training dataset of 2000 simulations are shown in Figure 1b. 2.3. Neural Networks The LSTM forward propagation process is described mathematically: ft = σ(Wf ∗[ht−1, xt] + bf ) (3) it = σ(Wi ∗[ht−1, xt] + bi) (4) eCt = tanh(Wc ∗[ht−1, xt] + bc) (5) Ct = ft ∗Ct−1 + it ∗eCt (6) ot = σ(Wo ∗[ht−1, xt] + bo) (7) ht = ot ∗tanh(Ct) (8) (8) where x and h are the input and the output, W the weight matrices and b the biases; σ and tanh are the logistic sigmoid and Tanh activation functions. The LSTM cell can decide whether to discard or keep the past information using the variable “Cell State” (C). With a value of C equal to 1, the information is completely kept, while a 0 means that the information is discarded. The signals of the accelerometers embedded in the helmet are the inputs to the LSTM network, while the outputs are the components of the linear or rotational acceleration of the head. Two separate but identical networks were used for the linear and rotational components to improve the estimation results. where x and h are the input and the output, W the weight matrices and b the biases; σ and tanh are the logistic sigmoid and Tanh activation functions. The LSTM cell can decide whether to discard or keep the past information using the variable “Cell State” (C). With a value of C equal to 1, the information is completely kept, while a 0 means that the information is discarded. The signals of the accelerometers embedded in the helmet are the inputs to the LSTM network, while the outputs are the components of the linear or rotational acceleration of the head. Two separate but identical networks were used for the linear and rotational components to improve the estimation results. p p A simple Neural Network with a single LSTM layer did not provide good perfor- mances in the impact force history identification, as stated by Zhou [29]. Assuming that the same conclusion can be considered for the estimation of impact acceleration, the deep neural network architecture used by Zhou [29] was the starting point for this study. Some improvements were identified to maximize the performance for the specific problem, and the final architecture is shown in Figure 3. It consists of a Bidirectional Long Short-Term Memory (BLSTM) layer, two LSTM layers and two Fully Connected (FC) layers. 2.3. Neural Networks Recurrent neural networks [41] are one of the most known types of Artificial Neural Networks, capable of processing sequential data or time series data. The ability to use feedback loops, commonly described as "memory", ensures the output is influenced by both previous and current inputs. The process of carrying memory forward is described mathematically: ht = fh(U ∗xt + V ∗ht−1) (1) ot = fo(W ∗ht) (2) (1) ( ) (2) (2) where xt is the input at time step t, ht stores the values of the hidden units at time step t and where xt is the input at time step t, ht stores the values of the hidden units at time step t and Sensors 2022, 22, 5592 5 of 16 5 of 16 ot is the output at time step t. fh and fo are the hidden and output unit activation functions. They define how the weighted sum of the input is transformed into an output; commonly used activation functions are logistic sigmoid, rectified linear (ReLU) and Hyperbolic Tangent (Tanh). The weight matrices U, W and V are determined with supervised training of the RNN, but it requires a huge amount of data (Figure 2a). A relevant problem in RNNs concerns long-term memory: input information (xt′) persists for a short time, but it cannot be kept for a long period of time due to the vanishing gradient problem. + X X X tanh σ σ tanh σ σ t- t t t- Ct- Ct t- t t t t t t t t- t t+ . . V V W U W U W U V V t- t t+ t- t t+ (a) (b) Figure 2. (a) Recurrent Neural Network; (b) Long Short Term Memory (LSTM) cell. (a) (b) Figure 2. (a) Recurrent Neural Network; (b) Long Short Term Memory (LSTM) cell. The long-term dependency was improved with the Long Short-Term Memory (LSTM) structure, introduced by Hochreitener and Schmidhurber [42] for the first time in 1997. They replaced the typical recurrent unit of RNNs with a more complex gated recurrent unit. The LSTM used in this study is a standard LSTM, and it is shown in Figure 2b. 2.3. Neural Networks Basically, a BLSTM layer consists of a forward LSTM layer and a backward LSTM layer. This provides the opportunity to consider both past and future responses of sequential inputs. BLSTM and the two LSTM layers have 200 cells per layer, the first FC layer has 200 hidden units, but the last FC layer has a number of hidden units equal to the number of outputs. The over-fitting was prevented using the dropout operation to each non-recurrent connection, as shown in the dashed lines in Figure 3. From here onwards, this neural network architecture will be referred to as deep Artificial Neural Network (dANN). 6 of 16 Sensors 2022, 22, 5592 CELL CELL CELL CELL CELL CELL CELL CELL CELL CELL CELL CELL BLSTM Layer LSTM Layers FC Layers CELL LSTM cell Simple neural cell Connection with dropout Simple connection Figure 3. Deep Artificial Neural Network (dANN) architecture. CELL Figure 3. Deep Artificial Neural Network (dANN) architecture. 3. Identification Steps STEP 1—Development of simulation datasets. Three datasets of simulations repro- ducing real-world head impacts in urban crashes were implemented with the procedure explained in Section 2.1. The first dataset consisted of 2000 simulations, and the second and the third ones consisted of, respectively, 200 and 300 simulations. The first dataset was used for training the network, while the second and the third datasets were used, respectively, for validation and testing purposes. g p p STEP 2—Neural Networks design. Two deep neural networks with the same structure described in Section 2.3 were implemented. The networks had the same inputs, i.e., accelerations from the accelerometers embedded in the helmet’t outer shell, but the outputs differed: the three head linear acceleration components were used as outputs of the network A and the three head rotational acceleration components were the outputs of the network B. From here onwards, the neural networks estimating linear and rotational accelerations will be referred to as A and B, respectively. p y STEP 3—Data preparation. Accelerations of the helmet outer shell were filtered using a SAE108 filter, but both the linear and rotational head accelerations were filtered with a CFC1000 fil d b ISO 6487 2002 d d [43] p y STEP 3—Data preparation. Accelerations of the helmet outer shell were filtered using a SAE108 filter, but both the linear and rotational head accelerations were filtered with a CFC1000 filt t d b ISO 6487 2002 t d d [43] CFC1000 filter as suggested by ISO 6487:2002 standard [43]. STEP 4—Model training. Networks A and B were fed with the inputs and outputs described in STEP 2. Weights and biases were optimized using a back-propagation through time (BPTT) [44] algorithm. The Root Mean Square propagation (RMSprop) [45] was selected as the optimizer. RMSprop is a Gradient Descent-based Learning Algorithm, which adapts, at each iteration, the learning rate of each parameter individually using a subset of the training data. A different subset, called a mini-batch, is used at each iteration. A mini-batch size equal to 16 was chosen to implement this process. The initial learning rate was 0.001, and the maximum epochs used for the training were 1000. After each training epoch, the Root-Mean-Squared-Error (RMSE) on the validation data was monitored. If the RMSE did not decrease after 30 consecutive epochs, the training process was stopped. Every 45 epochs, the learning rate was reduced by a factor of two. 3. Identification Steps y p g y STEP 5—Model performance assessment. Helmet accelerations from the test dataset were used as input for both the NNs A and B, and the estimated linear and rotational head acceleration components were compared to the target ones. Three parameters were introduced to assess the model performance: peak error, Head Injury Criterion (HIC) [46] and Rotational Injury Criterion (RIC) [12]. The assessment parameters were applied to both the reference head accelerations (extracted from the simulations) and the head accelerations estimated with the dANN. For each of them, the Pearson coefficient R2 was calculated. STEP 5—Model performance assessment. Helmet accelerations from the test dataset were used as input for both the NNs A and B, and the estimated linear and rotational head acceleration components were compared to the target ones. Three parameters were introduced to assess the model performance: peak error, Head Injury Criterion (HIC) [46] and Rotational Injury Criterion (RIC) [12]. The assessment parameters were applied to both the reference head accelerations (extracted from the simulations) and the head accelerations estimated with the dANN. For each of them, the Pearson coefficient R2 was calculated. Sensors 2022, 22, 5592 7 of 16 HIC [46] is the most commonly used metric for evaluating head and brain injury risk; it is currently required by the UN/ECE 22.06 standard [7] used in helmet regulation. HIC = max  (t2 −t1)  1 t2 −t1 Z t2 t1 |a(t)dt| 2,5 (9) (9) where a is the magnitude of the resultant head linear acceleration, and t1 and t2 are, respectively, the initial and final integral times over which HIC is calculated (t1 and t2 are selected to maximize HIC). RIC was proposed by Kimpara et al. [12]; it was formulated similarly to HIC by replacing the linear acceleration term with angular acceleration. RIC = max  (t2 −t1)  1 t2 −t1 Z t2 t1 |α(t)dt| 2,5 (10) (10) where α is the magnitude of the resultant head linear acceleration. For t1 and t2, the same considerations made for the HIC are applicable. In this paper, no consideration about the correlation between the HIC / RIC and head injuries will be analyzed. These criteria were selected exclusively as parameters to compare the estimated and target curves. 4.1. Training Dataset Training, validation and testing datasets were generated as described in Section 2.1 and the five parameters used to define the impact simulations were combined using the LHS. Initially the 2000 values of each parameter were sampled uniformly within their ranges. With this solution, most of the impact points were in the top outer shell area, but the left, right, back and chin areas had a poor spatial sampling. Therefore, the parameters that contributed to spreading the impact points over the outer shell surface (i.e., α and β) were modified, as shown in Figure 4a for β. Angle distributions were modified to have lower frequencies between −20◦and 20◦than between −100◦and −60◦or 60◦and 100◦. The new distribution of the parameters created a uniform spatial sampling over the entire outer shell surface of the helmet, as shown in Figure 1. g Figure 4b,c show, respectively, the distribution of the impact speed and the angle between the impact speed direction and the ground. Most of the impact simulations are characterized by an impact speed magnitude within the range of 8–16 m/s and an inclination angle within the range of 16–45◦. 0 5000 10000 15000 alphay [1/s 2] 6DOF helmet Real helmet 0-4 4-8 8-12 12-16 16-20 Impact speed [m/s] 0 100 200 300 400 500 600 700 800 Number of simulations Number of simulations (a) (b) (c) Figure 4. Distribution of (a) β angle values, (b) the impact speed magnitude, (c) the angle between the impact speed direction and the ground for the training dataset simulations. (a) (a) Figure 4. Distribution of (a) β angle values, (b) the impact speed magnitude, (c) the angle between the impact speed direction and the ground for the training dataset simulations. The deep Artificial Neural Networks used to estimate the head linear acceleration components were trained, as described in STEP 4 of Section 3. LSTM and BLSTM layers Sensors 2022, 22, 5592 8 of 16 8 of 16 and the first fully-connected layer had 200 hidden units. Dropout probability was set equal to 0.5 and learning rate equal to 0.001. Two examples of the estimated linear acceleration are shown in Figure 5: the plot in the rightmost column shows the resultant accelerations obtained by combining the three components plotted in previous columns. 4.1. Training Dataset 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 ax [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 ay [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 az [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] 0 20 40 60 80 100 120 140 160 180 a [g] Target Predicted Target HIC = 1266 Predicted HIC = 1002 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 ax [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 ay [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 az [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] 0 50 100 150 200 250 300 a [g] Targeta Predicteda Target HIC = 1107 Predicted HIC = 944 CASE 2 CASE 1 Figure 5. Linear acceleration prediction using the deep neural network model. 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 ax [g] Targeta Predicteda CASE 1 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 az [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -50 0 50 100 150 200 250 ay [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] 0 50 100 150 200 250 300 a [g] Target HIC = 1107 Predicted HIC = 944 CASE 1 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 ax [g] Target Predicted CASE 2 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] 0 20 40 60 80 100 120 140 160 180 a [g] Target HIC = 1266 Predicted HIC = 1002 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 az [g] 0 0.05 0.1 0.15 0.2 0.25 0.3 Time [s] -200 -150 -100 -50 0 50 ay [g] CASE 2 Figure 5. Linear acceleration prediction using the deep neural network model. The dANN reproduces the acceleration component’s shape and the peak values in both cases well (Figure 5). Peak values and HIC were calculated on the entire testing dataset (300 simulations). 4.1. Training Dataset Scatter plots in Figure 6 show the comparison between peak values of the target and estimated acceleration components. Peaks of the Z-component had the best overall performance with R2 = 0.972. Parameters calculated on the resultant acceleration, i.e., peak value and HIC, had similar R2 high values, confirming the strong learning ability of dANNs (Figure 7). 0 200 400 600 800 Target Max Linear ax [g] 0 100 200 300 400 500 600 Predicted Max Linear a [g] x R2=0.952 0 200 400 600 800 0 100 200 300 400 500 600 R2=0.949 0 200 400 600 800 0 100 200 300 400 500 600 R2=0.972 Predicted Max Linear a [g] y Predicted Max Linear a [g] z Target Max Linear ay [g] Target Max Linear az [g] (a) (b) (c) Testing data Regression line Figure 6. Correlation between target and predicted peak linear acceleration: (a) ax; (b) ay; (c) az. 0 0 200 400 600 800 0 100 200 300 400 500 600 R2=0.972 Predicted Max Linear a [g] z Target Max Linear az [g] (c) Figure 6. Correlation between target and predicted peak linear acceleration: (a) ax; (b) ay; (c) az. 9 of 16 Sensors 2022, 22, 5592 0 200 400 600 800 1000 Target Max Resultant a (g) 0 100 200 300 400 500 600 700 800 900 Predicted Max Resultant a (g) R2=0.946 Testing data Regression line Symmetry axis (a) 0 200 400 600 800 1000 Target Max Resultant a (g) 0 100 200 300 400 500 600 700 800 900 Predicted Max Resultant a (g) R2=0.946 Testing data Regression line Symmetry axis 0 5,000 10,000 15,000 Target HIC 0 5,000 10,000 15,000 Predicted HIC R2=0.960 (a) (b) Figure 7. Correlation between (a) target and predicted peak for the resultant linear acceleration and (b) target and predicted HIC. 00 0 5,000 10,000 15,000 Target HIC 0 5,000 10,000 15,000 Predicted HIC R2=0.960 (b) Figure 7. Correlation between (a) target and predicted peak for the resultant linear acceleration and (b) target and predicted HIC. 4.3. Head Rotational Acceleration The dANN used to estimate the head rotational accelerations had the same architecture as the dANN described in Section 4.2. Figure 8 shows the rotational accelerations for the same cases reported in Figure 5. Rotational acceleration components of shape and peak values were estimated with a good approximation (Figure 9). Rotational peak values around the Y-axis had the best performances, with R2 = 0.908. R2 coefficients for the peak values around the X and Z axes were, respectively, 0.800 and 0.783. 0 5 15 -5 10 -10 -15 -20 0 5 15 -5 10 -10 -15 -20 0 5 15 -5 10 -10 -15 -20 10 12 14 20 8 16 18 6 2 4 0 y z 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 2 4 10 6 8 12 14 SCENARIO 2 SCENARIO 1 Target RIC = 3.1x10 Predicted RIC = 2.7x10 7 7 Target RIC = 6.5x10 Predicted RIC = 6.4x10 7 7 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 y z Figure 8. Rotational acceleration prediction using the deep neural network model. 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 z 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 y 2 4 10 6 8 12 14 Target RIC = 3.1x10 Predicted RIC = 2.7x10 7 7 SCENARIO 1 0 2 4 10 -2 6 8 -6 -4 -10 -8 -12 0 5 15 -5 10 -10 -15 -20 y 0 5 15 -5 10 -10 -15 -20 SCENARIO 2 SCENARIO 2 Figure 8. Rotational acceleration prediction using the deep neural network model. 5 10 15 30 45 20 25 35 40 60 80 20 40 60 80 20 40 60 80 20 40 5 10 15 30 45 20 25 35 40 5 10 15 30 45 20 25 35 40 (a) (b) (c) z =0.800 =0.908 =0.783 y y z Testing data Regression line Figure 9. Correlation between target and predicted peak rotational acceleration: (a) αx; (b) αy; (c) αz. 4.3. Head Rotational Acceleration 5 10 15 30 45 20 25 35 40 60 80 20 40 60 80 20 40 60 80 20 40 5 10 15 30 45 20 25 35 40 5 10 15 30 45 20 25 35 40 (a) (b) (c) z =0.800 =0.908 =0.783 y y z Testing data Regression line Figure 9 Correlation between target and predicted peak rotational acceleration: (a) α ; (b) α ; (c) α (a) Figure 9. Correlation between target and predicted peak rotational acceleration: (a) αx; (b) αy; (c) αz. Sensors 2022, 22, 5592 10 of 16 10 of 16 Peak values of the resultant acceleration and RIC have an R2 correlation coefficient, respectively, of 0.771 and 0.687. (Figure 10). For both parameters, the regression line and the symmetry axis largely diverge for high values of acceleration. Testing data Regression line Symmetry axis =0.772 =0.687 (a) (b) Figure 10. Correlation between (a) target and predicted peak for the resultant rotational acceleration and (b) target and predicted RIC. Testing data Regression line Symmetry axis =0.772 (a) ( =0.687 b) Figure 10. Correlation between (a) target and predicted peak for the resultant rotational acceleration and (b) target and predicted RIC. 5. Discussions The indirect head kinematics estimation, through sensors embedded in protective devices and far from the head, is the only way to enable the real-time evaluation of TBIs without reducing the acceptability of the protective devices by the end users. This study presents the development and assessment of a new methodology, based on deep neural networks applied to a sensor system, to estimate the head kinematics with sensors attached to the inner surface of a helmet’s outer shell. Different designs for the neural network used to estimate linear and rotational accel- erations were tested. The analyzed architectures differed in the number of BLSTM and LSTM layers. Table 1 reports the designs of the networks and their performances for the rotational acceleration. R2 for peak values and RIC were used to compare the performances of the different deep neural networks investigated. DANN0 has the architecture and hyperparameters suggested by Zhou [29]; it was trained using the Adam optimizer, i.e., the same used by Zhou in his study. Network performances were not acceptable since values of R2 ranged between 0.168 and 0.278 for the estimation of peak values. A set of changes (network architecture integrated with an FC layer, learning rate decreased to 0.001, hidden units decreased to 100, and the Adam optimizer replaced with the RMSprop one) led to great improvements. Progressive increase in Hidden Unit values (100, 200 and 300; DANN1-DANN3) generated a slight improvement in the performances against an increase in training time. For instance, using 300 hidden units instead of 200 did not produce considerable improvement (as described in the next paragraphs, results are mainly affected by the lack of data for high acceleration values); therefore, 200 hidden units were used to analyze the learning rate influence. The worst results were obtained by decreasing or increasing the learning rate, as shown, respectively, by model DANN4 and DANN5. Other more complex architectures were investigated (models DANN6-DANN9), but none of them considerably improved the performances to justify the adoption of a more complex architecture against an increase in training time. BLSTM, 2LSTM and 2FC architecture were the most suitable for head kinematics estimation, and it was selected as the preferred one. DANN2 (Table 1) was used both to present the results shown in Section 4 and for the considerations listed below. Sensors 2022, 22, 5592 11 of 16 Table 1. 5. Discussions Effect of learning rate, hidden units and architecture in the peak values and RIC estimation. Architecture Learning Rate Hidden Units R2 Peak Value R2 RIC αx αy αz Resultant DANN0 BLSTM and 2LSTM [29] 0.005 128 0.248 0.278 0.168 0.192 0.404 DANN1 BLSTM and 2LSTM and 2FC 0.001 100 0.776 0.882 0.795 0.755 0.672 DANN2 BLSTM and 2LSTM and 2FC 0.001 200 0.800 0.908 0.783 0.772 0.687 DANN3 BLSTM and 2LSTM and 2FC 0.001 300 0.802 0.927 0.827 0.783 0.681 DANN4 BLSTM and 2LSTM and 2FC 0.01 200 0.775 0.849 0.759 0.746 0.679 DANN5 BLSTM and 2LSTM and 2FC 0.0001 200 0.689 0.016 0.231 0.529 0.514 DANN6 BLSTM and 3LSTM and 2FC 0.001 200 0.817 0.901 0.834 0.782 0.710 DANN7 2BLSTM and 3LSTM and 2FC 0.001 200 0.791 0.917 0.834 0.791 0.705 DANN8 3BLSTM and 2LSTM and 2FC 0.001 200 0.804 0.890 0.765 0.772 0.650 DANN9 4LSTM and 2FC 0.001 200 0.782 0.900 0.780 0.772 0.643 The overall correlation between the reference and estimated head accelerations was higher for the linear accelerations than the rotational ones (i.e., R2 = 0.946 vs. R2 = 0.772 for the resultant). Specifically, rotational acceleration estimation worsens as the absolute value of the acceleration peak increases. As shown in Figure 10, the regression line and the symmetry axis diverge for high values of acceleration. This did not happen for the parameters of the resultant linear acceleration (Figure 7), as the regression line and the symmetry axis remained almost parallel, even for high values of acceleration. The increase in the identification error was expected since rotational acceleration is strongly influenced by friction between head and helmet and the system behavior is more complex to model. Looking at the comparison between the target and estimated peak values of the resultant rotational acceleration (Figure 10a), the network underestimates the target values starting from a target peak of 40 krad/s2. This is confirmed by a smaller slope of the regression line compared to the symmetry axis. The network performance is affected by the small number of impacts with high rotational acceleration in the datasets (143 out of 2000 in the training dataset, 28 out of 300 in the testing dataset with a peak of rotational acceleration exceeding 40 krad/s2). 5. Discussions This result is a consequence of the procedure used to generate the databases: the position of the helmet with respect to the ground and the impact speed is defined, and the accelerations of the head–helmet system are a consequence of these parameters. Nonetheless, the network performances need to be properly framed within the physiological limits of the human body and take into account the overall objective of the research (i.e., early detection and treatment of TBIs). From this context, acceleration values, which cause immediate and permanent brain damage, should be clearly excluded. Brain injury tolerance based on head rotational acceleration was investigated in numer- ous studies Pike [47] proposed a peak angular head acceleration of 9 krad/s2 associated Table 1. Effect of learning rate, hidden units and architecture in the peak values and RIC estimation. Architecture Learning Rate Hidden Units R2 Peak Value R2 RIC αx αy αz Resultant DANN0 BLSTM and 2LSTM [29] 0.005 128 0.248 0.278 0.168 0.192 0.404 DANN1 BLSTM and 2LSTM and 2FC 0.001 100 0.776 0.882 0.795 0.755 0.672 DANN2 BLSTM and 2LSTM and 2FC 0.001 200 0.800 0.908 0.783 0.772 0.687 DANN3 BLSTM and 2LSTM and 2FC 0.001 300 0.802 0.927 0.827 0.783 0.681 DANN4 BLSTM and 2LSTM and 2FC 0.01 200 0.775 0.849 0.759 0.746 0.679 DANN5 BLSTM and 2LSTM and 2FC 0.0001 200 0.689 0.016 0.231 0.529 0.514 DANN6 BLSTM and 3LSTM and 2FC 0.001 200 0.817 0.901 0.834 0.782 0.710 DANN7 2BLSTM and 3LSTM and 2FC 0.001 200 0.791 0.917 0.834 0.791 0.705 DANN8 3BLSTM and 2LSTM and 2FC 0.001 200 0.804 0.890 0.765 0.772 0.650 DANN9 4LSTM and 2FC 0.001 200 0.782 0.900 0.780 0.772 0.643 Table 1. Effect of learning rate, hidden units and architecture in the peak values and RIC estimation. The overall correlation between the reference and estimated head accelerations was higher for the linear accelerations than the rotational ones (i.e., R2 = 0.946 vs. R2 = 0.772 for the resultant). Specifically, rotational acceleration estimation worsens as the absolute value of the acceleration peak increases. As shown in Figure 10, the regression line and the symmetry axis diverge for high values of acceleration. This did not happen for the parameters of the resultant linear acceleration (Figure 7), as the regression line and the symmetry axis remained almost parallel, even for high values of acceleration. 5. Discussions The increase in the identification error was expected since rotational acceleration is strongly influenced by friction between head and helmet and the system behavior is more complex to model. Looking at the comparison between the target and estimated peak values of the resultant rotational acceleration (Figure 10a), the network underestimates the target values starting from a target peak of 40 krad/s2. This is confirmed by a smaller slope of the regression line compared to the symmetry axis. The network performance is affected by the small number of impacts with high rotational acceleration in the datasets (143 out of 2000 in the training dataset, 28 out of 300 in the testing dataset with a peak of rotational acceleration exceeding 40 krad/s2). This result is a consequence of the procedure used to generate the databases: the position of the helmet with respect to the ground and the impact speed is defined, and the accelerations of the head–helmet system are a consequence of these parameters. Nonetheless, the network performances need to be properly framed within the physiological limits of the human body and take into account the overall objective of the research (i.e., early detection and treatment of TBIs). From this context, acceleration values, which cause immediate and permanent brain damage, should be clearly excluded. Brain injury tolerance based on head rotational acceleration was investigated in numer- ous studies. Pike [47] proposed a peak angular head acceleration of 9 krad/s2 associated Sensors 2022, 22, 5592 12 of 16 12 of 16 with a 10% risk of Mild Traumatic Brain Injury (MTBI) based on 27000 head impacts recorded from American football players at the collegiate level. Zhang et al. [48] proposed a maximum resultant rotational acceleration peak of 7.9 krad/s2 for an 80% probability of sustaining an MTBI. Rotational acceleration peak was also connected to specific TBI such as concussion and DAI. The first type of injury was analyzed by Ommaya et al. [8], which sug- gested an angular acceleration tolerance of 1.8 krad/s2 for a 50% probability of concussion. Rowson et al. [20] proposed a tolerance value of 6383 krad/s2 for the same probability of concussion. Finally, Margulies and Thibault [10] proposed an angular acceleration of 16 krad/s2 as tolerance to moderate to severe DAI for the human head subjected to a lateral motion. 5. Discussions These results support the exclusion of impacts with peak rotational acceleration above 40 krad/s2 from the assessment of the proposed method, as this threshold is more than double the highest value cited in [10]. With the redefined dataset, the estimation of peak values is improved, and R2 exceeded 0.9 for each component. The new scatter plots for peak values of the acceleration components, the resultant acceleration and RIC are shown in Figures 11 and 12. Considering Figures 7 and 12, a slight systematic underestimation of the accelerations is still evident when the absolute peak value increases. Future exploitation of this method in a real-time system implemented into a helmet should include a proper correction to adjust for these errors. Testing data Regression line y y z z (a) (b) (c) =0.923 =0.922 =0.913 Figure 11. Correlation between target and predicted peak rotational acceleration for the reduced testing dataset (exclusion of impacts with peak rotational acceleration above 40 krad/s2): (a) αx; (b) αy; (c) αz. y y (b) =0.922 Testing data Regression line (a) ( =0.923 z z c) =0.913 Figure 11. Correlation between target and predicted peak rotational acceleration for the reduced testing dataset (exclusion of impacts with peak rotational acceleration above 40 krad/s2): (a) αx; (b) αy; (c) αz. =0.925 Testing data Regression line Symmetry axis =0.914 (a) (b) =0.925 Testing data Regression line Symmetry axis =0.914 (a) (b) Figure 12. Correlation between (a) target and predicted peak for the resultant rotational acceleration and (b) target and predicted RIC for the reduced testing dataset (exclusion of impacts with peak rotational acceleration above 40 krad/s2). Figure 12. Correlation between (a) target and predicted peak for the resultant rotational acceleration and (b) target and predicted RIC for the reduced testing dataset (exclusion of impacts with peak rotational acceleration above 40 krad/s2). A considerable amount of data is usually needed to obtain acceptable results from a trained deep neural network. Zhou et al. [29], in the identification of the impact load history acting on a composite plate, have used 10,000 signals to train their dANN, compared to 2000 acceleration signals used in this study. All simulations were run on a 72-cpu cluster and each simulation took approximately 20 min, for 28 days of total computational time. 5. Discussions Sensors 2022, 22, 5592 13 of 16 As well as the performances of the dANNs, calculation time is really important for a future industrial application of this method. Results proved that the indirect identification of head kinematics history through dANNs, fed with data from accelerometers embedded in the helmet, can be successfully accomplished using a training dataset of limited size. The use of the LHS method to combine the factors described in Section 2.1 and create a well-distributed dataset of simulations for the training process was a relevant contribution to the methodology, as it avoided data overfitting and selection bias. 6. Conclusions In this paper, a novel method to identify the head linear and rotational acceleration time signals from acceleration data, acquired by single-axis sensors embedded in the outer shell of a helmet and processed with deep learning techniques, is presented. The results support the following major findings: • Neural Networks: LSTM Neural Networks are capable of reproducing the underlying non-linear behavior of the model. A specific network was defined to solve the problem, and the related hyperparameters were determined. This result enables real-time prediction of head kinematics and paves the road to the application of specific metrics for TBI estimation. – The training, validation and testing datasets can be obtained from a virtual environment using state-of-the-art tools (e.g., Finite Elements). The approach used for the generation of datasets greatly reduces time requirements and costs compared to experimental tests; – An adequate size for the datasets was determined, which may be used for guid- ance in further applications. The networks can be trained with a limited amount of data because of the well-distributed dataset generated using the LHS method to combine the parameters defining our case study; p g y – Both the use of virtual testing tools and the application of the LHS method to generate the simulation inputs facilitate the industrial application of the method- ology. • Prediction: The results proved the high accuracy of the trained networks, as a high correlation coefficient was obtained for all the parameters used in the assessment stage. To the best knowledge of the authors, this is the first application of neural networks for the estimation of a body (head) acceleration with sensors embedded in a second body (helmet), considering that the two bodies are not fully connected. The results proved the feasibility of the proposed methodology. The next steps will focus on the experimental validation of the findings highlighted in this study. Author Contributions: Conceptualization, A.B. and N.B.; methodology, A.B. and N.B.; software, A.B.; validation, A.B.; formal analysis, A.B.; investigation, A.B.; data curation, A.B.; writing—original draft preparation, A.B.; writing—review and editing, A.B. and N.B; visualization, A.B.; supervision, N.B. 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. Abbreviations TBIs Traumatic Brain Injuries PTW Powered Two Wheeler ANNs Artificial Neural Networks VRUs Vulnerable Road Users MEMS MicroElectroMechanical Systems HITS Head Impact Telemetry System CoG Centre of Gravity 6DOF 6 Degrees of Freedom GTF gForce Tracker RNNs Recurrent Neural Networks LHS Latin Hypercube Sampling HIII Hybrid III ReLU Rectified linear function Tanh Hyperbolic Tangent function LSTM Long Short Term Memory BLSTM Bidirectional Long Short Term Memory FC Fully Connected dANN deep Artificial Neural Network BPTT Back Propagation Through Time RMSprop Root Mean Square propagation RMSE Root Mean Square Error HIC Head Injury Criterion RIC Rotational Injury Criterion MTBI Mild Traumatic Brain Injury References 1. Santiago, L.A.; Oh, B.C.; Dash, P.K.; Holcomb, J.B.; Wade, C.E. A clinical comparison of penetrating and blunt traumatic brain injuries. Brain Inj. 2012, 26, 107–125. [CrossRef] [PubMed] 2. Daugherty, J.; Waltzman, D.; Sarmiento, K.; Xu, L. 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Dever, A.; Powell, D.; Graham, L.; Mason, R.; Das, J.; Marshall, S.J.; Vitorio, R.; Godfrey, A.; Stuart, S brain injury: A systematic review. Sensors 2022, 22, 1480. [CrossRef] 4. World Health Organization and others. Global Status Report on Road Safety 2018: Summary; World Health Organization: Geneva, Switzerland 2018. 5. Gabler, L.F.; Crandall, J.R.; Panzer, M.B. Assessment of Kinematic Brain Injury Metrics for Predicting Str d d [ f] 5. Gabler, L.F.; Crandall, J.R.; Panzer, M.B. Assessment of Kinematic Brain Injury Metrics for Predicting Strain Responses in Diverse Automotive Impact Conditions. Ann. Biomed. Eng. 2016, 44, 3705–3718. [CrossRef] 5. Gabler, L.F.; Crandall, J.R.; Panzer, M.B. Assessment of Kinematic Brain Injury Metrics for Predicting Strain Responses in Diverse Automotive Impact Conditions. Ann. Biomed. Eng. 2016, 44, 3705–3718. [CrossRef] p g 6. United Nations. Uniform Provisions Concerning the Approval of Protective Helmets and Their Visors for Drive 6. United Nations. Uniform Provisions Concerning the Approval of Protective Helmets and The Cycles and Mopeds; UN ECE 22.05; United Nations: San Francisco, CA, USA, 2002. Cycles and Mopeds; UN ECE 22.05; United Nations: San Francisco, CA, USA, 2002. y p ; UN C 05; U e N o s S c sco, C , US , 00 7. United Nations. 6. Conclusions Institutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable Informed Consent Statement: Not applicable Informed Consent Statement: Not applicable Data Availability Statement: The data that support the findings of this study are available from the authors upon reasonable request. Acknowledgments: This study utilized a model derived from a model licensed from Biomechanics Consulting and Research, LC (Biocore). The development of this model was made possible by a grant from Football Research, Inc. (FRI) and the National Football League, with input from the NFLPA. The views expressed are solely those of the authors and do not represent those of Biocore, FRI, or Sensors 2022, 22, 5592 14 of 16 14 of 16 any of its affiliates or funding sources. The authors acknowledge Dainese SpA for supporting this research through the availability of the FE helmet model. any of its affiliates or funding sources. The authors acknowledge Dainese SpA for supporting this research through the availability of the FE helmet model. Conflicts of Interest: The authors declare no conflict of interest. References Head imp football players. J. Biomech. 2011, 44, 2673–2678. [CrossRef] [PubMed] 19. Beckwith, J.G.; Greenwald, R.M.; Chu, J.J. Measuring head kinematics in football: Correlation between the head impact telemetry system and Hybrid III headform. Ann. Biomed. Eng. 2012, 40, 237–248. [CrossRef] y y g 20. Rowson, S.; Duma, S.M.; Beckwith, J.G.; Chu, J.J.; Greenwald, R.M.; Crisco, J.J.; Maerlender, A.C. 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Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom
Frontiers in microbiology
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ORIGINAL RESEARCH published: 22 March 2019 doi: 10.3389/fmicb.2019.00590 Citation: Citation: Zhang Y, Lin X, Shi X, Lin L, Luo H, Li L and Lin S (2019) Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom. Front. Microbiol. 10:590. doi: 10.3389/fmicb.2019.00590 Zhang Y, Lin X, Shi X, Lin L, Luo H, Li L and Lin S (2019) Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom. Front. Microbiol. 10:590. doi: 10.3389/fmicb.2019.00590 Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom Yaqun Zhang1, Xin Lin1, Xinguo Shi1,2, Lingxiao Lin1, Hao Luo1, Ling Li1 and Senjie Lin1,3* 1 State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, China, 2 College of Biological Science and Engineering, Fuzhou University, Fuzhou, China, 3 Department of Marine Sciences, University of Connecticut, Groton, CT, United States Diatoms and dinoflagellates dominate coastal marine phytoplankton communities as major players of marine biogeochemical cycles and their seasonal succession often leads to harmful algal blooms (HABs). What regulates their respective dominances and the development of the HABs remains elusive. Here we conducted time- sequential metatranscriptomic profiling on a natural assemblage that evolved from diatom dominance to a dinoflagellate bloom to interrogate the underlying major metabolic and ecological drivers. Data reveals similarity between diatoms and dinoflagellates in exhibiting high capacities of energy production, nutrient acquisition, and stress protection in their respective dominance stages. The diatom-to-dinoflagellate succession coincided with an increase in turbidity and sharp declines in silicate and phosphate availability, concomitant with the transcriptomic shift from expression of silicate uptake and urea utilization genes in diatoms to that of genes for light harvesting, diversified phosphorus acquisition and autophagy-based internal nutrient recycling in dinoflagellates. Furthermore, the diatom-dominant community featured strong potential to carbohydrate metabolism and a strikingly high expression of trypsin potentially promoting frustule building. In contrast, the dinoflagellate bloom featured elevated expression of xanthorhodopsin, and antimicrobial defensin genes, indicating potential importance of energy harnessing and microbial defense in bloom development. This study sheds light on mechanisms potentially governing diatom- and dinoflagellate- dominance and regulating bloom development in the natural environment and raises new questions to be addressed in future studies. *Correspondence: Senjie Lin Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 12 December 2018 Accepted: 07 March 2019 Published: 22 March 2019 Keywords: harmful algal blooms, metatranscriptome, Prorocentrum donghaiense, rhodopsin, energy generation, diatoms Zhang Y, Lin X, Shi X, Lin L, Luo H, Li L and Lin S (2019) Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom. Front. Microbiol. 10:590. doi: 10.3389/fmicb.2019.00590 Edited by: Hongbin Liu, The Hong Kong University of Science and Technology, Hong Kong Edited by: Hongbin Liu, The Hong Kong University of Science and Technology, Hong Kong Reviewed by: Giovanna Romano, Stazione Zoologica Anton Dohrn, Italy Gwenael Piganeau, Biologie Intégrative des Organismes Marins (BIOM), France *Correspondence: Senjie Lin senjie.lin@uconn.edu Reviewed by: Giovanna Romano, Stazione Zoologica Anton Dohrn, Italy Gwenael Piganeau, Biologie Intégrative des Organismes Marins (BIOM), France *Correspondence: Senjie Lin senjie.lin@uconn.edu *Correspondence: Senjie Lin senjie.lin@uconn.edu Field Campaign, Environmental Measurements, and Sample Collection Field Campaign, Environmental Measurements, and Sample Collection gy q Numerous physioecological studies have shown that diatoms and dinoflagellates have distinct ecological niches (Irwin et al., 2012). Diatoms are generally known for a high capacity to assimilate silicate and nitrogen and prefer lower temperatures, as such tend to thrive in cool nitrate-rich environments (Lomas and Glibert, 2000; Falkowski et al., 2004; Malviya et al., 2016). In contrast, dinoflagellates tend to bloom in warm waters, which are usually more nutrient poor (Smayda, 1997). Some studies also demonstrate that dinoflagellate red tide blooms increased as the N:P ratio fell (Anderson et al., 2002), while some other studies show that species differentiate in nutrient uptake efficiency and favorable nutrient conditions (Glibert and Burkholder, 2011). The ample ecological observations and experimental data constitute the core of our understanding on the relationship between diatoms and dinoflagellates (Anderson et al., 2002; Falkowski and Oliver, 2007). Yet it is still difficult to pinpoint which metabolic processes underpin the differential niches, govern their respective dominances and regulate the diatom-to-dinoflagellate succession leading to the dinoflagellate bloom, largely because in situ metabolic profiles are generally difficult to measure and contributions of different taxa are difficult to partition (Alexander et al., 2015a). Measurements, and Sample Collection Because regime shift from diatom dominance to a dinoflagellate bloom (most frequently of P. donghaiense) has occurred in the April–May period in the ECS coastal water since 1990s (Gao and Shao, 2011; Sun et al., 2017; Supplementary Figure S1), a research cruise was launched in the high HAB risk area of the Yangtze River Estuary, ECS. Surface seawater (0–2 m) samples were collected from April 30 to May 20 in 2014 when the phytoplankton community underwent major changes. Station 7 (29◦1′0′′N, 122◦9′27′′E) was sampled on April 30 (T0) and May 20 (T3) whereas station 7A (29◦3′19′′N. 122◦16′30′′E) on May 13 (T1) and May 15 (T2) (Supplementary Figure S1). Fifty- mL seawater samples were collected for each time point and fixed in Lugol’s solution (2%) for subsequent microscopic species identification and enumeration in Sedgwick-Rafter counting chamber. For metatranscriptomic analyses, 4–13L seawater samples were prefiltered through a 200 µm nylon mesh to remove large particles and zooplankton, then filtered through 3 µm pore-size 144 mm diameter polycarbonate membrane (Merck Millipore, MA, United States) using a vacuum pump under low vacuum pressure (<10 PSI). Field Campaign, Environmental Measurements, and Sample Collection The filters were cut into four even pieces, and each was immediately transferred to a 2 mL tube; two tubes containing 1 mL TRI Regent buffer for RNA extraction and two containing 1 mL DNA lysis buffer (100 mM Tris-Cl, 50 mM EDTA, pH = 8) for DNA analysis to be reported elsewhere. To minimize changes of gene expression and community structure in the process, total sample processing time, from arrival on deck to the buffers, was no more than 15 min. The samples for RNA were frozen in liquid N2 where they were kept until the end of the cruise, and once transported back to our laboratory the samples were stored at −80◦C until RNA extraction. The samples for DNA were stored at −20◦C until DNA extraction. Temperature, salinity, and turbidity were measured using CTD (SBE 17plus V2, Sea-Bird Scientific, United States) at each sampling event. Concentrations of dissolved inorganic nitrogen (DIN) (ammonium, nitrate, and nitrite), silicate and phosphate were determined using continuous flow analyzer, all measurements were performed on triplicate samples San++ (SKALAR, Breda, Holland) (Li et al., 2017). The metatranscriptomic approach has proven to be powerful in characterizing metabolic configurations in dinoflagellates (Lin et al., 2010; Cooper et al., 2014; Zhuang et al., 2015; Gong et al., 2017), diatoms (Alexander et al., 2015a), and other lineages (Alexander et al., 2015b) in the natural marine environment. In this approach, the relative expression levels of genes in the community are indicative of relative activities of the biochemical or physiological phenotypes regulated by the proteins or enzymes encoded by those genes. Furthermore, these genes can be traced to the organisms existing in the community by sequence comparison so that the contributing taxa to the active phenotypes can be identified and the relative contribution of each taxon can be assessed. Here we employed time-sequential (month long) metatranscriptomics to characterize the evolution of a phytoplankton community from diatom dominance (dominantly Skeletonema) to dinoflagellate (Prorocentrum donghaiense) bloom, an annual event observed in the studied area in East China Sea (ECS) for more than a decade (Liu et al., 2016; Xiao et al., 2017). INTRODUCTION Diatoms and dinoflagellates are two ecologically important groups of eukaryotic phytoplankton in the ocean, representing major contributors of marine primary production, which participates in the global carbon fixation and O2 production. They are major players in biogeochemical cycles of elements (including biological pump of carbon for deep-sea sequestration) and coastal March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org Molecular Regulation of Phytoplankton Community Dominance Zhang et al. seasonal succession (Margalef, 1978; Falkowski et al., 2004). While diatoms normally dominate, dinoflagellates can outgrow them seasonally or on occasions of environmental perturbations, often resulting in harmful algal blooms (HABs) that exact devastating impacts on the marine ecosystem, economy, and public health. How these two keystone phytoplankton groups are so successful and how their “seesaw” dynamics tips toward a bloom are fundamental yet poorly understood marine ecology questions. in response to environmental variations; and (3) Competitive advantage-conferring biochemical pathways may be facilitative for dinoflagellates to outgrow diatoms and form blooms. Field Campaign, Environmental Measurements, and Sample Collection The metatranscriptomic investigation was conducted to examine three hypotheses regarding the diatom-to-dinoflagellate regime shift: (1) Diatoms and dinoflagellates may use the same biological processes (e.g., energy production, nutrient acquisition, and stress protection) while maintaining their respective dominance in the natural assemblages; (2) diatoms and dinoflagellates may use distinct metabolic pathways in their respective dominance Frontiers in Microbiology | www.frontiersin.org RNA Extraction and Illumina High-Throughput Sequencing Frozen samples were thawed and plankton cells were washed offthe filters using pipettes in RNase-free petri dish and then moved back into the 2 mL centrifuge tube and centrifuged at 14000 × g for 15 s, and most of the supernatant was removed into a fresh tube leaving only about 200 µL behind. Next, a 1:1 mixture of 0.5 mm and 0.1 mm-diameter glass beads (Biospec, March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 2 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. Bartlesville, OK, United States) were added to the 200 µL sample, with the volume of beads being approximately equal to the cell pellets. Then the samples with beads were loaded onto a FastPrep- 24 bead mill (MP Biomedicals, Solon, OH, United States) for bead-beating at the rate of 6 m/s, performed three times with 1 min intervals when the samples were placed on ice. Two microliters of homogenate were checked microscopically to verify complete cell breakage. Then, the removed supernatant was combined with the homogenized component, and RNA was extracted following the TRI Reagent protocol coupled with the Direct-zolTM RNA columns as reported previously (Lin et al., 2010). RNA concentration was measured using a NanoDrop ND-2000 Spectrophotometer (Thermo Fisher Scientific, Walthman, MA, United States), while integrity was assessed using RNA 6000 Nano LabChip Kit in microcapillary electrophoresis Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). The RNA integrity number (RIN) of the samples was all above the recommended value (6.0) for metatranscriptome sequencing. For Illumina RNA-seq sequencing, 1 µg total RNA from each sample was used to isolate mRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, United States). mRNA was then fragmented with First Strand Synthesis Reaction Buffer and Random Primer Mix (2×) at 94◦C for 10 min, and first strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The double- stranded cDNA purified, end repaired, and ligated to adaptors. Fragments of about 400 bp (with the approximate insert size of 250 bp) were selected and sequenced on Illumina HiSeq instrument (Illumina, San Diego, CA, United States). package, with TMM normalization (Robinson et al., 2010), and significance of differential expression was assigned with edgeR’s exactTest function following previous reports (Marchetti et al., 2012; Shi et al., 2017). Transcriptome Analysis and Gene Expression Quantification p After Illumina RNA-seq sequencing, low quality reads (cut- offaverage score value of 20) and adaptors were removed using Trimmomatic V0.30 (Bolger et al., 2014), and then two different ways were taken to process the clean reads. First, to determine the taxonomic origin of the transcripts, clean reads from the time-serial samples were mapped to a “local database” consisting of all the assembled transcriptomes of eukaryotic algae generated by the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP, downloaded in March, 2015) and P. donghaiense transcriptome (Shi et al., 2017) using Bowtie2 version 2.2.1 (parameters: -sensitive) (Langmead and Salzberg, 2012; Alexander et al., 2015a). Reads mapped to diatoms and to P. donghaiense were separated for further analysis. Expression levels of these mapped reads were then quantified using HTSeq python 3.4.3 package (Anders et al., 2014), and normalized to total transcriptomic reads of the taxonomic group using FPKM value (Fragment Per Kilo bases per Million reads). Differential gene expression for diatoms and dinoflagellates, separately, was both analyzed between bloom group (T1, T2, T3, which were treated as triplicates for the bloom condition) and non-bloom group (T0). Count-based differential expression for metatranscriptome was analyzed using edgeR Bioconductor Phylogenetic Analyses Phylogenetic analysis was conducted to assess taxonomic affiliation of particular genes. Deduced protein sequences and selected reference sequences were aligned using ClustalW in MEGA 6 (Larkin et al., 2007; Tamura et al., 2013). ProtTest 3.4.2 were used to find the best model of protein evolution (Darriba et al., 2011). Phylogenetic trees were inferred using Maximum likelihood method in MEGA 6 using the model with rates and parameters estimated from ProtTest, with 1000 bootstrap replicates performed to obtain statistical support for the tree topology. The resulting tree file from MEGA 6 was then uploaded to the iTOL to make further modifications (Letunic and Bork, 2007). 1ftp://ftp.ncbi.nlm.nih.gov/blast/db/ [accessed 03, 2015] RNA Extraction and Illumina High-Throughput Sequencing To strengthen reliability of differential gene expression profile found from the dataset, which lacked replicates at T0, NOISeq-sim (Tarazona et al., 2015) (Tarazona et al., 2011) was also conducted to independently identify the differential expressed genes (q = 0.9). Only the genes identified by both edgeR and NOISeq-sim as differentially expressed were accepted as differential expressed genes. The criteria for defining statistically significant differential expression were fold changes >2 and FDR <0.05 (adjusted P-value, determined by the Benjamini and Hochberg multiple-testing correction implemented in the “p. adjust” method in R). Second, to retrieve all expressed genes, including those not represented in the MMETSP database, our RNA-Seq reads were assembled de novo using Trinity (r2013-02-25) (parameter: -default), and the assembled sequences were clustered to remove redundancy using software TGICL (TIGR Gene Indices clustering tools, V2.1) (Pertea et al., 2003). Open reading frames were predicted and confirmed based on Markov model. The resulting unigenes were used for BLAST search and annotation against the NCBI non-redundant (nr) database1 and Swiss- Prot with a 1e-5 value cutoff, and also annotated against eggNOGV4.1 database (for COG annotation) with a 1e-5 value cutoffand PFAM database with a 1e-3 value cutoff. KEGG mapping and analysis were carried out on KEGG Automatic Annotation Server (KAAS). Frontiers in Microbiology | www.frontiersin.org Common Metabolic Pathways Underlying Diatom- and Dinoflagellate-Dominance A research cruise was conducted from April 23 till May 23, 2014 to follow the regime shift from diatom dominance to a dinoflagellate bloom in the ECS coastal area, covering a series of stations along three transects (Supplementary Figure S1). At T0 (April 30) no bloom was noticed and microscopic examination indicated a diverse phytoplankton community dominated by the diatoms Skeletonema and Pseudo-nitzschia. A bloom was visible from T1 through T3 (May 13–20) and microscopic check indicated that the bloom was caused by the dinoflagellate P. donghaiense (Supplementary Figure S1). We chose four time points to represent a regime shift from dominance by diatoms (T0) to a bloom predominated by the dinoflagellate P. donghaiense (T1, T2, and T3, on May 13, 15, and 20, respectively). Total eukaryotic phytoplankton concentration at T0 was 5.23 × 105 cells L−1, 88.64% of which was contributed by diatoms (64.98% by Skeletonema and 20.62% by P. nitzschia), whereas that at T1, T2, and T3 was 2.54 × 106, 4.89 × 106, and 10.34 × 106 cells L−1, respectively, with 79.74, 77.45, and 90.96% accounted for by dinoflagellates, mostly P. donghaiense (69.32%, 67.26%, 86.04%, respectively) (Supplementary Figure S2). As a major nutrient for diatom growth, silicate concentration decreased markedly, from 83.11 µM at T0 to <20 µM at T1, T2, and T3 (t-test, P < 0.05, n = 3) (Figure 1A). DIN remained at a high concentration (>10 µM) throughout the study period (Figure 1A). In contrast, PO43−concentration decreased dramatically from 1.16 µM at T0 to 0.34 µM or lower Dinoflagellate-Dominance HiSeq RNA sequencing generated 69–96 million clean reads from T0, T1, T2, and T3 samples, which were, respectively, assembled into 416,844, 263,750, 249,364, and 179,699 unigenes (Supplementary Table S2). To assess the expression levels of genes in each major lineage of phytoplankton, we mapped the metatranscriptome clean reads to eukaryotic algal and P. donghaiense transcriptome databases (Keeling et al., 2014; Shi et al., 2017), and calculated the proportion of reads mapped to that lineage out of total reads that were mapped to the database. The number of reads mapped to diatoms was 3.87 × 106, 1.06 × 105, 1.47 × 105, 1.92 × 104 for T0, T1, T2, and T3 samples, and the number of reads mapped to dinoflagellates was 1.24 × 106, 2.75 × 107, 2.60 × 107, 4.34 × 107 at these four time points. Data Availability FIGURE 1 | The dynamics of the phytoplankton community and the environmental conditions from T0 to T3. (A) Concentrations of major nutrients in the ambient environment, including various forms of nitrogen, silicate, and phosphate. (B) Taxonomic distribution (fractions) of transcripts in the metatranscriptome (for the mapped reads) at each time point. Raw metatranscriptome sequencing reads in our study were deposited in NCBI Sequence Read Archive under accession numbers SAMN06849052, SAMN06849053, SAMN06849054, and SAMN06849072. subsequently (t-test, P < 0.05, n = 3) (Figure 1A). The turbidity of the water increased remarkably (from 0.51FTU to 11.02FTU, surface seawater temperature increased from 16.50 to 19.19◦C from April 30 to May 13–20, and salinity decreased slightly from 30.63 to 28.51 PSU in this period, Supplementary Figure S3). Reverse Transcription Quantitative PCR (RT-qPCR) of Rhodopsin We used calmodulin and actin as reference genes to normalize the expression of the selected genes as reported (Shi et al., 2013) (Supplementary Table S1). RT-qPCR was performed on iCycle iQ Real-Time PCR Detection System using Bio-Rad iQ SYBR Green Supermix Kit (Bio-Rad Laboratories, Hercules, United States). Relative expression was calculated using the comparative Ct method (2−11Ct) (Livak and Schmittgen, 2001). Significant differences of environmental factors (e.g., silicate, PO43−) between the means of T0 and T1, T2, T3 and RT-qPCR between the means of the four samples were determined using independent samples t-test (two sided). Reverse Transcription Quantitative PCR (RT-qPCR) of Rhodopsin As rhodopsin was found to be one of the highly expressed genes during the bloom, P. donghaiense rhodopsin expression was quantified for the field samples and additional samples collected from cultures for comparison. P. donghaiense cultures (triplicate) were grown at 20◦C under a photon flux of 100 µE m−2 s−1 with a 14:10 h light/dark cycle in L1 medium (without silicate) with 36 µM phosphate concentration. When the cultures entered the exponential growth stage, samples were collected at the middle of light period. RNA extraction was carried out as described above. For both the field and the culture samples, 300 ng of total RNA of March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 3 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. FIGURE 1 | The dynamics of the phytoplankton community and the environmental conditions from T0 to T3. (A) Concentrations of major nutrients in the ambient environment, including various forms of nitrogen, silicate, and phosphate. (B) Taxonomic distribution (fractions) of transcripts in the metatranscriptome (for the mapped reads) at each time point. each sample were reverse-transcribed in a final volume of 20 µL using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology, Dalian, China) including a gDNA removal step. Specific primers of rhodopsin were designed based on the unigene sequences obtained from P. donghaiense transcriptome (Supplementary Table S1). We used calmodulin and actin as reference genes to normalize the expression of the selected genes as reported (Shi et al., 2013) (Supplementary Table S1). RT-qPCR was performed on iCycle iQ Real-Time PCR Detection System using Bio-Rad iQ SYBR Green Supermix Kit (Bio-Rad Laboratories, Hercules, United States). Relative expression was calculated using the comparative Ct method (2−11Ct) (Livak and Schmittgen, 2001). Significant differences of environmental factors (e.g., silicate, PO43−) between the means of T0 and T1, T2, T3 and RT-qPCR between the means of the four samples were determined using independent samples t-test (two sided). each sample were reverse-transcribed in a final volume of 20 µL using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology, Dalian, China) including a gDNA removal step. Specific primers of rhodopsin were designed based on the unigene sequences obtained from P. donghaiense transcriptome (Supplementary Table S1). Common Metabolic Pathways Underlying Diatom- and Dinoflagellate-Dominance Furthermore, the T0 library mainly binned with Skeletonema, which accounted for 65% of the mapped reads, while T1, T2, and T3 libraries were dominated by dinoflagellates, with the proportion of Prorocentrum transcripts growing from 4% at T0 to 54–80.32% later (Figure 1B). The large proportion of diatom transcripts at T0 and that of dinoflagellate transcripts at the T1-T2-T3 period were consistent with the trend of diatom and dinoflagellate abundances shown above, indicating coincidence between transcriptional activity and numerical abundance of the lineages. March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 4 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. and cell proliferation in some dinoflagellates (Toulza et al., 2010; Zhuang et al., 2013). Using both edgeR and NOISeq analysis in combination, an analysis strategy to enhance the reliability of identified differentially expressed genes (DEGs), 7,267 (15.87%) genes out of 45,788 mapped genes in diatoms and 14,508 (12.77%) genes out of 11,3599 mapped genes (Bowtie2, parameters: - sensitive) in P. donghaiense were identified as DEGs (Figure 2A) between diatom-dominant assemblage and dinoflagellate-bloom assemblages. However, from the functional perspective, the whole phytoplankton community showed relatively stable COG functional group landscape over time (Figure 2B). Furthermore, there was a high similarity among T1, T2, and T3 in ambient environmental conditions, phytoplankton community structure, and the metatranscriptomic profile. Principal component analysis (PCA) of FPKM across the four time points also showed that gene expression profile at T0 was markedly different from that at T1, T2, and T3, while profiles from T1, T2, and T3 were similar (Supplementary Figure S4). Therefore, differential gene expression was further analyzed by treating data from these three time points as replicates and this time period is referred to as T123 hereafter. Interestingly, diatoms at T0 and dinoflagellates at T123 showed a similar set of up-regulated gene repertoires, e.g., ones that are involved in energy generation, carbohydrate metabolism, nutrient uptake and assimilation, and cell proliferation (Figure 2C). This result provides circumstantial support to our first hypothesis that diatoms and dinoflagellates may use the same biological processes to maintain their dominance. However, as will be elaborated later, diatoms and dinoflagellates appeared to use distinct metabolic pathways in respective dominant or bloom condition (Figure 2C and Supplementary Dataset 1), in support of our second hypothesis. Common Metabolic Pathways Underlying Diatom- and Dinoflagellate-Dominance We found that the most strongly regulated synthase and redox genes at T0 were those encoding damage repair and antioxidants in diatoms, e.g., geranylgeranyl diphosphate reductase (CHLP), peptide methionine sulfoxide reductase (MsrA), superoxide dismutase (SOD), and thioredoxin (Supplementary Figure S5a and Supplementary Dataset 2). Many of these supply reducing power for detoxifying lipid hydroperoxides or regulate repair of oxidized proteins (Ramel et al., 2009). P. donghaiense bloom metatranscriptome also contained anti-stress genes (antioxidants and UV filters) at T123 (Supplementary Figure S5b and Supplementary Dataset 2). Some of these, e.g., CHLP, SOD, and thioredoxin, were shared with the T0-diatom community (Supplementary Dataset 2 and Supplementary Figure S5), while others, e.g., 2-epi-5-epi-valiolone synthase for the biosynthesis of the UV-filtering mycosporine-like amino acid (MAA) shinorine (Lemmers et al., 2010) and flavonol synthase for the biosynthesis of UV combating flavonoids (Samanta et al., 2011) were unique in the P. donghaiense bloom (Supplementary Dataset 2). The elevated expression of the anti-stress genes during both diatom and dinoflagellate dominance is consistent with the importance of anti-oxidative stress in N metabolism and many cellular activities (Rosenwasser et al., 2014). Unique Transcriptomic Signatures in Diatom Dominance We further looked for specific metabolic pathways in diatoms that may be associated with their dominance to gain more support for our second hypothesis. As described below, a set of pathways were found to be strongly associated with diatom dominance. Both the diatom-dominant and dinoflagellate-bloom assemblages exhibited up-regulation, relative to their respective non-dominant periods, of genes regulating uptake and assimilation of N nutrient, such as ammonium transporter, nitrate transporter (NRT), nitrate/nitrite reductase, and glutamine synthetase (GS) (Figure 2Ci). This suggests that there might be DIN input allowing utilization by the phytoplankton community while keeping the ambient concentration relatively stable in the study period. A notable exception was homologs of NRT and GS from P. nitzschia sp., which showed up-regulation at T123. This, along with the up-regulation of a cyclin-dependent kinase, is indicative of active nutrient uptake and growth of this diatom at T123. This attests to the recently documented differential niches among diatom species in the natural assemblages (Alexander et al., 2015a). Frontiers in Microbiology | www.frontiersin.org Up-Regulated Si and Urea Uptake Mechanisms p g p Two types of silicate transporter genes in diatoms were identified, which shared high sequence similarity with Cylindrotheca fusiformis SiTs (SiT gene clade A) and Skeletonema costatum SiTs (SiT gene clade E) (Durkin et al., 2016). Both of them were more highly expressed at T0 than T123 (Figure 2Ci). It was previously shown that most types of SiTs in C. fusiformis (SiT gene clade A) were up-regulated to a maximum level around 2 h and 20 min after silicon addition (Hildebrand et al., 1998). Thus the high expression of SiTs indicated the active silicate uptake at T0. Besides, diatoms exhibited elevated capacity of urea uptake at T0, as indicated by the markedly up-regulated genes of urea active transporter 1 and urease (Figure 2Ci). An algal bloom can result from cell proliferation or grazing depression, the relative importance of which is often debated (Irigoien et al., 2005). For both the diatom dominance and the P. donghaiense bloom, cell proliferation was speculated to be active because genes encoding cell cycle proteins such as PCNA were all up-regulated in diatoms at T1 and in P. donghaiense at T123 (Figure 2Cii and Supplementary Dataset 1). Besides, G2/mitotic-specific cyclin-B was also up- regulated in P. donghaiense at T123. PCNA and G2/mitotic- specific cyclin-B have been shown to be associated with mitosis High Expression of Carbohydrate Metabolic Pathway For diatoms and P. donghaiense, genes involved in carbohydrate metabolism were both upregulated during their dominance (diatoms at T0 compared to T123, P. donghaiense at T123 compared to T0) (Figure 2Ciii). However, the expression level of genes involved in carbohydrate metabolism in diatoms at T0 was much higher (FPKM >103, Figure 3A) than the expression level of their homologs in P. donghaiense at T123 (FPKM = 83.93– 128.66). A previous study also showed overwhelming dominance March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 5 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. FIGURE 2 | Metatranscriptomic outlook in diatoms and P. donghaiense. (A) Number of differential expressed genes determined by edgeR and NOISeq. (B) Clusters of Orthologous Groups comparison over the four time points. (C) T0 vs. T123 fold changes in transcript abundances in four major metabolic pathways (FDR <=0.05). Data on top were most strongly expressed at T0 whereas those at bottom most strongly expressed at T123. Solid triangles, P. donghaiense; hollow circles, diatoms. Up-Regulated Si and Urea Uptake Mechanisms Color depicts a gene or gene group. Symbol size depicts the expression level (log CPM [counts per million]). Gene abbreviations: (i) AMC1/2, metacaspase-1/2; Amt, ammonium transporter; AP, alkaline phosphatase; AT, amino acid transporter; ATG, autophagy-related protein; CYN, cyanate hydratase; GOSR2, glogi SNAP receptor complex member 2; GS, glutamine synthetase; HPT, high-affinity phosphate transporter; NiR, nitrite reductase; NR, nitrate reductase; FIGURE 2 | Metatranscriptomic outlook in diatoms and P. donghaiense. (A) Number of differential expressed genes determined by edgeR and NOISeq. (B) Clusters of Orthologous Groups comparison over the four time points. (C) T0 vs. T123 fold changes in transcript abundances in four major metabolic pathways (FDR <=0.05). Data on top were most strongly expressed at T0 whereas those at bottom most strongly expressed at T123. Solid triangles, P. donghaiense; hollow circles, diatoms. Color depicts a gene or gene group. Symbol size depicts the expression level (log CPM [counts per million]). Gene abbreviations: (i) AMC1/2, metacaspase-1/2; Amt, ammonium transporter; AP, alkaline phosphatase; AT, amino acid transporter; ATG, autophagy-related protein; CYN, cyanate hydratase; GOSR2, glogi SNAP receptor complex member 2; GS, glutamine synthetase; HPT, high-affinity phosphate transporter; NiR, nitrite reductase; NR, nitrate reductase; PhnG, 6-phosphofructokinase 7; NRT, nitrate transporter 2.5; PhnI, uncharacterised protein (PhnI domain); PhnW, 2-aminoethylphosphonate-pyruvate transaminase; PiT, phosphate transport protein; SiT, silicon transporter; STX, syntaxin; UT, urea transporter; VAMP, synaptobrevin homolog. (ii) CCN, Cyclin; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen. (iii) ALDO, Fructose-bisphosphate aldolase class 1; CS, citrate synthase; CSY2, citrate synthase 2 mitochondrial; ENO: enolase; ENOSF, mitochondrial enolase superfamily; FBA2, fructose-bisphosphate aldolase 2, chloroplastic; FH, fumarate hydratase, mitochondrial; FRD, fumarate reductase; FumB, fumarate hydratase class (i) anaerobic; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; GpmA, 2, 3-bisphosphoglycerate-dependent phosphoglycerate mutase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase mitochondrial; PDC: pyruvate dehydrogenase [NADP(+)], mitochondrial; PDHA1, pyruvate dehydrogenase, mitochondrial; PGD, 6-phosphogluconate dehydrogenase decarboxylating 2; PGK, phosphoglycerate kinase; PGK1, phosphoglycerate kinase chloroplastic; PK, pyruvate kinase; (Continued) FIGURE 2 | Metatranscriptomic outlook in diatoms and P. donghaiense. (A) Number of differential expressed genes determined by edgeR and NOISeq. (B) Clusters of Orthologous Groups comparison over the four time points. (C) T0 vs. T123 fold changes in transcript abundances in four major metabolic pathways (FDR <=0.05). Data on top were most strongly expressed at T0 whereas those at bottom most strongly expressed at T123. Solid triangles, P. donghaiense; hollow circles, diatoms. Color depicts a gene or gene group. Up-Regulated Si and Urea Uptake Mechanisms Symbol size depicts the expression level (log CPM [counts per million]). Gene abbreviations: (i) AMC1/2, metacaspase-1/2; Amt, ammonium transporter; AP, alkaline phosphatase; AT, amino acid transporter; ATG, autophagy-related protein; CYN, cyanate hydratase; GOSR2, glogi SNAP receptor complex member 2; GS, glutamine synthetase; HPT, high-affinity phosphate transporter; NiR, nitrite reductase; NR, nitrate reductase; PhnG, 6-phosphofructokinase 7; NRT, nitrate transporter 2.5; PhnI, uncharacterised protein (PhnI domain); PhnW, 2-aminoethylphosphonate-pyruvate transaminase; PiT, phosphate transport protein; SiT, silicon transporter; STX, syntaxin; UT, urea transporter; VAMP, synaptobrevin homolog. (ii) CCN, Cyclin; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen. (iii) ALDO, Fructose-bisphosphate aldolase class 1; CS, citrate synthase; CSY2, citrate synthase 2 mitochondrial; ENO: enolase; ENOSF, mitochondrial enolase superfamily; FBA2, fructose-bisphosphate aldolase 2, chloroplastic; FH, fumarate hydratase, mitochondrial; FRD, fumarate reductase; FumB, fumarate hydratase class (i) anaerobic; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; GpmA, 2, 3-bisphosphoglycerate-dependent phosphoglycerate mutase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase mitochondrial; PDC: pyruvate dehydrogenase [NADP(+)], mitochondrial; PDHA1, pyruvate dehydrogenase, mitochondrial; PGD, 6-phosphogluconate dehydrogenase decarboxylating 2; PGK, phosphoglycerate kinase; PGK1, phosphoglycerate kinase chloroplastic; PK, pyruvate kinase; (C ti d) (Continued) March 2019 | Volume 10 | Article 590 6 Frontiers in Microbiology | www.frontiersin.org Molecular Regulation of Phytoplankton Community Dominance Zhang et al. FIGURE 2 | Continued RPE, ribulose-phosphate 3-epimerase; SQR, succinate dehydrogenase flavoprotein subunit 1 mitochondrial; TAL, transaldolase; TPI, triosephosphate isomerase. (iv) CA, Carbonic anhydrase; CytC, cytochrome c6; FBP1, fructose-1 6-bisphosphatase; Fdx, ferredoxin; FNR, ferredoxin-NADP+ reductase; ISP, cytochrome b6-f complex iron-sulfur subunit, chloroplastic; LHC, light harvesting protein complex; PRK, phosphoribulokinase; PSBO, oxygen-evolving enhancer protein 1; PSI/PSII, photosystem I/II proteins; Rubisco, ribulose-1, 5-bisphosphate carboxylase/oxygenase; TKT, transketolase. FIGURE 3 | Expression levels of representative highly expressed genes in (A) diatoms and (B) P. donghaiense between T0 and T123. AtpG, Atp synthase gamma chain; PetC, cytochrome b6-f complex iron-sulfur subunit; PsbA, photosystem II D1; FD, ferredoxin; FCPA, fucoxanthin-chlorophyll a-c binding protein; ALDO, fructose-bisphosphate aldolase; TKT, transketolase; PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; FNR, ferredoxin-nadp reductase embryo isozyme; TR, trypsin; TR-II, trypsin 5g1; AtpE, chloroplast ATP synthase subunit C; PCP, chloroplast peridinin-chlorophyll a-binding protein precursor; PsbE, cytochrome b559 subunit alpha; PetD, cytochrome b6-f complex subunit IV; PsbC, photosystem II CP43 reaction center protein; PsbD, photosystem II protein D2; PsbB, photosystem II CP47 reaction center protein; MBNP, major basic nuclear protein. FIGURE 3 | Expression levels of representative highly expressed genes in (A) diatoms and (B) P. donghaiense between T0 and T123. Unsuspected Highly Active Expression of Trypsin Unsuspected Highly Active Expression of Trypsin The most conspicuous and surprising signature of the diatom dominance is the very high expression of trypsin (Figure 3A). Two major groups of trypsin (trypsin I and trypsin II) cDNAs were retrieved from the metatranscriptomes, and their representative members were found to phylogenetically affiliate with homologs in Skeletonema and Thalassiosira (Supplementary Figure S6). They collectively accounted for 1% of the total diatom transcript reads at T0 (FPKM = 8441) while only 0.1–0.6% at T123 (Supplementary Dataset 3). Notably, we found 19 trypsin genes in the genome of the diatom Thalassiosira pseudonana (Armbrust et al., 2004) but only two in the dinoflagellate Fugacium kawagutii (Lin S. et al., 2015) despite the much smaller genome in the former. The gene copy number and expression level in concert suggest that trypsin may play important roles in diatom growth. Abundant protease genes have been associated with silica nanostructure development in diatoms (Otzen, 2012), which is essential for diatom proliferation. Mainly cleaving peptide bonds with arginine or lysine, trypsin can produce amine-rich residues to promote the production Unique Transcriptomic Signatures in P. donghaiense Bloom With the changes of environmental conditions, increased transcriptional activities of photoenergy acquirement and alternative nutrient assimilation pathways in the dinoflagellate subcommunity distinguished the T123 assemblage from the T0 assemblage (Figure 3). This provides additional support to our second hypothesis about distinct metabolic pathways that might be associated with the dominant states of diatoms and dinoflagellates. Furthermore, this result revealed up-regulated genes of P. donghaiense during the dinoflagellate bloom, with predicted functions likely conferring competitive advantages for this dinoflagellate to outgrow diatoms and form a bloom, which is in support of our third hypothesis. Up-Regulated Si and Urea Uptake Mechanisms AtpG, Atp synthase gamma chain; PetC, cytochrome b6-f complex iron-sulfur subunit; PsbA, photosystem II D1; FD, ferredoxin; FCPA, fucoxanthin-chlorophyll a-c binding protein; ALDO, fructose-bisphosphate aldolase; TKT, transketolase; PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; FNR, ferredoxin-nadp reductase embryo isozyme; TR, trypsin; TR-II, trypsin 5g1; AtpE, chloroplast ATP synthase subunit C; PCP, chloroplast peridinin-chlorophyll a-binding protein precursor; PsbE, cytochrome b559 subunit alpha; PetD, cytochrome b6-f complex subunit IV; PsbC, photosystem II CP43 reaction center protein; PsbD, photosystem II protein D2; PsbB, photosystem II CP47 reaction center protein; MBNP, major basic nuclear protein. of carbohydrate metabolism in natural diatom populations (Alexander et al., 2015a). of polyamines, which are required in silica morphogenesis in diatoms (Sumper and Kröger, 2004). This possibility, albeit consistent with the up-regulated expression of Si transporter at T0, needs to be investigated in the future. Frontiers in Microbiology | www.frontiersin.org Proton-Pump Rhodopsin donghaiense photosynthesis genes were highly expressed and up-regulated at T123, e.g., carbonic anhydrase, a crucial enzyme for inorganic carbon uptake being up-regulated by 101-folds, and Ribulose-1, 5-bisphosphate carboxylase/oxygenase, an essential enzyme for carbon fixation, up-regulated by twofolds (Figures 2Civ, 3B and Supplementary Dataset 4). Meanwhile, in the P. donghaiense bloom as many as 375 rhodopsin genes were found (Supplementary Dataset 5), mainly the proton- pump types proteorhodopsin and xanthorhodopsin (Figure 4A) containing proton pumping and absorbing spectrum tuning residues (Figure 4B). Xanthorhodopsins were most highly represented in the T123 metatranscriptomes (Figure 4A). One of the unigenes (CL1Contig24959), identical to the previously reported P. donghaiense xanthorhodopsin (Shi et al., 2015), showed a notable ∼48-fold up-regulation at T123 (Figure 4A, P < 0.05). RT-qPCR verified the higher expression pattern and also showed much higher expression at T3 than in cultures (Figure 4C). In xanthobacter, retinal and the carotenoid salinixanthin are used as photoantenna to aid in harvesting light energy for xanthorhodopsin (Balashov et al., 2005). We found strong up-regulation of P. donghaiense genes involved in the synthesis of retinal and carotenoid at T123 (Supplementary Dataset 6). The heightened xanthorhodopsin expression during the bloom may be associated with bloom development in the turbid and phosphate-deficient ambient environment, as suggested by results from P. donghaiense cultures (Shi et al., 2017). Also highly active during the dinoflagellate bloom were ATPases (Figure 2Civ and Supplementary Dataset 4), enzymes responsible for ATP synthesis and hydrolysis. bloom (Cui et al., 2016). Besides, we detected PhnI and PhnG at T123 in P. donghaiense, suggesting that this species is possibly able to scavenge some types of phosphonates from the environment, as these genes belong to the Phn operon known to regulate utilization of phosphonates in Escherichia coli (Fenner et al., 2013). All these reveal a potentially multi-faceted strategy of P. donghaiense to cope with phosphate-deficiency for continued growth. Comparatively, diatoms exhibited a lower phosphate uptake capacity, with only one type of inorganic phosphate transporter highly expressed at T0 (Figure 2Ci and Supplementary Dataset 7). pp y Our metatranscriptomic data suggest that recycling of intracellular components via autophagy might be an important source of nutrients to support P. donghaiense growth. Autophagy is a lysosome-dependent cellular catabolic mechanism, which composes vesicle-associated membrane protein, syntaxin and Glogi SNAP receptor complex member 2 (Renna et al., 2011). Proton-Pump Rhodopsin Activation of autophagy requires autophagy-related protein 8 (ATG8) in algae (Pérez-Pérez et al., 2010); ATG12 and ATG3 also play an essential role in autophagosome formation (Pérez- Pérez et al., 2010). These genes were more highly expressed in P. donghaiense at T123 (Figure 2Ci and Supplementary Dataset 7). In comparison, although diatom autophagy-related genes were up-regulated at T0, the number of genes and the magnitude of expression increase were lower than dinoflagellates at T123 (Figure 2Ci). More diversified phosphate acquisition strategy and the recycling of intracellular components via autophagy found in our study potentially in part define the ecological niche to acquire nutrients for P. donghaiense to form the bloom. Antimicrobial Defense An effective defense mechanism may be important for population growth and bloom formation (Potin et al., 2002; Gobler et al., 2011). We found nine P. donghaiense genes in the bloom metatranscriptome annotated as antimicrobial peptides (Supplementary Dataset 8). Among these, macrophage migration inhibitory factor (MIF) is known in humans and animals as a pivotal regulator of innate immunity and an integral component of the host antimicrobial alarm system and stress response (Kleemann et al., 2000). Plant defensins are generally recognized as host defense peptides against some pathogenic fungi and bacteria (Broekaert et al., 1995). Synaptobrevin is one of the SNARE proteins involved in the formation of SNARE complexes while pleurodin and SNARE are peptides that have antibacterial activity in invertebrates and vertebrates (Collins et al., 2003; Smith et al., 2010). The expression of all these gene homologs was undetectable at T0 but substantial at T123 as well as in the other bloom mentioned above (Supplementary Dataset 8), indicative of potential importance of antimicrobial defense in natural bloom formation. It is noteworthy that a functionally similar (but with distinct gene sets) antimicrobial defense mechanism has been identified in the genome of Aureococcus anophagefferens prone to form massive blooms (Gobler et al., 2011). Whether these mechanisms equip the bloom algae to fight offalgicidal bacteria will be of high interest to investigate in the future. Proton-Pump Rhodopsin Energy acquisition via electron transport (photosynthetic reaction centers) in photosynthetic organisms is well known March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 7 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. but a photosystem-independent pathway through proton translocation (by the action of rhodopsins) has been discovered more recently (Hohmann-Marriott and Blankenship, 2011). The putative rhodopsin-based light energy acquiring mechanism is known only in bacteria (Béja et al., 2001) and several eukaryotic lineages (Lin et al., 2010; Marchetti et al., 2012). Many of the P. donghaiense photosynthesis genes were highly expressed and up-regulated at T123, e.g., carbonic anhydrase, a crucial enzyme for inorganic carbon uptake being up-regulated by 101-folds, and Ribulose-1, 5-bisphosphate carboxylase/oxygenase, an essential enzyme for carbon fixation, up-regulated by twofolds (Figures 2Civ, 3B and Supplementary Dataset 4). Meanwhile, in the P. donghaiense bloom as many as 375 rhodopsin genes were found (Supplementary Dataset 5), mainly the proton- pump types proteorhodopsin and xanthorhodopsin (Figure 4A) containing proton pumping and absorbing spectrum tuning residues (Figure 4B). Xanthorhodopsins were most highly represented in the T123 metatranscriptomes (Figure 4A). One of the unigenes (CL1Contig24959), identical to the previously reported P. donghaiense xanthorhodopsin (Shi et al., 2015), showed a notable ∼48-fold up-regulation at T123 (Figure 4A, P < 0.05). RT-qPCR verified the higher expression pattern and also showed much higher expression at T3 than in cultures (Figure 4C). In xanthobacter, retinal and the carotenoid salinixanthin are used as photoantenna to aid in harvesting light energy for xanthorhodopsin (Balashov et al., 2005). We found strong up-regulation of P. donghaiense genes involved in the synthesis of retinal and carotenoid at T123 (Supplementary Dataset 6). The heightened xanthorhodopsin expression during the bloom may be associated with bloom development in the turbid and phosphate-deficient ambient environment, as suggested by results from P. donghaiense cultures (Shi et al., 2017). Also highly active during the dinoflagellate bloom were ATPases (Figure 2Civ and Supplementary Dataset 4), enzymes responsible for ATP synthesis and hydrolysis. but a photosystem-independent pathway through proton translocation (by the action of rhodopsins) has been discovered more recently (Hohmann-Marriott and Blankenship, 2011). The putative rhodopsin-based light energy acquiring mechanism is known only in bacteria (Béja et al., 2001) and several eukaryotic lineages (Lin et al., 2010; Marchetti et al., 2012). Many of the P. P Acquisition and Nutrient Recycling q y g Consistent with the diminishing PO43− in the ambient environment at T123 (Figure 1B), P. donghaiense markedly up-regulated genes involved in P uptake, including regular and high-affinity PO43−transporters and numerous genes potentially facilitating the utilization of dissolved organic phosphate (Figure 2Ci and Supplementary Dataset 7). Alkaline phosphatase (AP), hydrolyzing phosphoesters to release PO43− for phytoplankton use (Lin X. et al., 2015), was expressed in P. donghaiense at T123 but not detectable at T0 (Figure 2Ci and Supplementary Dataset 7). We also detected up-regulation at T123 of apyrase (Figure 2Ci and Supplementary Dataset 7), a calcium-activated enzyme known in plants to catalyze the hydrolysis of ATP to AMP and phosphate (Thomas et al., 1999). Genes known to facilitate utilization of phosphonates (C-P bond organophosphate), such as 2-aminoethylphosphonate (AEP) – pyruvate transaminase (PhnW), PhnI domain-containing protein, and 6-phosphofructokinase 7 (Phn G) were also detected in P. donghaiense with up-regulation at T123 (Figure 2Ci and Supplementary Dataset 7), suggesting intracellular phosphorus recycling to cope with the phosphate-deficiency during the March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 8 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. FIGURE 4 | Diversity and expression profile of rhodopsin genes represented in the metatranscriptomes. (A) Phylogenetic analysis of rhodopsin genes. Circles at nodes represent relative bootstrap values, the larger the circles, the higher the bootstrap value (only values >60% are shown); PR: proteorhodopsin; XR: xanthorhodopsin; SR: sensory rhodopsin; highlighted in red is assembled genes in this study; P. donghaiense rhodopsin is highlighted in large font size. The heatmap imposed outer of the tree shows the relative expression of each type of rhodopsin in T0, T1, T2, and T3. The outermost dark blue column shows the average FPKM of each type of rhodopsin over the entire T0 to T3 period. (C) Relative expression of rhodopsin in the field samples and cultures measured using (Continued) FIGURE 4 | Diversity and expression profile of rhodopsin genes represented in the metatranscriptomes. (A) Phylogenetic analysis of rhodopsin genes. Circles at nodes represent relative bootstrap values, the larger the circles, the higher the bootstrap value (only values >60% are shown); PR: proteorhodopsin; XR: xanthorhodopsin; SR: sensory rhodopsin; highlighted in red is assembled genes in this study; P. donghaiense rhodopsin is highlighted in large font size. P Acquisition and Nutrient Recycling FIGURE 5 | Schematic summary of changes in expression of key genes or metabolic processes in diatoms and the dinoflagellate P. donghaiense in the course of the diatom-dominance to dinoflagellate-bloom regime shift. RHO, rhodopsin; ETP, electron transfer proteins; Genes or pathways with FPKM ranging from 0 to 100 are shown in light blue (low activity), from 100 to 1000 in orange, over 1000 in red (high activity). Generally, FPKM of pathways involving multiple genes are based on the gene with the highest FPKM. DIP (dissolved inorganic phosphate) is indicated in light blue in diatoms during diatom-dominance because it only composed one type of transporter. FIGURE 5 | Schematic summary of changes in expression of key genes or metabolic processes in diatoms and the dinoflagellate P. donghaiense in the course of the diatom-dominance to dinoflagellate-bloom regime shift. RHO, rhodopsin; ETP, electron transfer proteins; Genes or pathways with FPKM ranging from 0 to 100 are shown in light blue (low activity), from 100 to 1000 in orange, over 1000 in red (high activity). Generally, FPKM of pathways involving multiple genes are based on the gene with the highest FPKM. DIP (dissolved inorganic phosphate) is indicated in light blue in diatoms during diatom-dominance because it only composed one type of transporter. FIGURE 5 | Schematic summary of changes in expression of key genes or metabolic processes in diatoms and the dinoflagellate P. donghaiense in the course of the diatom-dominance to dinoflagellate-bloom regime shift. RHO, rhodopsin; ETP, electron transfer proteins; Genes or pathways with FPKM ranging from 0 to 100 are shown in light blue (low activity), from 100 to 1000 in orange, over 1000 in red (high activity). Generally, FPKM of pathways involving multiple genes are based on the gene with the highest FPKM. DIP (dissolved inorganic phosphate) is indicated in light blue in diatoms during diatom-dominance because it only composed one type of transporter. Metabolism Regulated by Novel and Unique Genes Six novel genes were identified as some of the most highly expressed genes in P. donghaiense (Supplementary Dataset 9). Four of these showed 10- to over 100-fold elevation of expression during T123 (Unknown I), and were also expressed actively in the other P. donghaiense bloom mentioned above (Supplementary Datasets 9, 10). Two of them showed an opposite expression pattern, dramatically up-regulated at T0 (Unknown II, Supplementary Dataset 9). P Acquisition and Nutrient Recycling The heatmap imposed outer of the tree shows the relative expression of each type of rhodopsin in T0, T1, T2, and T3. The outermost dark blue column shows the average FPKM of each type of rhodopsin over the entire T0 to T3 period. (C) Relative expression of rhodopsin in the field samples and cultures measured using (Continued) FIGURE 4 | Diversity and expression profile of rhodopsin genes represented in the metatranscriptomes. (A) Phylogenetic analysis of rhodopsin genes. Circles at nodes represent relative bootstrap values, the larger the circles, the higher the bootstrap value (only values >60% are shown); PR: proteorhodopsin; XR: xanthorhodopsin; SR: sensory rhodopsin; highlighted in red is assembled genes in this study; P. donghaiense rhodopsin is highlighted in large font size. The heatmap imposed outer of the tree shows the relative expression of each type of rhodopsin in T0, T1, T2, and T3. The outermost dark blue column shows the average FPKM of each type of rhodopsin over the entire T0 to T3 period. (C) Relative expression of rhodopsin in the field samples and cultures measured using (Continued) Frontiers in Microbiology | www.frontiersin.org March 2019 | Volume 10 | Article 590 9 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. FIGURE 4 | Continued RT-qPCR normalized by calmodulin and actin. (B) Secondary structure analysis of rhodopsin proteins represented in the bloom metatranscriptome dataset. The 7-transmembrane structure resulted from ProteinPredict run. Highlighted in purple are residues that form retinal binding pocket; in green (7th transmembrane domain) is lysine that links rhodopsin to retinal; in red (the 5th transmembrane domain) is keto-carotenoids binding. The 3rd transmembrane domain contains residues that determine the function and absorbing light spectra: the residue in blue functions for spectral tuning, with L (lucine) specifying green absorbing, which accounted for 70% of the rhodopsin cDNA retrieved, and Q (20%) specifying blue light absorbing (upper right pie chart); the two residues in black and the T in purple in the middle forms a motif that determines the function of the rhodopsin, with D-T-E (aspartate-threonine-glutamate) specifying proton pump, which accounted for 61.1% of the rhodopsin cDNA retrieved (upper left pie chart). The function of motif with I-T-E (isoleucine-threonine-glutamate) and motif with T-T-E (threonine-threonine-glutamate) are still unknown, they accounted for 16.7 and 5.4%, respectively (upper left pie chart). AUTHOR CONTRIBUTIONS SL and YZ conceived the study. SL, YZ, XL, and XS drafted and edited the manuscript and figures. YZ, LXL, and XS performed the bioinformatics analyses. YZ, HL, and LL performed the experiments. All authors read and approved the final version of the manuscript. ACKNOWLEDGMENTS We are indebted to Ms. Chentao Guo and Ms. Shanshan Meng (Xiamen University) for their logistic assistance in this project. We also thank Prof. Mingjiang Zhou (Institute of Oceanology, Chinese Academy of Sciences) and Da-Zhi Wang (Xiamen University) for providing the opportunity to join the research cruise, and the crew of R/V Yanping #2 for assistance throughout the cruise. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00590/full#supplementary-material P Acquisition and Nutrient Recycling These genes are likely unique genes important in regulating dinoflagellate population proliferation and bloom formation (Unknown I) or population maintenance (Unknown II). It is noteworthy that major basic nuclear proteins (MBNPs) were also highly expressed at T123 (Figure 3B and Supplementary Dataset 9). The function of March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 10 Molecular Regulation of Phytoplankton Community Dominance Zhang et al. MBNPs in dinoflagellates is unclear but is thought to replace histones in packaging chromosomal DNA (Kato et al., 1997). The high expression of MBNPs at T123 suggests some important role in cell proliferation. Many of the highly expressed genes reported here have never been detected, or never found to be highly expressed, in culture studies, underscoring the need for more in situ studies in order to fully understand underlying mechanisms of competition and succession of phytoplankton lineages in the natural assemblages. studies, underscoring the need for more in situ studies in order to fully understand underpinning mechanisms of competition and succession of phytoplankton lineages in the natural assemblages. FUNDING Our results provide insights into molecular inner working of a phytoplankton community during a shift from diatom (Skeletonema)-dominance to a dinoflagellate (P. donghaiense)- bloom. Diatoms and dinoflagellates may use the same biological processes when at high relative abundance in their common coastal environment. This is evident from the similar set of most active metabolic processes (energy and nutrient acquisition, anti-stress) to promote growth during dominance (Figure 5). More importantly, our data also reveal distinct metabolic pathways between diatoms (carbohydrate metabolism, trypsin-based metabolism) and dinoflagellates (proton-pump rhodopsin-based energy acquisition, antimicrobial defense) and many highly expressed unique complements of functional genes, potentially defining distinct ecological niches for these two lineages. P. donghaiense possessed more diversified light energy and phosphate acquisition strategy and antimicrobial defense, which might lead them to outgrow diatoms and form blooms (Figure 5). “Omic” studies often lead to discoveries of unsuspected physiologies or biochemistries underpinning certain ecological phenotype, but typically the discoveries remain to be proven in subsequent experimental studies. With no exception, many of the findings reported here stand as new questions and hypotheses for further experimental inquiries, with functions of key genes to be demonstrated using a functional genetic tool. While no functional genetic tool is yet available, efforts to develop one are underway in various laboratories. Finally, many of the highly expressed genes reported here have never been detected, or never found to be highly expressed in culture This work was supported by the National Key Research and Development Program of China grant 2016YFA0601202, the National Natural Science Foundation of China grants NSFC 41330959 (SL), 41776116 (SL), 41706116 (XL), and 41606121 (XS), Fundamental Research Funds for the Central Universities 20720180101 (XL), and China Scholarship Council 201603310181 (YZ). SL was also in part supported by the Gordon and Betty Moore Foundation’s Marine Microbial Initiative GBMF grant #4980. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org March 2019 | Volume 10 | Article 590 REFERENCES Cyclin B gene and its cell cycle- dependent differential expression in the toxic dinoflagellate Alexandrium fundyense Atama Group I/Clade I. Harmful Algae 26, 71–79. doi: 10.1016/j.hal. 2013.04.002 Sumper, M., and Kröger, N. (2004). Silica formation in diatoms: the function of long-chain polyamines and silaffins. J. Mater. Chem. 14, 2059–2065. doi: 10.1039/B401028K Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Sun, K., Qiu, Z., He, Y., Fan, W., and Wei, Z. (2017). Vertical development of a Prorocentrum donghaiense bloom in the coastal waters of the East China Sea: coupled biophysical numerical modeling. Acta Oceanol. Sin. 36, 23–33. doi: 10.1007/s13131-016-0965-z Copyright © 2019 Zhang, Lin, Shi, Lin, Luo, Li and Lin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Zhang, Lin, Shi, Lin, Luo, Li and Lin. 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. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725– 2729. doi: 10.1093/molbev/mst197 Tarazona, S., Furió-Tarí, P., Turrà, D., Pietro, A. D., Nueda, M. J., Ferrer, A., et al. (2015). Data quality aware analysis of differential expression in RNA-seq March 2019 | Volume 10 | Article 590 Frontiers in Microbiology | www.frontiersin.org 13
https://openalex.org/W2570057063
http://jurnalalqalam.or.id/index.php/Alqalam/article/download/341/256
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IMPLEMENTASINILAI-NILAIPENDIDIKAN DALAM SURAH ALI 'IMRAN PADA MTsN WATAMPONE
Al Qalam - Balai Penelitian Lektur Keagamaan Ujung Pandang/Al-Qalam
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cc-by-sa
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M. Amir H.M. M. Amir H.M. Sekolah Tinggi Agama Islam Negeri Watampone Jl. Hos Cokroaminoto Tlp./Faks. (0481)21395/23928 E il h i h @ il Sekolah Tinggi Agama Islam Negeri Watampone Jl. Hos Cokroaminoto Tlp./Faks. (0481)21395/23928 Email: hm amir hm@gmail com Abstrak Penelitian ini bertujuan untuk menemukan wujud nilai-nilai pendidikan dalam surah Ali 'Imran dan impelementasinya pada Madrasah Tsanawiyah Negeri Watampone, yang secara ideal semua proses pendidikan pada madrasah tersebut berdasar pada Alquran, sebagai dasar normatif pendidikan Islam yang salah satu di dalamnya adalah surah Ali 'Imran. Untuk menemukan data yang akurat dipergunakan berberapa metode di antaranya, metode pendekatan pedagogis dan psikologis, sedangkan pengumpulan datanya dilakukan melalui library research da.n field research, dan diolah dengan metode kualitatif serta dianalisis dengan content analysis dan concept analysis.. Hasil penelitian ditemukan tujuan, fungsi dan metode pendidikan yang terdapat dalam surah Ali 'Imran. Lalu dikaitan dengan implementasinya pada Madrasah Tsanawiyah Negeri Watampaone. Para guru dan siswa yang ditunjuk sebagai sampel searah pendapatnya bahwa sesungguhnya nilai-nilai pendidikan dalam surah Ali 'Imran telah terimplementasi dengan baik pada madrasah tersebut, namun masih perlu penyempurnaan, terutama pada mata pelajaran tertentu yang bukan mata pelajaran pendidikan agama Islam (PAI). Kata kunci: implementasi, nilai-nilai, pendidikan, surah Ali 'Imran Abstract This study aims to find educational values in Surah Ali Imran and its implementation in MadrasahTsanawiyahWatampone. In order to find accurate data, several methods were used, among others were the pedagogical and psychological approach. Data were also collected through library research and field research. Data processed using qualitative methods and analyzed with content and concept analysis. Result shows that surah Ali Imran contains educational values and schools goal, and methods of education have been in line with though still need further improvement, especially in non-Islamic education subjects Keywords: implementation, values, educational, surah Ali 'Imran Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 43 PENDAHULUAN atau nilai-nilai yang paling sahih adalah Alquran dan al-Hadis yang kemudian dikembangkan oleh hasil ijtihad para ulama atau cendekiawan muslim. Nilai-nilai yang bersumber dari pemikiran, adat istiadat atau tradisi serta ideologi sangat rentan dan kondisional, sebab kesemuanya merupakan produk budaya manusia yang kebenarannya bersifat relatif, lokal dan situasional. Sedangkan nilai-nilai Alquran, yaitu nilai-nilai yang bersumber dari Alquran bersifat mutlak dan universal (Said Agil Husain al- Munawwar, 2005: 3). S e m d S ecara filosofis, nilai sangat terkait dengan masalah etika. Itulah sebabnya etika sering disebut dengan filsafat nilai, yangmengkajinilai- nilai moral sebagai tolok ukur tindakan dan perilaku manusia dalam berbagai aspek kehidupannya. Sumber-sumber etika dan moral, selain dari agama juga boleh jadi bersumber dari pemikiran, adat istiadat atau tradisi serta ideologi. Dalam konteks nilai-nilai pendidikan dalam Islam, sumber etika terampil, disiplin, etos kerja, profesional, inovatif dan produktif (al-Munawwar, 2005: 7-9). Karena itu, pendidikan Islam seyogianya menjadi pilihan pertama dan utama bagi masyarakat sebagai perwujudan untuk mencerdasakan mereka, baik kecerdasan intelektual, kecerdasan emosional, dan kecerdasan spiritual. Bahkan Menurut Omar Muhammad Al-Tomy Al-Syaibany seperti yang dikutip oleh Ramyulis bahwa pendidikan Islam tidak hanya bertujuan untuk menumbuhkan, mengembangkan dan membangun segala aspek kepribadian umat manusia dan segala potensi serta dayanya. Juga mengembangkan segala segi kehidupan dalam masyarakat, seperti sosial budaya, ekonomi, politik, termasuk memberi solusi atas segala permasalahan yang dihadapi umat manusia masa kini dan yang akan datang serta memelihara sejarah dan peradabannya(Ramayulis, 2011: 35). Ketiga dimensi tersebut, mencerminkan tentang nilai-nilai pendidikan dalam Alquran yang tidak hanya berorientasi pada kepentingan kehidupan umat manusia di dunia tetapi juga untuk kepentingan kehidupan di akhirat. Bahkan pendidikan dalam Alquran tidak hanya berfungsi untuk mencerdaskan akal pikiran umat manusia yang lebih dikenal dengan kecerdasan itelektual, tetapi juga kecerdasan emosional, dan sipiritual, Terkait dengan hal tersebut, Ali Abd. al- A'azhim mengemukakan bahwa Alquran merupakan kitab yang sangat komprehensif yang di dalamnya tercakup persoalan filsafat, penalaran ilmiah, dan problema sosial yang mensinkronkan antara urusan dunia dan akhirat, antara ritus dengan perbuatan nyata, serta mengkombinasikan antara realisme dan idealisme. Islam memberi kesempatan kepada penganutnya untuk hidup dan berkarya di bumi, namun tetap memperhatikan serta memikirkan tentang apa yang ada di langit (Ali Abd. al-Azhim, 1989:75-76). Karena itu, untuk menemukan makna- makna yang tercakup dalam Alquran termasuk nilai-nilai pendidikan diperlukan keseriusan dan ketelatenan serta didasari dengan keyakinan bahwa Alquran bukan kitab ilmiah yang tersusun berdasarkan standar kitab/buku ilmiah yang disusun oleh manusia, tetapi Alquran mengandung makna ilmiah yang mendalam. 44 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 45 PEMBAHASAN peserta didik menulis, baik tulisan itu untuk kepentingan pribadinya, maupun orang lain. Hal ini tergambar pada ayat 4 surah al-Alaq tersebut. Kelima, bahwa manusia dituntut agar senantiasa menambah wawasan serta ilmu pengetahuannya, melalui penelitian dan pengkajian untuk menemukan ilmu pengetahunan yang belum diketahui sebagaimana janji Allah swt pada ayat keliam suarah Al-Alaq tersebut. Sekilas Tentang Surah Ali 'Imran Sekilas Tentang Surah Ali Imran Surah Ali 'Imran, dinamakan demikian karena di dalamnya ditemukan kisah keluarga 'Imran yakni Isa, Yahya dan Maryam dan ibu beliau. Sedangkan 'Imran adalah ayah Maryam as. atau kakek Nabi Isa. Surah ini terdiri dari 200 ayat, sekitar 80 ayat pertama berkaitan dengan kedatangan serombongan pendeta Kristen dari Najran (sebuah lembah di perbatasan Yaman dan Saudi Arabiyah) di masjid Madinah pada tahun sembilan hijriyah untuk berdiskusi dengan Nabi Muhammad saw. tentang Nabi Isa as. berkaitan dengan keesaan Allah swt. Diskusi dimaksud berjalan beberapa hari, namun tidak memperoleh kesepakatan. Kalau ayat-ayat yang pertama diturunkan oleh Allah swt, yakni surah al-Alaq ayat 1-5 mengandung berbagai unsur pendidikan, maka tentu surah yang lainpun demikian adanya termasuk surah Ali Imran, hanya saja keterbatasan kemampuan umat manusia menemukannya. Bahkan, Sepanjang penelusuran peneliti tidak menemukan hasil penelitian yang secara utuh mengungkap nilai- nilai pendidikan dalam surah Ali 'Imran, namun ditemukan beberapa referensi yang mendukung penelitian ini seperti kitab tafsir dan beberapa buku pendidikan. Karena itu, penelitian ini dengan segala keterbatasannya bertujuan mengungkap beberapa nilai-nilai pendidikan yang terdapat dalam surah Ali Imran, lalu melihat impelementasinya pada madrasah tsanawiyah negeri Watampone. Tujuan utama surah Ali 'Imran (keluarga 'Imran) ini adalah sebagai pembuktian tentang tauhid, keesaan dan kekuasaan Allah swt serta penegasan bahwa dunia, kekuasaan, harta dan ank- anak yang terlepas dari nilai-nilai ilahiyah, tidak akan bermanfaat di akhirat kelak. Demikian pula hukum-hukum alam yang melahirkan kebiasaan- kebiasaan, pada hakikatnya diatur oleh Allah swt Yang Maha Hidup dan Qayyum (Maha menguasai), sebagaimana peristiwa-peristiwa yang dialami oleh keluarga Ali 'Imran. Melalui merekalah Allah swt menunjukkan keesaan, kekuasaan dan penguasaan- Nya terhadap seluruh alam. Serta membuktikan pula bagaimana suatu keluarga yakni ayah, ibu, anak suami dan istri, kesemuanya tunduk, patuh dan percaya kepada Allah Yang Maha Esa. Metode Penelitian Penelitian ini berlokasi pada Madrasah Tsanawiyah Negeri Watampone yang merupakan jenis penelitian diskriptif analitik dengan menggunakan pendekatan pedagogis dan psikologis. Kemudian datanya selain diperoleh melalui library research yakni buku-buku kepustakaan baik yang bersifat primer seperti kitab-kitab tafsir dan buku-buku pendidikan maupun yang bersifat skunder seperti kamus. Juga diperoleh melalui field research yakni data yang diperoleh dari lapangan melalui obsevasi wawancara yakni percakapan yang diarahkan pada suatu masalah tertentu antara interviewer (pengejar informasi) dengan interviewee (pemberi informasi). (Kartini Kartono, 1986: 171). Karena banyaknya jumlah guru dan siswa, maka untuk mendapatkan data yang valid digunakan sampel dengan teknik pengunaan proposional random sampling. Kemudian semua data yang diperoleh diolah dengan mengunakan metode kualitatif dan dianalisis melalui analisis isi (contnt analysis) dipakai untuk menganalisis makna yang terkandung dalam Alquran yang berkaitan dengan pendidikan serta analisis konsep (concept analysis), yakni menganalisis kata-kata kunci atau pokok yang mewakili sebuah gagasan atau konsep. Penempatan urutan surah ini sungguh amat tepat, kerena sur ah al- Fatihah yang merupakan surah pertama merangkul seluruh ajaran Islam secara singkat. Sedangkan al-Baqarah yang merupakan surah kedua dari segi urutan, menjelaskan lebih rinci tentang ajaran-ajaran agama. Sementara surah Ali 'Imran yang merupakan urutan ketiga menekankan sesuatu yang menjadi dasar dan sendi utama tuntunan tersebut, yakni Tauhid. Tanpa tauhid, maka seluruh amalan yang dilakukan oleh umat manusia tidak akan bernilai di sisi Allah swt (Shihab, 2005: 3-4). Nilai-nilai pendidikan yang dimaksud dalam surah Ali 'Imran antara lain: PENDAHULUAN Fungsi dan tujuan pendidikan tersebut akan dapat teraplikasi dalam kehidupan manusia apabila pendidikan diarahkan untuk menggali dan menyelami nilai-nilai pendidikan dalam Alquran. Karena Alquran diturunkan kepada Nabi Muhammad saw, untuk mengangkat derajat umat manusia dari lembah kegelapan, kezhaliman dan kebodohan, menuju alam yang terang benderang, berperadaban dan bermartabat. Sejarah menunjukkan bahwa masyarakat sebelum datangnya Islam atau yang sering disebut masyarakat jahiliah, yang tidak memiliki peradaban dan arah tujuan hidup, Nabi Muhammad saw dalam kurun waktu yang relatif singkat sekitar 23 tahun berhasil mengantar mereka ke dalam kehidupan baru yang berperadaban dan maju, yakni kehidupan yang disemangati dengan nilai-nilai pendidikan serta didasari dengan iman dan takwa kepada Allah swt. Demikian pentingnya pendidikan menurut Alquran, maka ayat pertama yang diturunkan oleh Allah swt kepada Nabi Muhammad saw adalah surat al-Alaq (96: ayat 1-5) memuat beberapa nilai pendidikan yang seharusnya diterjemahkan oleh umat Islam dalam kehidupan kesehariannya. Pertama, perintah membaca sebagai bagian dari proses pendidikan tergambar pada ayat pertama suarah al-Alaq tersebut. Kedua, hubungan antara sesama manusia dalam arti bahwa sebuah pendidikan, akan berjalan dengan baik apabila ada interaksi antara subjek dan objek pendidikan. Hal ini tergambar pada ayat kedua surah al-Alaq tersebut. Ketiga, bahwa pendidikan akan berhasil apa bila mereka yang terlibat dalam proses sebuah pendidikan berusaha menjaga hubungan baik dengan Allah saw baik melalui ibadah mahdah, maupun doa, karena dengan berkat kemurahan- Nya, manusia memperoleh ilmu pengetahuan. Hal ini tergambar ayat ketiga surah al-Alaq tersebut. Keempat, bahwa salah satu hasil dari pendidikan, terutama pendidikan formal adalah kemampuan Bagi umat Islam, Alquran merupakan asas dan landasan sekaligus inspirator seluruh kehidupan umat manusia, termasuk intelektualnya (M. Taufik, 2012: 45), yang oleh Fazlur Rahman dikatakan bahwa sumber definisi Islam, baik secara doktrinal maupun secara praksis, adalah Alquran dan Sunnah (Fazlur Rahman, 1987: 2-3). Karena itu, ketika berbicara tentang nilai-nilai pendidikan tidak bisa melepaskan diri dari petunjuk Alquran yang secara normatif tujuan yang ingin dicapai dalam proses aktualisasi nilai-nilai pendidikan dalam Alquran meliputi tiga dimensi atau aspek kehidupan yang harus dibina dan dikembangkan. Pertama, dimensi spiritual, yakni iman, takwa dan akhlakmulia. Kedua, dimensi budaya, yakni keperibadian yang mantap dan mandiri, tanggung jawab kemasyarakatan dan kebangsaan. Ketiga, dimensi kecerdasan yang membawa kepada kemajuan yang cerdas, kreatif, 44 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 Peningkatan ketauhidan kepada Allah swt Tauhid adalah berasal dari bahasa Arab I X - I ^ J J (wahhada, yuwahhidu, tauhidan) yang berati mengesakan, mengimani bahwa tiada Tuhan selain Allah dan menyatukan. Kemudian dalam bentuk Ism al-Masdar-nya (tauhidan) berarti keyakinan atas keesaan Allah (Munawwir, 1984: 1646-1647). Karena itu, pendidikan hendaknya diarahkan peserta didik memahami dan meyakini kemahaesaan Allah yang terus menerus mengurus makhluk-Nya (QS. Ali Imran/3: 2). Menurut Ibnu Kastir bahwa yang dimaksud potongan ayat ^ ^ L ^ V 4—151 J J - c . <JJ A J I jl (tidak ada agama yang diterima di sisi Allah kecuali agama Islam), yaitu tidak ada agama di sisi-Nya yang diterima kecuali agama Islam yang merupakan agama oleh para rasul yang diutus oleh Allah swt sampai ajaran mereka ditutup dengan kedatangan Muhammad saw. Dengan kehadiran beliau semua ajaran atau syariat yang berbeda dengan syariat Nabi Muhammad saw tidak akan diterima di sisi Allah swt (Abiy al-Fidai Isma'il ibn Katsir al-Qurasyiy sl- Dimisyqiy, t.th.: 354). Term 4—1) pada ayat tersebut berakar kata dari huruf hamzah, lam dm ha berarti sembahan. Kalau dikaitkan dengan Allah menunjukkan bahwa Dialah yang berhak disembah, (Abiy al-Husain Ahmad bin Faris bin zakariya.jilid I, 1999: 127), Term yH»Jl berarti memiliki sifat hidup, (Shihab, Volume 2, 2005: 5) Bila dikaitkan dengan Allah, -diartikan dengan Maha Hidup. Term f J-J-SJI berasal dari kata f > 2 (qawama) berakar kata dari huruf qaf, wau dan mim yang mengandung pengertian terlaksananya sesuatu dengan kesempurnaannya. Allah dinamakan Qayyum karena Dia yang mengatur segala sesuatu yang merupakan kebutuhan makhluk, sehingga terlaksana secara sempurna dan berkesinambungan (M. Quraish Shihab, Volume 2, 2005: 6). Dari keterangan di atas dipahami bahwa semua agama yang dibawa oleh para rasul pada dasarnya adalah Islam dalam arti mengandung makna keselamatan, penyerahan diri dan ketundukkan kepada yang menciptakannya, karena semua agama mengandung ajaran tauhid. Tetapi makna Islam pada ajaran mereka hanya sebatas sifat, bukan nama sebagai agama. Apalagi kalau ditelusuri ayat-ayat dalam Alquran tidak ada yang ditemukan kata Islam yang menunjuk kepada suatu agama kecuali yang menyangkut tentang kesempurnaan ajaran Muhammad saw. Karena itu, Allah tidak menerima agama kecuali agama Islam yang dibawa dan diajarkan oleh Nabi Muhammad saw (QS.Ali Imran/3: 85). Dengan demikian, ayat tersebut memberi pemahaman bahwa Allah adalah Yang Maha Esa, tidak ada Tuhan selain Dia, Yang Maha Hidup tidak berawal dan tidak berakhir (mati), Yang Maha Kuasa, kekuasaan-Nya meliputi segala yang ada. Karena itu, Dialah yang berhak disembah dimintai pertolongan segala kebutuhan. Peningkatan ketauhidan kepada Allah swt Tauhid adalah berasal dari bahasa Arab Keyakinan seperti ini harus menjadi tujuan utama dalam proses pendidikan, sehingga nantinya setiap anak didik memiliki bekal untuk tidak bersifat angkuh dan takabbur karena prestasi dan prestise yang dimilikinya kelak setelah memperoleh ilmu pengetahuan. Dari ayat di atas dipahami bahwa siapa pun yang mencari agama setelah kehadiran Nabi Muhammad saw, selain agama yang diajarkan oleh beliau yakni Islam, tidak akan diterima oleh Allah swt Karena itu, makna al-Islam pada kedua ayat di atas, sebagai nama sebuah agama adalah agama yang dibawa oleh Nabi Muhammad saw dan berlaku sepanjang zaman. Walaupun diyakini bahwa semua agama yang datang sebelum Nabi Muhammad saw. juga dari segi pemaknaan mengandung makna Islam dan berimplikasi kepada ajaran tauhid, tetapi kebenarannya berlaku hanya sebatas setiap kurun Tujuan pendidikan Tujuan pendidikan yang terdapat dalam surah Ali Tmran tidak terlepas dari pembentukan manusia seutuhnya yakni sehat jasmani dan rohani, memahami tugasnya sebagai hamba dari Allah swt dan memahami fungsinya sebagai makhluk sosial, serta memahami peranannya sebagai makhluk yang harus menjaga kelestarian lingkungan hidup. Karena itu dalam surah Ali Imran ditemukan beberapa tujuan pendidikan, yakni: ketaatan, perhitungan, balasan, dan agama. Sedangkan term (^-"-1-f) menurut asy-Syarawi seperti yang dikutip oleh M. Quraish Shihab, bahwa pengertian Islam tidak terbatas hanya risalah yang dibawah oleh Muhammad saw Tetapi Islam adalah ketundukan makhluk kepada Tuhan Yang Maha Esa dalam ajaran yang dibawa oleh para rasul. Hanya saja kata Islam untuk ajaran para nabi sebelum M u h a m m a d saw, merupakan sifat, sedangkan umat Nabi Muhammad saw. memiliki keistimewaan dari segi kesinambungan sifat bagi agama umat Muhammad sekaligus menjadi tanda dan nama baginya (Shihab, volume 2, 2005: 40-41). 46 | Jurnal "Al-Qalam" Volurrve 20 Edisi Khusus Desember 2014 Peningkatan keyakinan terhadap kebenaran agama Islam Ajaran Islam meyakinkan kepada pemeluknya bahwa adalah suatu kealfaan ketika seseorang yang mencari agama selain Islam, karena sesungguhnya agama di sisi Allah adalah Islam (QS. Ali-Imran/3: 19). Term (u> pada ayat tersebut memiliki beberapa pengertian, antara lain; ketundukkan, 46 | Jurnal "Al-Qalam" Volurrve 20 Edisi Khusus Desember 2014 waktu masing-masing para rasulnya. Dari keempat sifat tersebut menunjukkan bahwa orang yang mendalam ilmunya berarti pada dasarnya mereka telah memiliki keempat kecerdasan seperti yang telah disebutkan terdahulu dan sekaligus mewujudkan fungsi pendidikan yang harus terwujud dalam sebuah proses pendidikan. Karena itu, salah satu tujuan pendidikan Islam, adalah terwujudnya manusia-manusia yang memperjuangkan keselamatan, ketaatan dan tunduk kepada khaliknya, yang dilandasi dengan keyakinan tentang kebenaran ajaran agama yang dibawa oleh Muhammas saw yakni agama Islam, yang menangkal seluruh kebenaran agama yang datang sebelumnya. Dengan demkian, untuk memiliki ilmu pengetahuan yang mendalam seperti yang dimaksudkan pada potongan ayat ^ jj^iJjllj ?• 1» H memerlukan proses dan sistem yang tidak mudah, dan disinilah pendidik harus berfungsi maksimal memberi bimbingan dan arahan kepada peserta didik dan pada gilirannya mereka menyandang predikat tersebut, sehingga mereka memiliki wewenang dan kemampuan memahami ayat-ayat Allah, terutama ayat-ayat mutasyabih yakni ayat yang memiliki pengertian bersayap. Atau jika salah satu dari dua perkara mirip dengan yang lainnya sehingga tidak mampu dibedakan. (Muhammad Ali al-Hasan, 2007: 165). Bagi mereka memiliki pengetahuan yang kokoh dan mendalam tentu mereka peroleh melalui proses pendidikan, baik pendidikan formal, informal dan non formal serta hidayah atau petunjuk dari Allah swt. Fungsi Pendidikan Islam Fungsi pendidikan Islam yang dimaksudkan adalah terbentuknya peserta didik melalui bimbingan dan arahan, sehingga mereka memiliki kecerdasan intelektual, kecerdasan emosional, dan kecerdasan spiritual dalam menata kehidupannya berdasarkan dengan petunjuk agama. Dalam surah Ali Imran terdapat beberapa ayat yang berkaitan dengan fungsi pendididkan Islam, antara lain: Meningkatkan pengetahuan Bahwa mereka yang dapat memahami secara mendalam tentang makna-makna yang terdapat dalam Alquran terutama ayat-ayat yang memerlukan penakwilan adalah mereka yang mendalam ilmu pengetahuannya (QS. Ali 'Imran/3: 7 ). mplementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 47 Peduli terhadap sesama umat manusia Term (-»JJ-»-AJI berarti sesuatu yang baik menurut pandangan u m u m suatu masyarakat selama sejalan dengan al-khair yakni nilai universal yang diajarkan oleh Alquran dan Sunnah. Sedangkan >£-j-«Jl adalah sesuatu yang dinilai buruk oleh suatu masyarakat serta bertentangan dengan nilai-nilai Ilahi (Shihab, volume 2, 2 0 0 5 : 1 7 5 ) . yang menyadarkannya tentang hubungan dirinya dengan penciptanya (Tuhan), sehingga muncul dalam dirinya bahwa semua kebutuhannya sangat tergantung dengan kehendak dan redhah dari Allah swt sebagai pencipta, pengatur dan pemilik segala sesuatu (QS. Ali 'Imran/3: 1 8 9 ). Allah swt m a m p u melaksanakan ancaman- Nya itu, karena milik Allah kerajaan langit dan bumi, Dia yang menciptakan, memiliki dan mengaturnya, serta mengetahui segala rincian yang terjadi pada keduanya (Shihab,volume 2, 2 0 0 5 : 3 0 5 ) . Karena Allah adalah pemilik segala yang ada di langit dan di bumi, maka kepada-Nyalah akan kembali segala sesuatu (QS. Ali l m r a n / 3 : 1 0 9 ) . Dengan demikian, ayat tersebut menggambarkan bahwa salah satu ciri umat (Islam) yang terbaik adalah mereka yang senantiasa memerintahkan kebaikan (<-*jj-«-^l) dan mencegah kemungkaran (j ^ ' « ^)- Mereka yang dapat melakukannya adalah yang beriman dengan benar. Ciri keberimanan mereka tampak di wajahnya dan dibuktikan dengan kreativitasnya yang selalu mengarah kepada kebaikan, dan menghindarkan dirinya dari perbuatan jelek yang bertentangan dengan ajaran agama. Padahal umat sebelum datangnya umat Islam, mereka hanya melakukan kejelekan, permusuhan di antara mereka dan tidak mengindahkan perintah amar ma'ruf dan nahi mungkar (Ahmad Mushthafa al-Maragiy, Juz IV: 1 9 4 6 : 2 9 ) . Bahwa Allah swt adalah pemilik segala sesuatu, yang mengatur semua urusan mereka sesuai dengan sunnatullah yang sangat bijaksana, dan tidak berubah karena perubahan situasi dan kondisi (al-Maragiy, juz 4 , 1 9 4 6 : 2 7 ) .Allah swt menjelaskan bahwa Dialah pemilik segala yang ada, maka Dia berhak penuh mengatur, mewujudkan dan meniadakannya. Kekuasaan Allah tidak ada yang dapat menandinginya, itulah sebabnya semua persualan sekecil apapun pada akhirnya kembali kepada-Nya, dan tidak ada yang tersembunyi bagi- Nya (QS. Ali Imran/3: 5 ) . Karena itu, kepedulian sosial pada diri suatu umat menjadi penting, seperti memberi bantuan baik material, maupun spiritual. Keinginan untuk saling membantu antara satu sama lain harusnya tumbuh pada diri seseorang sejak umur usia dini, sehingga sikap itu melekat dan perwujudannya menjadi kebiasaan bagi mereka. Peningkatan Pendekatan Spiritual Kebutuhan manusia terhadap aspek spiritual merupakan kebutuhan fitrah yang pemenuhannya berkaitan langsung dengan kesempurnaan masa pertumbuhan dan kedewasaan kepribadian seseorang (G. Lindsay C.S. Hall, and R.E Thompson 1 9 7 6 : 3 6 1 ) . Aspek spiritual terdiri dari; berpegang teguh kepada ketakwaan, mencintai kebaikan, kebenaran, keadilan, keindahan, dan membenci keburukan, kebatilan dan kezaliman (Najit, 2 0 0 5 : 3 5 ) . Peduli terhadap sesama umat manusia Term UJ ^ 1111 >H pada ayat tersebut adalah bentuk plural dari kata tunggalnya berasal dari fi'il al-madi j-^j berakar kata dari huruf ra, sin dan kha berarti tetap (Abiy al-Husain Ahmad bin Faris bin zakariya, jilid II, 1999: 395), juga berarti kokoh pada tempatnya, menguatkan, berurat berakar (Abidin Bisri dan Munawwir A. Fatah, 1999: 248). M. Quraish Shihab mengartikannya dengan sesuatu yang berat ditempatkan pada suatu tempat yang lunak. Seperti besi yang berat ditempatkan pada suatu tempat yang lunak (lembek), pasti besi itu masuk ke dalam sehingga tidak mudah goyah. Bahkan boleh jadi besi tersebut tidak tampak di permukaan, kemudian di artikan dengan mendalam. Bagi mereka yang mendalam ilmunya semakin mantap dan kokoh keimanan dan ketakwaannya kepada Allah. Lebih lanjut beliau mengemukakan bahwa ada sifat yang harus disandang oleh mereka yang mendalam ilmunya, yaitu; 1) takwa antara dirinya dengan Allah, 2) kerendahan hati antara dirinya dengan manusia, 3) zuhud yakni meninggalkan kenikmatan duniawi pada hal ia mampu memilikinya, karena ingin mendekatkan diri kepada Allah, dan 4) mujahadah, kesungguhan mengolah dirinya menghadapi nafsunya (M. Quraish Shihab volume 2, 2005: 16-17). Salau satu tugas manusia sebagai khalifah Allah di atas persada bumi, adalah menjaga kemaslahatan antara sesama umat manusia, antara lain dengan berwasiat kepada kebaikan dan mencegah kemungkaran (QS. Ali 'Imran/3:110). Term yang terdapat pada ayat tersebut berasal dari fi'il al-madi jl£ berakar kata dari huruf kaf, harf al-'Illah (al-wawu) dan nun yang berarti berita tentang kejadian sesuatu (Abiy al-Husain Ahmad bin Faris bin zakariya, jilid VI, 1999: 148). Karena itu, yang dimaksud kuntum pada ayat di atas adalah kamu dijadikan atau diciptakan (Ahmad Mushthafa al-Maragiy, Juz 4, 1946: 28). Sedangkan yang dimaksud dengan ^->i digunakan untuk semua kelompok yang dihimpun oleh sesuatu, seperti agama yang sama, waktu dan tempat yang sama, baik penghimpunannya secara terpaksa, maupun atas kehendak sendiri (Shihab, volume 2, 2005: 185) yang dimaksud umat pada ayat di atas adalah umat Islam. Term UJJ-* ^ adalah fi'il al-mudari dari fi'il al-madi amara yanmg berakar kata dari huruf hamzah, mim dan ra berarti perintah tentang suatu persoalan, atau lawan dari larangan (Abiy al-Husain Ahmad bin Faris bin Zakariya, jilid I, 1999: 137), Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 47 juga berarti memerintahkan. (Shihab, volume 2, 2 0 0 5 : 1 7 4 ) . Peduli terhadap sesama umat manusia Hal ini dapat terwujud, ketika pendididkan dapat berfunsi secara maksimal terhadap penbentukan kepribadian anak, karena salah satu fungsi pendidikan adalah merubah moralitas peserta didik dari yang tidak baik menjadi baik dan dari yang baik menjadi lebih baik. Bahwa Allah swt mengatur makhluk-Nya, maka tentu saja tidak ada yang tersembunyi bagi- Nya, baik makhluk yang berada di bumi, maupun makhluk yang berada di langit dengan seluruh tingkatannya (Shihab, volume 2, 2 0 0 5 : 9 ) . Kalau segala sesuatu tidak ada yang tersembunyi bagi Allah swt maka seharusnya umat manusia ketika meniatkan atau pun melakukan sesuatu, menyadari dengan sungguh-sungguh bahwa ia dilihat dan diawasi serta dinilai, bahkan dibalas oleh Allah swt. Salah satu upaya agar keyakinan seperti tersebut, melekat pada diri seseorang melalui dengan pendidikan. Katena itu, salah satu fungsi pendidikan adalah memberi kesadaran kepada peserta didik tentang perlunya berkomunikasi dengan Allah melalui dengan ibadah, sehingga pendekatan spiritual dalam pendidikan menjadi sesuatu yang sangat penting agar terwujud manusia yang sadar tentang keberadaannya untuk mengabdi dan beribadah kepada Allah swt. 48 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 Metode Pendidikan dan pengajaran dalam Surah Ali Imran Metode berarti: 1) cara teratur yang digunakan untuk melaksanakan suatu pekerjaan agar tercapai sesuai dengan yang dikehendaki, 2) cara kerja yang Keterangan di atas, menunjukkan bahwa yang dimaksud dengan spiritual adalah suatu keadaan jiwa yang terdapat dalam diri manusia 48 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 bersistem untuk memudahkan pelaksanaan suatu kegiatan guna mancapai tujuan yang ditentukan. (Departemen Pendidikan Nasional, 2002: 740). Dalam bahasa Arab metode dikenal dengan istilah thariqah yang berarti langkah-langkah strategis dipersiapkan untuk melakukan sesuatu pekerjaan (Ramayulis, 2008: 2-3). diskusi/dialog mereka itu, Allah memastikan kepada mereka bahwa tidak ada yang mustahil bagi- Nya kalau sekiranya Dia menghendakinya. Dengan demikian, metode diskusi/dialog dan tanya jawab perlu diterapkan dalam pembelajaran sebagai upaya untuk merangsang peserta didik berflkir atau mengeluarkan pendapatnya sendiri mengenai persoalan-persoalan yang dihadapi dan memerlukan pemecahaannya. Karena itu, metode ini tidak hanya berorientasi pada percakapan dan debat, tetapi yang lebih penting adalah pemecahan masalah, seperti yang ditunjukkan oleh Allah swt pada ketiga ayat tersebut. Dengan demikian, yang dimaksud dengan metode pendidikan dan pengajaran adalah suatu cara yang di tempuh oleh pendidik ketika terjadi proses belajar mengajar, sehingga tujuan pembelajaran tercapai sesuai dengan sasaran yang diinginkan. Dalam Alquran terkhusus surah Al-Imran ditemukan berbagai metode pendidikan yang seharusnya menjadi landasan normatif dalam proses pendidikan dan pengajaran. Metode Pemberian Hukuman Kata hukuman berasal dari kata hukum yang dalam Kamus Besar Bahasa Indonesia diartikan dengan; 1) peraturan atau adat yang secara resmi dianggap mengikat, yang dikukuhkan penguasa atau pemerintah, 2) Undang-undang, peraturan untuk mengatur pergaulan hidup masyarakat, 3) patokan (kaedah, ketentuan) mengenai peristiwa (alam dan sebagainya) yang tertentu, 4) kegunaan atau pertimbangan yang ditetapkan oleh hakim (dalam pengadilan), vonis. Setelah mendapat akhiran an menjadilah hukuman yang berarti; 1) siksaan dan sebagainya, yang dikenakan kepada orang yang melanggar undang-undang dan sebagainya, 2) keputusan yang dijalankan oleh hakim, 3) hasil atau akibat menghukum (Departemen Pendidikan Nasional, 2002: 410-411). Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 49 Metode Perumpamaan berakar kata dari huruf qaf dan shad yang berarti mengikuti sesuatu, mengikuti secara berturut-turut, mengikuti jejaknya, juga dapat berarti memotong (Abiy al-Huain Ahmad bin Faris bin zakariyah, juz V, 1999: 11). Karena itu, yang dimaksud metode kisah adalah metode yang dilakukan oleh guru ketika memberikan pelajaran, mereka juga menyampaikan kisah-kisah yang berkaitan pembahasan meteri pelajarannya. Metode ini selain membuka wawasan siswa, juga mengurangi ketegangan, sehingga siswa semakin rileks mengikuti materi pelajaran, tanpa mengurangi tujuan pembelajaran yang ingin dicapai. Metode kisah merupakan salah satu metode pendidikan yang cukup penting diterapkan, karena pada umumnya kisah menyentuh jiwa pendengarnya, terutama kalau yang menyampaikan itu didasari dengan keikhlasan dan penghayatan (al- Jamil, 1995:125). Kisah Maryam ketika dipersiapkan melahirkan Isa, cukup menarik untuk dijadikan salah satu metode pendidikan, sebagaimana yang digambarkan oleh Allah dalam (QS. Ali Tmran/3: 42- 47). Metode perumpamaan adalah sebuah metode dalam pendidikan dengan mengemukakan perbandingan-perbandingan atau perumpamaan sesuatu dengan suatu yang lain. Metode ini menarik perhatian peserta didik ketika dikemukakan perumpamaan yang mudah dicerna akal, yang sebelumnya boleh jadi dalam bentu abstrak, lalu diambil perumpamaan yang berbentuk konkret (nyata), sehingga mudah dipahami dan diterima oleh akal. Allah swt mengemukakan perumpamaan dengan mengambil contoh yang konkret dari hal- hal yang bersifat abstrak. Allah swt menjadikan perumpamaan orang yang membelanjakan hartanya hanya kepentingan dunia, seperti angin yang menimpa tanaman lalau merusaknya (QS. Ali 'Imran/3: 117). Term J-J-» matsal yang terdapat pada ayat tersebut berakar kata dari huruf min, tsa dan lam yang berarti membandingkan sesuatu dengan sesuatu yang lain (Abiy al-Husain Ahmad bin Faris binzakariya,jilidIII, 1999:296). Penyebutan kalimat L±j xJl i\ j ^ \\ SIA ^ mengisyaratkan bahwa infak yang mereka lakukan itu, bertujuan semata-mata untuk kepentingan kehidupan dunia. Harta mereka benar-benar dimanfaatkan untuk memperoleh kelezatan hidup, memperoleh prestasi dan prestise di dunia, sehingga harta mereka tidak dimaanfatkan di jalan Allah. Perumpamaan mereka seperti angin yang sangat dingin yang menghancurkan sawah dan ladang suatu kaum (Ahmad Mushthafa al-Maraghy, Juz 4, 1946: 40). Karena itu, ayat tersebut dipahami bahwa Allah swt menjadikan perumpamaan orang- orang yang membelanjakan hartanya, semata-mata untuk kepentingan dunia, tidak ada niat untuk kepentingan akhirat, karena memang mereka tidak meyakini tentang adanya balasan di akhirat. Perbuatan mereka diibaratkan angin yang sangat dingin sehingga merusak tanaman. Maksudnya harta yang mereka infaqkan itu sama sekali tidak berguna bagi mereka. Dari beberapa ayat tersebut, menggambarkan tentang kisah Maryam yang mendapatkan informasi dari malaikat tentang kelebihannya, dan keharusannya tunduk menyembah dan taat kepada Allah. Metode Perumpamaan Demikian pula informasi dari malaikat tentang kesediaannya memiliki anak yang bernama Isa. Lalu Maryam heran mendengar informasi itu, karena secara akal dan kebiasaan tidak mungkin ia memilki anak, karena tidak pernah bersentuhan dengan seorang laki-laki. Lalu malaikat berkata terimalah kenyataan ini karena kalau Allah menghendaki sesuatu pasti terjadL Kisah tersebut menarik disampaikan kepada siswa ketika mengajarkan materi pelajaran yang berkaitan masalah akidah terutama ketika membahas sifat-sifat Allah swt, sifat-sifat nabi dan rasul serta reproduksi manusia. Penyampaian metode kisah dalam proses pendidikan sangat penting untuk dikembangkan dan diterapkan, karena pada umumnya kisah- kisah itu menyentuh akal pikiran, hati dan jiwa manusia, sehingga kisah yang disamapaikan itu mempermudah pemahaman siswa terhadap meteri pelajaran yang diajarkan pada waktu itu, terutama kalau kisah itu ada relevansinya dengan materi pelajaran. Dengan demikian, metode pendidikan perumpamaan sangat berguna untuk mempercepat daya tangkal dan daya ingat siswa tentang sesuatu terutama yang bersifat abstara (tidak dirasakan langsung panca indra) lalu kemudian diangkat perumpamaannya dengan hal-hal yang konkret (dapat dirasakan langsung panca indra). Metode Diskusi/Dialog, dan Tanya Jawab Dalam pengertian yang umum, diskusi ialah suatu proses yang melibatkan dua atau lebih individu yang berintegrasi cecara verbal dan saling berhadapan muka mengenai tujuan atau sasaran yang sudah tertentu melalui cara tukar menukar informasi (information sharing), mempertahankan pendapat (self maintenance), atau pemecahan masalah (problem sovling) (Ramayulis, 2008: 289). Karena itu, yang dimaksud metode dialog/diskusi adalah suatu metode yang dilakukan dengan bertukar pikiran antara guru dengan peserta didik atau antara sesama peserta didik, guna mengambil kesimpulan dari suatu masalah. Atau guru memberi kesempatan kepada para peserta didik untuk mengadakan pembicaraan ilmiah guna mengambil kesimpulan atau membuat alternatif-alter natif pemecahan terhadap beberapa masalah yang harus dipecahkan bersama. Karena itu, pemberian hukuman kepada peserta didik dimaksudkan sebagai balasan terhadap perbuatan mereka, dan tidak ada alternati lain kecuali diberikan hukuman, agar mereka menyadari kesalahan yang mereka lakukan. Dalam pendidikan Islam metode pemberian hukuman atau mengancam memberikan hukumam kepada mereka yang melanggar adalah sesuatu tindakan yang dibenarkan selama dilakukan dengan motivasi mendidik bukan balas dendam dan sebagainya. Allah swt mengancam orang kikir yang tidak mau menginfatkan hartanya, lalu hartanya itu Allah menjadikan kalung, kemudian memasangkannya di lehernya di had kemudian (QS. Ali-Imran/3: 180). Salah satu kandungan surah Ali Imran adalah masalah dialog/diskusi, seperti yang dialami oleh Zakariya dan Maryam ketika berdialog/diskusi dengan Allah swt tentang kemungkinannya memperoleh anak (QS. Ali Tmran/3:40-41 dan 47). Ketiga ayat tersebut memberikan gambaran tentang contoh metode dialog/diskusi yang ditunjukkan oleh Allah swt untuk memberi pemahaman tentang sesuatu yang dimaksud kepada lawan berbicara, yakni Zakariya (ayat 40-41) dan Maryam (ayat 47), yang pada prinsipnya ketiga ayat tersebut membicarakan tentang kemungkinannya mereka memiliki anak, yang secara empirik tidak mungkin lagi mereka memilikinya. Zakariya dan istrinya sudah tua sedangkan Maryam tidak ada laki-laki yang pernah menyentuhnya. Dari hasil Memberi ancaman hukuman kepada peserta didik yang melanggar merupakan salah satu metode yang seharusnya diterapkan. Bahkan bukan saja bersifat ancaman, tetapi betul-betul dilaksanakan ketika mereka melanggar, agar tindakan itu menjadi pelajaran, tidak hanya bagi yang bersangkutan, tetapi juga kepada peserta didik lain. 50 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 Implementasi Nilai-nilai Pendidikan dalam Surah Ali 'Imran pada Madrasah Tsanawiyah Watampone Untuk memperoleh data yang akurat tentang implementasi nilai-nilai pendidikan dalam surah Ali Tmran pada Madrasah Tsanawiyah Negeri Watampone, peneliti terlebih dahulu melakukan wawancara dengan para guru dan siswa madrasah tersebut yang diyakin dapat mewakili teman- temannya dan dapat memberikan informasi yang akurat. Menurut al-Maragiy, ^-ij-* (zurriyyatan) berarti anak, kata ini dipakai untuk bentuk mufrad dan jamak. Sedangkan V ' j ^ (thayyibatan) Hal-hal yang nyaman dipandang, baik dari segi perilakunya (al-Maragiy, Juz 3, 1946: 142), maupun akhlaknya. Menurut Ibn Katsir, yang dimaksud dengan * .' j ^ (zurriyyatan thayyibatan) adalah anak saleh. Ketika Nabi Zakariya mengetahui keindahan tingkah laku dan ilmu pengetahuan yang dimiliki Maryam tentang Allah swt maka beliau berharap semoga dikaruniai oleh Allah anak saleh seperti Maryam, cerdas serta memikat hati bagi yang melihatnya, sehingga mereka berharap agar dikaruniai anak yang cerdas seperti Maryam (al-Dimisyqiy, fuz 3, t.th: 360). Menurut M. Quraish Shihab yang dimaksud ^ (di sanalah), yakni di Mihrab tempat Maryam berada dan menjawab pertanyaan Nabi Zakariya tentang sumber rezki Maryam, maka muncul keinginan Zakariya memperoleh anak, yang sebelumnya selalu dipendam karena ia sadar bahwa dirinya dan istrinya sudah tua. Melihat apa yang terjadi pada diri Maryam dan mendengar serta menyadari ucapan Maryam bahwa Allah memberi rezki kepada siapa yang Dia kehendaki. Ketika itulah Zakariyah bermohon kepada Allah untuk mendapatkan anak saleh (Shihab, volume 2:2005: 84). Dari segi tujuan pendidikan yang meliputi peningkatan ketauhidan dan keyakinan terhadap kebenaran agama pada umumnya interview (pemberi informasi) baik dari guru maupun siswa menyatakan bahwa sesungguhnya tujuan pendidikan yang dimaksudkan itu telah terlaksana dalam setiap proses belajar mengajar di madrasah ini, walaupun masih ada kekurangan, ketika mata pelajaran di luar mata pelajaran Pendidikan Agama Islam (PAI), misalnya matematika, bahasa Inggris, bahasa Indonesia, karena keasikannya menjelaskan materinya mereka lupa menyinggung hal-hal yang berkitan dengan kebesaran Allah dan kebenaran agama, namun pada umumnya guru membaca Basmalah sebelum memulai pelajaran. Sebagaimana yang dikemukakan oleh Sabbang, guru bahasa Inggeris bahwa setiap memlulai pertemuan selalu diawalibacaanbasamalah,hanyasajatidakselamanya mengajak seluruh siswanya membaca Basmalah. Walaupun disadari bahwa membaca Basmalah adalah bagian dari upaya mengimplementasikan nilai-nilai ketauhidan kepada Allah swt, Ada pun upaya peningkatan kesadaran tentang kebenaran agama, hampir semua pertemuan selalu tersentuh masalah agama dari berbagai sudut pandang, termasuk masalah kemahaesaan Allah swt karena selalu mengemukakan contoh-contoh kalimat bahasa Inggris yang ada kaitannya dengan makna agama, misalnya mereka disuruh membuat kalimat dari kata sembahyang dan Tuhan (wawancara, 1/10/2014). Metode Kisah Dalam bahasa Arab, keteladanan diistilahkan dengan uswah atau qudwah yang menurut al- Term kisah berasal dari bahasa arab qsahashun adalah ism al-masdar dari kata kerja qashsha yang 50 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 berbakti kepada kedua orang tuanya. Dengan demikian metode keteladanan dalam pendidikan menjadi penting sebagai upaya untuk mewujudkan tujuan pendidikan. Pendidik yang dapat memberi contoh yang baik, seperti bertutur kata, bertata krama, bersopan santun sampai kepada pelaksanaan syariat agama secara utuh sangat berpengaruh secara fisik dan psikologi para siswa. berbakti kepada kedua orang tuanya. berbakti kepada kedua orang tuanya. Dengan demikian metode keteladanan dalam pendidikan menjadi penting sebagai upaya untuk mewujudkan tujuan pendidikan. Pendidik yang dapat memberi contoh yang baik, seperti bertutur kata, bertata krama, bersopan santun sampai kepada pelaksanaan syariat agama secara utuh sangat berpengaruh secara fisik dan psikologi para siswa. Asfahani berarti suatu keadaan ketika seorang manusia mengikuti manusia lain, apakah dalam kebaikan, kejelekan, kejahatan atau dalam kemurtadan (Al-Asfahani, t.th: 105). Keteladanan yang dimaksud dalam pendidikan Islam adalah sesuatu yang diikuti oleh peserta didik dari pendidik atau orang yang dituakan baik dari perktaan maupun perbuatan. Keberhasilan pendidikan pada zaman Rasulullah saw adalah keteladanan yang ditunjukkan oleh Rasululah sebagai uswah. Rasulullah ternyata banyak memberikan keteladan dalam mendidik para sahabatnya (Armai Arief, 2002: 116). Zakariya adalah teladan yang baik ketika beliau selalu berdoa agar mendapatkan keturunan yang baik (QS. Ali 'Imran/3: 38). Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. | 51 Implementasi Nilai-nilai Pendidikan dalam Surah Ali 'Imran pada Madrasah Tsanawiyah Watampone Sikap Nabi Zakariya tersebut, yakni memohon kepada Allah swt untuk mendapatkan anak yang saleh, berakhlak muliah, memikat hati orang yang melihatnya, merupakan suatu sikap yang perlu diteladani. Sebagai orang tua seharusnya selalu berdoa kepada Allah semoga mendapat anak yang saleh, atau semoga anak yang telah dilahirkan selalu mendapat hidayah dari Allah swt agar menjadi anak saleh, yang selalu beribadah kepada Allah swt serta Lain halnya dengan informasi yang disampaikan oleh Aminuddin seorang guru matematika bahwa setiap mengajar selalu diawali dengan pemberian salam, tentu dijawab oleh siswa. Hanya saja ketika menyampaikan materi jarang sekali menyinggung masalah yang bersifat spiritual, selain karena keterbatasan waktu, juga memang materinya pada umumnya hanya meningkatkan kecerdasan intelektual. Namun disadari bahwa sesungguhnya pelajaran matematika inklusif di dalamnya nilai-nilai spiritual, misalnya ketika memahami makna angka 1 (satu) sampai angka 10 (sepuluh), akhirnya angka 1 (satu) kembali muncul, artinya bahwa semua yang terjadi di alam ini berproses dari yang satu (Yang Masa Esa) kemudian kembali kepada asalnya yakni yang satu. Pemahaman seperti ini tidak selamanya terungkap dalam semua pertemuan, karena boleh jadi suatu pertemuan ada hal-hal yang harus diselesaikan dalam bentukperhitungan (Wawancara, 3/10/2014). demikian, pada dasarnya tujuan dan fungsi nilai- nilai pendidikan dalam surah Ali 'Imran telah terimplementasi dengan baik. Dari segi metode pendidikan dan pengajaran yang tersirat dalam surah Ali 'Imran, seperti metode diskusi/dialog dan tanyak jawab, hukuman, perumpamaan, Kisah, dan keteladanan, menurut M. Taufik guru bidang studi Alquran Hadis mengemukakan bahwa pada dasarnya semua metode tersebut selalu diperktekkan ketika mengajar, tetapi tidak sekaligus, tergantung materi yang diajarkan, boleh jadi pertemuan pertama digunakan metode kisah dan perumpamaan, tetapi pertemuan berikutnya yang digunakan metode hukuman dan diskusi/dialog atau tanyak jawab, demikianlah terus menerus dalam setiap pertemuan pasti metode tersebut ada yang digunakan, karena disadari bahwa bagaimanapun bagusnya materi pelajaran kalau tidak ditopan dengan metode pengajaran yang baik, pada umunya tidak berhasil maksimal, bahkan menimbulkan kejenuhan para siswa, yang berakibat pada ketidak pahaman mereka terhadap materi yang diajarkan (Wawancara, 5/10/2014). Alamsyah, seorang guru bidang studi komputer, mengemukakan bahwa setiap masuk ruangan selalu di awali dengan salam', dijawab oleh siswa dengan baik, sehingga suasana di kelas terasa akrab, sehingga ketika memulai pelajaran tidak ada perasaan tegang, baik bagi saya sebagai guru, maupun di kalangan siswa. Sebelum pelajaran dimulai, semua siswa diwajibkan berdoa dan membaca basmalah sebagai pernyataan sikap bahwa ada yang Maha Kuasa yang diharapkan memberikan pertolongan dalam semua aktivitas manusia. terutama ketika membuka komputer atau laptop. 52 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 PENUTUP Surah Ali 'Imran merupakan surah ketiga dalam Alquran yang di dalamnya terdapat nilai- nilai pendidikan yang seharusnya dikembangkan terus dalam kehidupan umat manusia, terutama ketika dipahami tujuan utama surah tersebut yaitu pemahaman tentang ketuhidan, keesaan dan kekuasaan Allah swt serta penegasan ba,hwa dunia, harta dan anak-anak yang terlepas dari nilai-nilai ilahiyah tidak akan bermanfaat di akhirat kelak. Pemahaman dan keyakinan seperti ini harusnya terpatri pada diri seseorang sejak kecil melalui jalur pendidikan, baik pendidikan formal, informal maupun non formal. Implementasi Nilai-nilai Pendidikan dalam Surah Ali 'Imran pada Madrasah Tsanawiyah Watampone Ketika memperkenalkan cara pengoprasikannya, selalu mengingatkan mereka tentang keutamaan ilmu pengetahuan, sehingga alat seperti ini dapat terwujud. Sekali pun demikian, di balik dari alat ini harus disadari ada kekuasaan Allah swt karena alat ini tidak ada gunanya kalau tidak di topan dengan kekuatan akal manusia, sejak pembikinannya sampai pengoprasionalannya. Kalau Allah swt tidak menghendaki bermanfaat, Allah swt memutus satu saja urat saraf manusia, tentu tidak ada lagi yang dapat mengoperasionalkannya (Wawancara, 1/10/2014). Keterangan di atas searah dengan padangan Martan Aziz, pengasuh mata pelajaran IPA bahwa tanpa metode pengajaran proses belajar mengajar tidak akan dapat berhasil dengan baik. Karena itu, metode pengajaran yang terdapat pada surah Ali 'Imran tersebut pada prinsipnya saya telah mengimplementasikannya dalam setiap pertemuan, sekalipun tidak sekaligus, tergantung dengan objek pembahasan, apalagi materi pelajaran IPA terkadang ada hal-hal yang spesifikasi tidak memerlukan metode, hanya diperlukan kecakapan dan keterampilan. Selain apresiasi dari guru, siswa juga memberikan apresiasi (Anisa Riadatul Jannah kelasa V, Khaeratul Hisan kelas VII, dan Rilna Nisa Alifiyah, kelas IX), mereka memiliki pandangan yang sama, sesuai dengan pandangan dan pengalamannya selama mengikuti proses belajar mengajar di Madrasah Tsanawiyah Negeri Watampone, bahwa semua guru senantiasa memberikan spirit keagamaan terutama yang berkaitan dengan keyakinan, ibadah, dan akhlak ketika mereka mengajar, tidak terkeculai guru yang mengajar selain mata pelajaran Pendidikan Agama Islam (PAI). Demikian pula pada umumnya mereka menggunakan metode pengajaran seperti yang terdapat pada surah Ali 'Imran, sekali pun mereka bervariasi dalam penerapannya (wawancara, 10/10/2014). Dapatlah dipahami bahwa sekalipun mereka bukan secara khusus membina mata pelajaran materi pendidikan agama Islam (PAI), tetapi mereka tetap berusaha memasukkan nilai-nilai spiritual dalam setiap menyampaikan meteri pelajarannya, walaupun dalam beberapa kesempatan terdapat masalah teknis yang tidak bisa dihindari. Namun 52 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 dasar normatif dalam pengembangan pendidikan di masa yang akan datang. Dari hasil wawancara tersebut, dapat dipahami bahwa pada dasarnya metode pendidikan dan pengajaran yang terdapat dalam surah Ali 'Imran telah terimplemnetasi dengan baik pada Madrasah Tsanawiyah Negeri Watampone, walau pun masih terdapat kekurangan-kekurangan yang masih memerlukan kesadaran serta perhatian baik dari kalangan guru, maupun pimpinan dan segenap yang berkompeten terhadap pelaksanan proses belajar mengajara pada madrasah tersebut, sehingga semakin hari semakin terasa peranan Alquran khusus surah Ali 'Imran sebagai dasar normatif tujuan, fungsi dan metode pendidikan Islam. Ucapan Terima Kasih Ucapan terima kasih disampaikan kepada pengelola Jurnal Al-Qalam atas kesediannya memuat tulisan ini. Ucapa yang sama disampaikan kepada bapak/ibu dan saudara selaku narasumber atas kesediaannya memberi informasi yang valid, sehingga penelitian ini dapat selesai sesuai dengan waktu yang direncenakan. Akhirnya ucapan terima kasih disampaiakan pula kepada civitas akademika STAIN Watampone atas motivasi dan bantuannya, demi terselesaikannya penelitian ini. Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. j 53 DAFTAR PUSTAKA Ahmad bin Faris bin zakariya, Abiy al-Husain. 1999. Mu'jam Maqyis al-Lugah. Bairut: Dar al-Jili. Arif, Armai. 2002. Pengantar Ilmu dan Metodologi Pendidikan Islam. Jakarta: Ciputat Pres. al-Asfahani, Al-Ragib. t.th. Mufradat alfazh al- Qur'an. Damsyiqy: Dar Al-Qalam. al-Abrasy, Mohammad Athiyah. 1969. Ruh al- Tarbiyyat wa al-Ta'lim. Qairo: Isa al-Baby al- Halaby. Tujuan dan fungsi pendidikan dalam surah Ali 'Imran tidak terlepas dari semangat peningkatan ketauhidan, yakni keyakinan bahwa segala sesuatu akan kembali kepada penciptanya yang mengatur dan memeliharanya, serta keyakinan tentang kebenaran agama Islam yang dibawa oleh Rasululan saw. Sedangkan fungsi pendidikan yang terdapat dalam surah Ali I m r a n antara lain; peningaktan ilmu pengetahuan, peduli terhadap sesama umat manusia dan peningkatkan nilai-nilai spiritual dalam segala segi kehidupan. Ada pun metode pendidikan yang terdapat dalam surah Ali 'Imran antara lain adalah metode diskusi/dialog dan tanyak jawab, metode pemberiah hukuman, metode perumpamaan, metode kisah, dan metode keteladanan. al-'Azhim Ali, Abd. 1989. Epistemologidan Aksiologi Ilmu, terj. Khalilullah Ahnas Masykur Hakim. Bandung: Rosda Karya. Bisri, Abidin, dan Munawwir A. Fatah. 1999. Ramus Indonesia Arab, Arab-Indonesia. Surabaya: Pustaka Progressif. C.S. Hall, G. Lindsay, and R.F. Thompson. 1976. Psyckology. New York: Worth Publishers, Inc. Departemen Pendidikan Nasional. 2002. Kamus Besar Bahasa Indonesia. Jakarta: Balai Pusataka. al-Jamali, Moh. Fadhil. 2002. Falsafah Pendidikan Dalam al-Qur'an, terj. Junaidi al-Falasany. Sjurabaya: Bina Ilmu. Kartone, Kartini. 1986. Pengantar Metodologi Research Sosial. Bandung: Penelitian Alumni. Tujuan, fungsi dan metode pendidikan dalam Alquran tersebut, pada dasarnya telah terimplementasi pada Madrasah Tsanawiyah Negeri Watampone, walapun masih terdapat beberapa guru yang belum melaksanakannya secara maksimal, karena mata pelajaran yang diajarkan itu tidak selamanya sesuai dengan metode-metode tersebut, tetapi setidaknya niali-nilai pendidikan yang terdapat dalam surah Ali 'Imran telah menjadi Munawwir, Ahmad Warson. 1984. Al-Munawwir Kamus Arab Indonesia. Yogyakarta: Pondok Pesanteren al-Muanawwir. al-Maragiy, Ahmad Miishthafa. 1946. Tafsir al- Maragiy. Mesir: Syakitaun wa Mathbaatun Musthafa al-Babiy al-Halabiy wa Auladuh. al-Munawwar, Said Agil Husain. 2005. Aktualisasi Implementasi Nilai-Nilai Pendidikan dalam Surah Ali 'Imran pada MTsN Watampone - M. Amir H.M. j 53 Nilai-Nilai Al-Qur'an Dalam Sistim Publishing Company. Pendidikan Islam. Ciputat: PT. Cipoutat Ramayulis. 2008. Metodologi Pendidikan Agama Press. Islam. Jakarta: Kalam Mulia. Najit, Muhammad Utsman. 2005. Ilmu Jiwa Dalam . 2011. Ilmu Pendidikan Islam. Jakarata: al-Qur'an. Jakarta: Pustaka Azzam. Kalam Muliah. al-Qurasyiy sl-Dimisyqiy, Abiy al-Fidai Ismail ibn Shihab, M. Quraish. 2005. Tafsir Al-Misbah Pesan, Katsir. t.th. Tafsir al-Qur'an al-Azhim, Juz I. Kesan dan Keserasian al-Qur'an, volume 2. Mesir: Dar al-Kutub. Jakarta: Lentera Hati. Rahman, Fazlur. 54 | Jurnal "Al-Qalam" Volume 20 Edisi Khusus Desember 2014 DAFTAR PUSTAKA 1987. Health and Medicine in the Taufik, M. 2012. Kreativitas Jalan Baru Pendidikan Islam Tradition. New Yok: The Crossroad Islam. Solo: Kurnia Kalam Semesta. Nilai-Nilai Al-Qur'an Dalam Sistim Pendidikan Islam. Ciputat: PT. Cipoutat Press. Najit, Muhammad Utsman. 2005. Ilmu Jiwa Dalam al-Qur'an. Jakarta: Pustaka Azzam. al-Qurasyiy sl-Dimisyqiy, Abiy al-Fidai Ismail ibn Katsir. t.th. Tafsir al-Qur'an al-Azhim, Juz I. Mesir: Dar al-Kutub. Rahman, Fazlur. 1987. Health and Medicine in the Islam Tradition. New Yok: The Crossroad . 2011. Ilmu Pendidikan Islam. Jakarata: Kalam Muliah. Najit, Muhammad Utsman. 2005. Ilmu Jiwa Dalam al-Qur'an. Jakarta: Pustaka Azzam. Najit, Muhammad Utsman. 2005. Ilmu Jiwa Dalam al-Qur'an. Jakarta: Pustaka Azzam. Shihab, M. Quraish. 2005. Tafsir Al-Misbah Pesan, Kesan dan Keserasian al-Qur'an, volume 2. Jakarta: Lentera Hati. al-Qurasyiy sl-Dimisyqiy, Abiy al-Fidai Ismail ibn Katsir. t.th. Tafsir al-Qur'an al-Azhim, Juz I. Mesir: Dar al-Kutub. al-Qurasyiy sl-Dimisyqiy, Abiy al-Fidai Ismail ibn Katsir. t.th. Tafsir al-Qur'an al-Azhim, Juz I. Mesir: Dar al-Kutub. Rahman, Fazlur. 1987. Health and Medicine in the Islam Tradition. New Yok: The Crossroad Taufik, M. 2012. Kreativitas Jalan Baru Pendidikan Islam. Solo: Kurnia Kalam Semesta. Taufik, M. 2012. Kreativitas Jalan Baru Pendidikan Islam. Solo: Kurnia Kalam Semesta.
https://openalex.org/W2753999658
https://kar.kent.ac.uk/83566/1/Garner_Incentive_Value_And_Spatial_Certainty_2020.pdf
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Distinct selection mechanisms for when predictions and rewards guide visual selective attention
bioRxiv (Cold Spring Harbor Laboratory)
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Licence for this version CC BY (Attribution) Additional information Versions of research works Versions of Record If this version is the version of record, it is the same as the published version available on the publisher's web site. Cite as the published version. Author Accepted Manuscripts If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding. Cite as Surname, Initial. (Year) 'Title of article'. To be published in Title of Journal , Volume and issue numbers [peer-reviewed accepted version]. Available at: DOI or URL (Accessed: date). The version of record is available from https://doi.org/10.3758/s13414-020-02124-w The version of record is available from https://doi.org/10.3758/s13414-020-02124-w Kent Academic Repository Kent Academic Repository Garner, Kelly, Bowman, Howard and Raymond, Jane (2020) Incentive value and spatial certainty combine additively to determine visual priorities. Attention, Perception, & Psychophysics . ISSN 1943-3921. Downloaded from https://kar.kent.ac.uk/83566/ The University of Kent's Academic Repository KAR Downloaded from This document version Publisher pdf DOI for this version Enquiries If you have questions about this document contact ResearchSupport@kent.ac.uk. Please include the URL of the record in KAR. If you believe that your, or a third party's rights have been compromised through this document please see our Take Down policy (available from https://www.kent.ac.uk/guides/kar-the-kent-academic-repository#policies). Attention, Perception, & Psychophysics https://doi.org/10.3758/s13414-020-02124-w Abstract How does the brain combine information predictive of the value of a visually guided task (incentive value) with information predictive of where task-relevant stimuli may occur (spatial certainty)? Human behavioural evidence indicates that these two predictions may be combined additively to bias visual selection (Additive Hypothesis), whereas neuroeconomic studies posit that they may be multiplicatively combined (Expected Value Hypothesis). We sought to adjudicate between these two alternatives. Participants viewed two coloured placeholders that specified the potential value of correctly identifying an imminent letter target if it appeared in that placeholder. Then, prior to the target’s presentation, an endogenous spatial cue was presented indicating the target’s more likely location. Spatial cues were parametrically manipulated with regard to the information gained (in bits). Across two experiments, performance was better for targets appearing in high versus low value placeholders and better when targets appeared in validly cued locations. Interestingly, as shown with a Bayesian model selection approach, these effects did not interact, clearly supporting the Additive Hypothesis. Even when conditions were adjusted to increase the optimality of a multiplicative operation, support for it remained. These findings refute recent theories that expected value computations are the singular mechanism driving the deployment of endogenous spatial attention. Instead, incentive value and spatial certainty seem to act independently to influence visual selection. Keywords Attention . Prediction . Expectation . Reward . Incentive Incentive value and spatial certainty combine additively to determine visual priorities K.G. Garner1,2 & H. Bowman1 & J.E. Raymond1 # The Author(s) 2020 # The Author(s) 2020 1 School of Psychology, University of Birmingham, Birmingham, UK 2 Queensland Brain Institute (79), University of Queensland, St Lucia, QLD 4072, Australia * K.G. Garner getkellygarner@gmail.com Introduction remains incomplete. Particularly unclear is how multiple con- current sensory cues, each associated with and therefore pre- dictive of specific consequent outcomes, are combined to in- fluence visual selection. A central tenet of many cognitive, computational, and neurobiological theories of visual selec- tion (Buschman & Kastner, 2015; see Itti & Koch, 2001; Moore & Zirnsak, 2017, for reviews), is that competition for high-level neural representation of external objects is flexibly biased by information pertaining to goal-directed outcomes. Indeed, multiple endogenous sources appear to exert parallel influences on visual selection (see Hutchinson & Turk- Browne, 2012, for a review), even when learned information is antithetical to the current goals as defined by the task-set (see Awh et al., 2012, for a review). In the current study, we specifically seek to understand how selection biases that stem from learned associations between sensory cues and reward outcomes (incentive value) are combined with biases driven by associations between sensory cues and the probable loca- tion of task-relevant target information (spatial certainty). Humans are good at learning that specific sensory informa- tion, or cues, can predict subsequent events. For example, we learn quickly that hearing a siren on the left predicts a speed- ing emergency vehicle appearing from that direction, or that seeing a smile predicts a likely future opportunity to gain social approval. Knowledge about where and when new, im- portant sensory information may appear or new reward oppor- tunities may arise is only useful, however, if such knowledge can influence how cognitive mechanisms prioritise informa- tion representation for the eventual control of behaviour. Yet, our understanding of how learning and experience modify such prioritisation of visual signals, i.e. visual selection, * K.G. Garner getkellygarner@gmail.com * K.G. Garner getkellygarner@gmail.com Recently, a large number of studies have shown that visual selection can be biased by the learned predictiveness and in- centive value signalled by sensory cues (see Le Pelley et al., Atten Percept Psychophys certainty are combined by a common, perhaps singular, mech- anism to exert influence on visual selection. Support for this theory comes from the finding that human saccadic initiation times are more tightly correlated with the relative expected value of potential upcoming target locations than with either the spatial certainty for the target location or its incentive value alone (Milstein & Dorris, 2007). Introduction Another large body of literature arising much earlier (in the 1980s) showed that sensory signals that predict where task- relevant information might appear also as bias visual selection (Posner 1980). When centrally presented, symbolic cues (e.g., arrows) are highly predictive of a target’s location (valid cues), response times (RTs) are faster and accuracy better compared to when spatial cues predict the wrong location (invalid cues). Central, symbolic spatial cues are often termed endogenous due to the assumption that their ability to bias visual selection largely stems from previously acquired and internally accessed knowledge. Our interest here is how en- dogenous (learned) spatial associations interact with other learned associations, specifically value associations to control visual selection. Strong evidence that spatial cueing effects depend on learning is that they scale with the reliability of the cue (Lanthier et al., 2015; Vossel et al., 2006, 2015), being stronger when cues are more predictive and weaker when predictability is low. Indeed, cueing effects appear to be a linear function of the certainty gained (in bits of information) by a spatial cue (Prinzmetal et al., 2015). However, preliminary support for this possibility has been reported by Stankevich and Geng (2014). They asked partic- ipants to detect as quickly and accurately as possible a simple target that could appear on the left or right within a coloured placeholder. In their experiment, placeholder colour indicated the magnitude of response-contingent rewards, as it does in the experiment we report here. However, they provided no explicit spatial cues as to target location. Instead, across blocks and without instruction, the probability of the target appearing on one side versus the other was varied. Greater performance benefits were observed when the target appeared in the more probable location and when that location corresponded to a high versus a low value placeholder. Critically, these benefits were additive, suggesting that incen- tive value and spatial certainty acted independently to influ- ence visual selection, according to additive-factors logic (Sternberg, 1998). However, in their experiment, spatial- certainty was established over many trials, allowing predic- tions about target location to be generated well before each Although the issue of how multiple endogenous influences act on goal-directed visual selection has been previously ad- dressed (Awh et al. 2012; Klink et al. 2014), it remains unclear how incentive value and spatial-certainty information provid- ed by pre-target cues are combined. Introduction Moreover, close correspon- dence has been shown between neurons that fire proportion- ally to the incentive values associated with sensory cues and those that change their response to correlate with the spatial certainty introduced by a directional cue, at least in macaque V1 (Stănişor et al., 2013). Indeed, this latter finding was interpreted as providing evidence for a singular neural mech- anism able to re-weight incentive values across the visual scene on-the-fly by updating computation of expected values, given post-cue spatial probabilities. 2016, for a review). Such studies typically present stimuli in a simple selection task that probabilistically predict the reward magnitude available for correct performance, allowing those stimuli to become value associated. Then, in a subsequent visual selection task, these stimuli are presented as targets or distractors. The central finding is that visual selection perfor- mance is better or worse, respectively, compared to when such stimuli are absent (Anderson et al., 2011; Chelazzi et al., 2014; Raymond & O’Brien, 2009; although see Sha & Jiang, 2016). Convergent evidence from the behavioural (Le Pelley et al., 2016), neuro-cognitive (e.g. Barbaro et al., 2017; Hickey et al., 2010; Raymond & O’Brien, 2009) and neuro- economic (e.g. Dorris & Glimcher, 2004; Platt & Glimcher, 1999; Stănişor et al., 2013) literature also provides a convinc- ing picture that value cues influence visual selection propor- tionally to their associated reward value, underscoring the role of prior experience. A second alternative is the Additive Hypothesis, which posits incentive value and spatial certainty exert independent, as opposed to multiplicative, influences on visual selection. Origins for this idea stem from computational theories propos- ing that variation in physical salience within a scene (a salien- cy map) for different stimulus dimensions (e.g., colour, mo- tion or orientation) are independently calculated, then weight- ed and summed to create a visual priority map (Zhao & Koch, 2012). Indeed, empirical investigations support the notion of additive effects for the combination of salience information based on different features, and report that nonlinear combi- nations across feature dimensions arise only when there is overlap between the underlying saliency mechanisms (Nothdurft, 2000). Although suggestive of the possibility that incentive value and spatial certainty might combine additively to control selection, it remains unclear whether additivity could apply to non-physical features, such as learned associations. Introduction One possibility, the Expected Value Hypothesis, is that visual selection is biased by the relative expected value, i.e. incentive value multiplied by spatial certainty for each outcome. Derived from economic theory (Morgenstern & Von Neumann, 1953), expected value is conventionally defined as reward magnitude multiplied by reward probability; in the context of selective attention in re- sponse to pre-target cues, such a computation would be the product of cue incentive value and spatial certainty. It is likely to be normalised across potential outcomes given the trial context (Padoa-Schioppa & Assad, 2008). Evidence for such an operation would suggest that incentive value and spatial Atten Percept Psychophys incentive value and spatial certainty, predicts an interaction of these two variables on performance (see Fig. 1). Specifically, it predicts a super-additive effect when spatial certainty is high and a sub-additive influence when it is low. In contrast, the Additive Hypothesis implies simple additivity of incentive value and spatial certainty. Evidence for this would be simple main effects on performance of incentive value and spatial certainty with no interaction effect. In Experiment 1, we var- ied spatial certainty over a wide range. In Experiment 2, re- wards for correct responses diminished as RT lengthened, creating conditions favouring the use of an expected value mode of cue combination. Results were evaluated using a Bayesian model selection approach. To anticipate, we found in both Experiments that the influence of incentive value and spatial certainty remains additive across all tested levels of spatial certainty, arguing against the Expected Value Hypothesis. trial began and certainly in advance of location-specific incen- tive information. This may explain why incentive information provided an additive “top-up” effect. Such effects might not occur when location-specific incentive information is avail- able first and spatial certainty cues providing task relevant information are presented subsequently. Accordingly, how two different endogenous cues (incentive and spatial) might be combined in humans when they are available only via new sensory information as each trial unfolds remains unknown. To address this issue, we conducted two experiments that combined the methods of relevant previous studies to directly test these two hypotheses. As described in Fig. 2, on each trial, participants viewed two spatially separated coloured place- holders that served as incentive cues for 400–500 ms; place- holder colour signalled the reward value for a subsequently presented letter target, should it appear in that placeholder and be correctly identified. Introduction Then, a central symbolic spatial cue was presented that signalled which placeholder would more likely encircle the upcoming target. Then, 400 ms after spatial cue onset, one letter briefly appeared in each placeholder. The task was to identify the target (as one of two possible letters) as fast and accurately as possible. Incentive value for the poten- tial target location was always either high or low on every trial; however, the reliability of the spatial cue (spatial certain- ty) was varied between blocks. The Expected Value Hypothesis, implicating a multiplicative relationship between Participants As larger samples protect against spurious findings (Button et al., 2013; Lorca-Puls et al., 2018), we opted to double the sample size of previous work correlating human performance with expected value (N=10) (Milstein & Dorris, 2007), and recruit a minimum of 20 participants. We calculated the stop- ping rule for data collection as the number of weeks where testing at maximum capacity would bring us to at least the minimum sample size (6 weeks with 4 people per week, allowing recruitment of > 20 participants in order to protect against dropouts). Participants were recruited if they were aged 18 years or over and reported normal or corrected-to- normal vision, with no history of psychiatric or neurological illness, injury, or disorder. Participants earned either course credit or payment (£7 per session), and any additional rewards accrued during the session (~£10). All procedures were ap- proved by the University of Birmingham Human Research Ethics Committee. A total of 23 participants were recruited. Of these, one was excluded due to technical failure and a second due to experimenter error. The remaining 21 partici- pants (19 female, 18 right-handed, mean age = 20.3 years, SD 4.5) completed all the procedures. Experiment 1 Five levels of spatial certainty were tested. Although it is conventional in attention-cueing experiments to use valid spa- tial cues on 80% and invalid cues on 20% of trials, we includ- ed conditions wherein the computation of spatial certainty is trivial, i.e., as the spatial cue approaches 100% validity. We Fig. 1 Illustration of theoretical predictions. Predicted response times (RTs) in arbitrary units, plotted by cue validity (columns), spatial certain- ty (sc, x-axis) and incentive value (iv, colours), according to an additive bias operation (y ~ -β1sc + -β2iv) or an expected-value model (y ~ -βsc*iv). (Note: The spatial certainties used in this simulation are the same as those chosen for Experiment 1) Fig. 1 Illustration of theoretical predictions. Predicted response times (RTs) in arbitrary units, plotted by cue validity (columns), spatial certain- ty (sc, x-axis) and incentive value (iv, colours), according to an additive bias operation (y ~ -β1sc + -β2iv) or an expected-value model (y ~ -βsc*iv). (Note: The spatial certainties used in this simulation are the same as those chosen for Experiment 1) Atten Percept Psychophys SOFREP-144 computer with a 23-in. Asus VG278HE moni- tor (1,920 x 1,080 pixels, 60-Hz refresh rate) viewed from 57 cm. reasoned that if, as the Expected Value Hypothesis posits, a single mechanism responds to both incentive value and spatial-certainty information, then more resources would be available to respond to value information under high versus low spatial-certainty conditions, leading to greater versus less- er effects of incentive value, thereby showing super-additivity. To test this idea, we more densely sampled valid trial proba- bilities between .90 and .96 as well as the more conventional probabilities of .8 and .6. Stimuli Two white (RGB: 200, 200, 200) vertical lines were presented in the centre of the screen. A darkening of one line (50, 50, 50) served as the endogenous spatial cue. The entire spatial cue display (i.e. the rectangle comprising both lines and the grey background displayed between them) was 0.5° w x 1° h. Comparable cues have been used in previous work examining the influence of dynamic cue reliabilities on the deployment of spatial attention (Vossel et al., 2015), and therefore seemed appropriate for the aims of the current study. Although this type of central spatial cue had a small lateralised element, possibly activating exogenous orienting mechanisms to some extent, they were centrally presented (with 3.75° between the outer edge of the central cue and the inner edge of the periph- eral target), and, critically, require some interpretation from the brain to map the relationship between the cue and target locations (Berger et al., 2005; Briand & Klein, 1987; Posner, 1980). Thus, the main characteristics of the cues used here resemble those used by previous researchers to study spatial certainty effects. Two coloured discs (2.2° in diameter), one in purple (87, 75, 80), the other in orange (120, 86, 1) (matched for luminance) served as value cues. They were aligned along the horizontal meridian and positioned 4.5° from the centre. Targets (‘H’ or ‘N’) and distractors [‘Z’ or ‘K’] were presented in light grey (90, 90, 90) Helvetica font, encompassed 1°, and were centred on the disc’s centre. This distance from the cen- tre allowed the viewer to discriminate the letters without mak- ing an eye movement, while remaining just within the locus of high-acuity vision (~6°). Feedback was presented in green (0, 255, 0) for high-reward values, amber (255, 191, 0) for low- reward values, and red (255, 0, 0) for errors. All stimuli were presented on a grey (RGB: 118, 118, 118) background. Procedure As shown in Fig. 2, each trial began with the simultaneous presentation of both incentive value cues and two centrally presented vertical lines. After a pseudo-randomly chosen du- ration of 400–500 ms, the left or right fixation line darkened for 300 ms. After a further 100 ms, the target and distractor were presented for 100 ms (target identity was equiprobable for each incentive value x spatial certainty x cue validity con- dition). Participants pressed the ‘V’ or ‘G’ key on a standard keyboard to indicate the target identity. After 500 ms, feed- back was presented for 750 ms; either the central fixation was replaced with the high-reward value, the low-reward value, an error signal (fixation lines turned red), or the fixation remained the same (no reward). Rewards were awarded on 80% of Method All the task and analysis code and data from the current study are available online.1 The trial sequence of the spatial- orienting task (Posner, 1980) used to assess the combined influence of spatial certainty and location-specific incentive cues, and the key manipulations for Experiments 1 and 2, are shown in Fig. 2. 1 https://github.com/kel-github/attention-value-certainty Apparatus All experimental procedures took place in a room with a sin- gle testing station, under conditions of low ambient light. All tasks were programmed in Matlab (Mathworks, Natick, MA, USA, 2013a), using the Psychophysics Toolbox extension (Brainard, 1997; Pelli, 1997). The tasks were run on a Stone Atten Percept Psychophys Fig. 2 Study method. Task procedure and feedback conditions for Experiment 1 and Experiment 2. (a) Trial structure: Participants monitored two different-coloured circular placeholders (incentive cues). Colour indicated the magnitude of a performance-contingent reward for correct target (‘H’ or ‘N’) identification, should the target subsequently appear within that placeholder. Then, one of two central bars darkened, indicating the more likely target location (left, right). Note that the trial depicted is an invalid trial. (b) Reward feedback structure: After response + 250 ms, performance feedback and response-contingent rewards were presented as shown. In Experiment 2, reward feedback was either inde- pendent of response time (fixed) or decremented exponentially after tar- get onset until response (decaying). (c) Spatial certainty was parametri- cally manipulated across blocks by increasing the information gained (in bits) from the spatial cue. (d) Logic of the decaying reward condition in Experiment 2. Figure shows reward value available as a function of time from target onset. As the expected value computation involves a multiplicative weighting of spatial certainty and incentive value, re- sponses should be super-additive or sub-additive depending on the spatial certainty/incentive value combination. As response times should reflect the inverse of this weighting, responses should be faster in a high-certain- ty/high-incentive scenario than responses based on an additive operation, and slower in a low-certainty/low-incentive scenario than an additive operation. Applying an exponential decay function to the incentive value at target onset means that the extra rewards accrued by being faster to- wards high-incentive value cues (change in the high (5,000) value on the y-axis, while moving leftward on the x-axis) would outweigh the losses accrued from being slower towards low-incentive value cues (change in the low (100) value on the y-axis, while moving rightward on the x-axis). Therefore, any operation that favours this response pattern would accrue greater total rewards than an additive operation, and therefore may emerge under such reward conditions Fig. 2 Study method. Task procedure and feedback conditions for Experiment 1 and Experiment 2. (a) Trial structure: Participants monitored two different-coloured circular placeholders (incentive cues). Apparatus Colour indicated the magnitude of a performance-contingent reward for correct target (‘H’ or ‘N’) identification, should the target subsequently appear within that placeholder. Then, one of two central bars darkened, indicating the more likely target location (left, right). Note that the trial depicted is an invalid trial. (b) Reward feedback structure: After response + 250 ms, performance feedback and response-contingent rewards were presented as shown. In Experiment 2, reward feedback was either inde- pendent of response time (fixed) or decremented exponentially after tar- get onset until response (decaying). (c) Spatial certainty was parametri- cally manipulated across blocks by increasing the information gained (in bits) from the spatial cue. (d) Logic of the decaying reward condition in Experiment 2. Figure shows reward value available as a function of time from target onset. As the expected value computation involves a Fig. 2 Study method. Task procedure and feedback conditions for Experiment 1 and Experiment 2. (a) Trial structure: Participants monitored two different-coloured circular placeholders (incentive cues). Colour indicated the magnitude of a performance-contingent reward for correct target (‘H’ or ‘N’) identification, should the target subsequently appear within that placeholder. Then, one of two central bars darkened, indicating the more likely target location (left, right). Note that the trial depicted is an invalid trial. (b) Reward feedback structure: After response + 250 ms, performance feedback and response-contingent rewards were presented as shown. In Experiment 2, reward feedback was either inde- pendent of response time (fixed) or decremented exponentially after tar- get onset until response (decaying). (c) Spatial certainty was parametri- cally manipulated across blocks by increasing the information gained (in bits) from the spatial cue. (d) Logic of the decaying reward condition in Experiment 2. Figure shows reward value available as a function of time from target onset. As the expected value computation involves a multiplicative weighting of spatial certainty and incentive value, re- sponses should be super-additive or sub-additive depending on the spatial certainty/incentive value combination. As response times should reflect the inverse of this weighting, responses should be faster in a high-certain- ty/high-incentive scenario than responses based on an additive operation, and slower in a low-certainty/low-incentive scenario than an additive operation. Data pre-processing and pi is the probability that the target appears at location i, given the cue. For example, with two locations, and a cue that is .8/.2 valid/invalid: All data were analysed using the R programming language (v3.3.2) (R Core Team, 2013), and R Studio (v1.0.44) (RStudio Team, 2016). RT data were rejected if they were greater than ± 2.5 SD from the mean for that participant in that condition. As participants were not explicitly informed when there was a change in spatial-certainty, we assumed that trials immediately subsequent to changes in spatial-certainty would be contaminated by learning effects. To remove the contaminated trials for each participant, we collapsed the data across spatial certainty blocks, and ordered the data according to trial number. We then fit piecewise linear regressions to find the break point that minimized the mean square error (MSE). Trials occurring prior to the breakpoint were removed (mean = 12.3, SD 8.0). However, when we performed the analyses without removing these trials, the pattern of results was the same. Hcue ¼ −:8 log2:8 ð Þ−:2 log2:2 ð Þ≈:72 As Hno information is 1 (corresponding to complete uncertain- ty, i.e. .5/.5), then the information gained by the cue is 1 - .72 ≈.28 bits. As Hno information is 1 (corresponding to complete uncertain- ty, i.e. .5/.5), then the information gained by the cue is 1 - .72 ≈.28 bits. After fitting all possible models to the RT data obtained in each experiment, we computed Bayes factors (BFs) to quan- tify evidence for each linear mixed-effects model against the null model (intercept plus random effects of participant) using the Bayes factor package (Morey et al., 2014), and implementing the default Jeffreys-Zeller-Siow (JZS) prior on slope estimates (Liang et al., 2008). We then identified the six best performing models. We report the BF of the winning model relative to the null model, and the BF ratios between the best model and the next five best models, to demonstrate the strength of evidence in favour of the winning model. We follow the guidelines of Kass and Rafferty (Kass & Raftery, 1995) when interpreting the strength of evidence. This was sufficient to determine whether the evidence favoured a model that included only main effects, or an incentive value x spatial certainty interaction. All BFs are reported along with the pro- portional error of the estimate. Apparatus Therefore, spatial certainty gained by an informative cue can be calculated as: completely unpredictable with regard to the target location. Therefore, spatial certainty gained by an informative cue can be calculated as: were also informed that their points would be exchanged for cash at the end of the session (1,000 = £1). At the start of the first session, participants practiced until they achieved at least 16/20 correct responses. Spatial certainty ¼ Hno information−Hcue ð1Þ ð1Þ Model specification and selection The aim of the study was to compare whether an additive model remained the best, given the data, even under condi- tions where an additive relationship could be expected to break down. The key aim of each analysis was to determine whether a model that included an interaction term between incentive value and spatial certainty was more probable, given the data, than one that only included main effects (i.e., an additive model). To quantify evidence, we used a Bayesian approach that provides the advantage of offering the ability to quantify evidence against a specific model, which is not pos- sible using null hypothesis significance-testing approaches (Nickerson, 2000; Wagenmakers, 2007). Additionally, Bayesian approaches protect against the problem of model complexity: although more complex models may predict with high likelihood a greater range of values, if these predictions are uninformative, this will result in a more diffuse marginal likelihood distribution when integrating across prior distribu- tions for the parameters, thereby penalising the resulting mod- el evidence. First, we fit all possible linear mixed models on the RT data, with the regressors being (a) incentive value of the target location, (b) cue validity, and (c) spatial certainty offered by the cue. Spatial certainty was computed in line with Prinzmetal et al. (2015). Specifically, Shannon’s (1948) mea- sure of entropy (H) measures the amount of uncertainty in a probability distribution and is at maximum when the cue is Statistical approach when H is defined in the standard manner: H ¼ −∑ipilog2pi ð2Þ ð2Þ H ¼ −∑ipilog2pi Data pre-processing For readers interested in confirming that a null hypothesis significance testing (NHST) approach yields the same conclusions, please refer to the online repository for this study. Accuracy data Accuracy data were analysed to ensure the results were not due to a speed-accuracy trade-off. For each experiment, we fitted all possible linear mixed models to the accuracy data, and selected the winning model. We then reported the fixed effects estimates for each relevant factor in the winning model. Apparatus Applying an exponential decay function to the incentive value at target onset means that the extra rewards accrued by being faster to- wards high-incentive value cues (change in the high (5,000) value on the y-axis, while moving leftward on the x-axis) would outweigh the losses accrued from being slower towards low-incentive value cues (change in the low (100) value on the y-axis, while moving rightward on the x-axis). Therefore, any operation that favours this response pattern would accrue greater total rewards than an additive operation, and therefore may emerge under such reward conditions four sessions so that we could have sufficient trial numbers to obtain a reliable estimate of performance for the low spatial- certainty conditions. Target-value contingencies were split equally within each set of valid and invalid trials for each cue-likelihood condition. correct trials to prevent feedback signals becoming redundant. The high- and low-incentive value cue locations and the target location were equally likely to be on the left or right; all con- ditions (cue value location, target location, and target identity) were fully crossed within each session. Colour/value pairings (e.g. purple = 50 points/orange = 1 point) as well as target- response mappings were counterbalanced across participants. Participants were explicitly instructed how many points were available should the target appear in the location of the high- and low-incentive value cues (50 vs. 1 point), and were instructed that the incentive cues signified that points were available most but not all of the time. They were also instructed that the darkened line was a clue to where the target could appear; however, they were not explicitly informed that the spatial cue’s reliability might vary. Participants were re- quested to keep their eyes at fixation, and to respond as accu- rately and as quickly as possible to the target. Participants Across blocks, the likelihood of cue validity was varied to be either .6 valid/.4 invalid, .8/.2, .9/.1, .92/.08, .96/.04, resulting in information gains (spatial-certainty) of .029, .29, .53, .6 and .86 bits. Each block contained 100 trials. At each of four sessions, participants completed four blocks for each lev- el of spatial certainty. Participants took between 4 days and 1.5 weeks to complete the experiment (block order was pseudo-randomised for each session). Participants completed Atten Percept Psychophys completely unpredictable with regard to the target location. Response time Group mean RT data (dots) and winning model fit (lines) are presented in Fig. 3a. The main effect of incentive value was to Atten Percept Psychophys Fig. 3 Results from Experiment 1. (a) Observed group mean response times (RTs) in ms for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels). Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model. The winning RT model did not involve any interaction of incentive value (iv) and spatial certainty (sc) supporting an Additive Hypothesis, al- though it did indicate an interaction of sc and validity (v). (b) BFs for the probability of the winning RT model (P[Win]: (v * sc) + AME) relative to the five next best models (Alternative, P[Alt], models, y-axis). The larger the BF value, the stronger the evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]; BF values between 3 (dashed line) and 10 constitute moderate evidence for the winning model, values > 10 provide strong evidence. Dark bars indicate P[Alt] contains only an additive influence of incentive value; light bars indicate P[Alt] involves a multiplicative influence of incentive value and either spatial certainty or validity. The Alt RT models were as follows: (1) ~v + iv, (2) ~AME, (3) ~(v * sc) + (sc * iv) + AME, (4) ~(v * sc) + (v * iv) + AME, (5) ~(v * sc) + (v * iv) + (sc * iv) + AME. (c) Observed group mean accuracy plotted as in panel (a). Importantly, the accuracy data show that the RT findings were not due to a speed-accuracy trade-off. BF = Bayes factor, rc = reward condition, AME = all main effects. Fig. 3 Results from Experiment 1. (a) Observed group mean response times (RTs) in ms for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels). Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model. The winning RT model did not involve any interaction of incentive value (iv) and spatial certainty (sc) supporting an Additive Hypothesis, al- though it did indicate an interaction of sc and validity (v). Response time (b) BFs for the probability of the winning RT model (P[Win]: (v * sc) + AME) relative to the five next best models (Alternative, P[Alt], models, y-axis). The larger the BF value, the stronger the evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]; BF values between 3 (dashed line) and 10 constitute moderate evidence for the winning model, values > 10 provide strong evidence. Dark bars indicate P[Alt] contains only an additive influence of incentive value; light bars indicate P[Alt] involves a multiplicative influence of incentive value and either spatial certainty or validity. The Alt RT models were as follows: (1) ~v + iv, (2) ~AME, (3) ~(v * sc) + (sc * iv) + AME, (4) ~(v * sc) + (v * iv) + AME, (5) ~(v * sc) + (v * iv) + (sc * iv) + AME. (c) Observed group mean accuracy plotted as in panel (a). Importantly, the accuracy data show that the RT findings were not due to a speed-accuracy trade-off. BF = Bayes factor, rc = reward condition, AME = all main effects. speed RT by approximately 50 ms ± 3 (SE) for high versus low incentives. Spatial certainty served to increase the effect of cue validity; the difference between valid and invalid trials increased by approximately 90 ms ± 29 (SE) across levels of certainty. Arguing against the Expected Value Hypothesis, the preferred model included only main effects of each factor (incentive value, spatial certainty, and cue validity), and a spatial certainty x cue validity interaction term (BF, relative to the intercept only null model, = 1.74E+58, ± .87%; see Fig. 3b). Importantly, there was positive evidence that this model was preferred over the next best model (BF = 3.8, ± 1.45%), which was identical to the winning model except that it also included an incentive value x spatial certainty interaction term. Therefore, the evidence favours a model that does not include an interaction between incentive value and spatial certainty. cues. This shows that as participants became slower on invalid and low-incentive value trials, they also became less accurate, arguing against the notion that effects were due to a speed accuracy trade-off (see Fig. 3c). The preferred model for these data, relative to the null model, included only main effects of incentive value and cue validity (BF = 8.83E+47, ±.56 %, relative to the null model). Discussion There are two noteworthy findings from Experiment 1. First, even though the optimal strategy in this experiment would be to ignore the spatial cues entirely, especially in blocks with low spatial certainty, clear effects of valid versus invalid cues were found. Second, models posing an additive influence of incentive value and spatial certainty outperform those allowing an interaction between the two. This goes against the Expected Value Hypothesis and suggests that visual selec- tion mechanisms do not integrate incentive value and spatial certainty, even when approaching the limits of certainty. Nevertheless, the second-best model to account for the RT 2 For a demonstration that participants were exposed to a sufficient range of the reward decay function to identify the reward structure, we refer the reader to the Online Supplementary Material: https://github.com/kel-github/ attention-value-certainty/blob/master/code-analysis-and-task/Supplement_ IncentiveValueAndSpatialCertainty.pdf Participants We calculated the stopping rule for data collection as the number of weeks where testing at maximum capacity would bring us over the minimal sample size (3 weeks with 10 peo- ple per week). Of the 28 participants recruited, one was ex- cluded due to technical difficulties with the eye tracker. A second participant was excluded as they did not meet the criterion required to terminate the practice. The remaining 26 (mean age = 19.5 years, SD = 1.03, 24 females, 26 right- handed) completed all the study procedures. Two of these participants had also completed Experiment 1. Experiment 2 The aim of Experiment 2 was to test whether the additive influence found in Experiment 1 would persist even when the reward structure of the task renders additivity to be sub- optimal. In Experiment 2, we leveraged the predictions made by the Additive and Expected Value operations to construct such a reward structure. As mentioned above, expected value computations are multiplicative, and should produce super- additive effects when both incentive values and spatial cer- tainty are high, and sub-additive effects when incentive value and spatial certainty are low. Thus, RTs driven by an expected value operation should be faster than those driven by an addi- tive operation when spatial certainty and incentive value are high and slower than an additive operation when spatial cer- tainty and incentive value are low. Therefore, conditions that preferentially reward fast RTs on high-incentive, high- certainty trials and outweigh the costs incurred for slow RTs on low-incentive/low-certainty trials should favour adoption of an expected value strategy over an additive strategy (see Fig. 2d). To produce these reward conditions, in Experiment 2, participants completed the same task as in Experiment 1 (albeit sampling fewer levels of spatial certainty), with an added condition wherein reward value exponentially decayed after target onset.2 Of course, the assumption that these reward conditions favour a multiplicative (i.e. Expected Value) oper- ation only holds if both spatial and incentive cues are lever- aged to bias information processing. For example, an alternate and arguably optimal strategy would be to ignore the spatial cues entirely and to attend only to the high value location. However, we know from Experiment 1 that both cues effec- tively influence performance, making safe the assumption that decaying reward conditions favour a multiplicative over an additive operation. Accuracy The probability of an accurate response on invalid trials was .16 ±.02 (SE) lower than on valid trials and was .05 ±.02 (SE) less for targets appearing in low- versus high-incentive value Atten Percept Psychophys Replicating the finding from Experiment 1, evidence sup- ports the Additive Hypothesis but not the Expected Value Hypothesis. data included an interaction between spatial certainty and in- centive value, suggesting that this interaction is not entirely implausible. Perhaps additive effects would fail to provide the best description of the data if another form of appropriate pressure was applied to visual selection. Indeed, previous studies show that learning can direct the sampling of sensory information to optimise reward accrual (Drugowitsch et al., 2015; Kiani & Shadlen, 2009; Serences, 2008), allowing the possibility that a reward structure favouring an expected value operation may be sufficient to modulate the additive influence of incentive value and spatial certainty. The aim of Experiment 2 was to provide this test. Apparatus In addition to that used for Experiment 1, an EyelinkⓇ1000 desktop-mounted eye-tracker (SR Research Ltd., Ottawa, Ontario, Canada) recorded movements of the left eye with a sampling frequency of 500 Hz. This was used to ensure that eye movements were not contributing to results, even though participants were clearly instructed not to move their eyes (replicating instructions used in Experiment 1, which were not, however, verified for compliance). Stimuli The stimuli were the same as in Experiment 1, except that the value cues were presented at 5.7 . This change was made to match the exact layout used in previous work (Stankevich & Geng, 2014). Response time Group mean RT data are shown in Fig. 4a. Correct responses were approximately 21 ms ± 17 (SE) slower for targets appearing in low- versus high-incentive value placeholders; valid versus invalid spatial cues speeded RTs on average by 33 ms ±12 (SE); and the decay-reward condition speeded RTs by 54 ms ±17 (SE) on average compared to the fixed-reward condition. Unlike Experiment 1, there was no detectable in- fluence of spatial certainty. (Note: We tested whether this could be attributed to a reduced sensitivity owing to the fewer levels of spatial certainty used in Experiment 2 relative to Experiment 1, by taking the data from Experiment 1 and extracting only the levels of spatial certainty that were used in Experiment 2). A repeated-measures ANOVA (incentive value x spatial certainty x cue validity) showed a significant spatial certainty x cue interaction (F(1, 20) = 4.84, MSE = .001, p = .047), suggesting that reducing the levels of spatial certainty is not sufficient to reduce detectability of such an interaction). As in Experiment 1, we identified the most likely model given the data and found that the winning model in- cluded main effects of cue validity, incentive value, and re- ward condition (BF = 2.18E+67 ±.69 %, relative to null mod- el), but did not include an influence of spatial certainty (al- though see Accuracy data). There was good evidence that this was the best model for the data, as it was positively preferred to the next best model, which included an additional incentive value x cue validity interaction term (BF = 4.65 ±2.43 %, see Fig. 4b). As spatial certainty was found to interact with cue validity in Experiment 1, we tested the evidence for the win- ning model against one that also included a spatial certainty x cue validity interaction term. Again, there was positive evi- dence that the winning model provided a better fit to the data (BF = 8.97 ±1.79%). Collectively, the results show that even when an additive operation is disadvantageous, an additive model is still a better account of the data. Participants completed 200 trials for each of four spatial certainty/reward contingency conditions (.29/fixed, .29/decay, .029/fixed, .029/decay; block order was counterbalanced across participants). Response time Although trial number per condition was substantially lower than in Experiment 1, adjustments to changes in spatial certainty in other cuing studies have shown clear effects with less than half the trial numbers we used here (Prinzmetal et al., 2015; Vossel et al., 2015). We included only these two levels of spatial certainty as we wanted to avoid any possible floor or ceiling effects when testing the influence of reward condition. We also tested the separate hypothesis that individuals may mentally represent the high- and low-incentive placeholders differently in terms of their relative value, when their value can be obtained more reliably (i.e. in the fixed reward-condi- tion, relative to the decay reward-condition), and that this may be expressed via physical placement on a linear space. Every 50 trials, participants were instructed to use a mouse to drag the two placeholders wherever they liked on a single line. However, we found no evidence that cue-likelihood influ- enced placement of the placeholders (p = .96), and this sepa- rate aspect of the study is discussed no further. Participants also completed a BIS/BAS questionnaire (Carver & White, 1994) that was used to test a hypothesis for a separate study not reported here. 1) ~(rc x iv) + v + iv + rc, 2) ~AME, 3) ~(rc x v) + iv + v + rc, 4) ~(v x sc) + AME, 5) (v x iv) + v + iv + rc. BF = Bayes factor, v = cue-validity, sc = spatial certainty, iv = incentive value, rc = reward condition, AME = all main effects Results condition (see below), and assuming ~85% accuracy, this provides ~136 rewarded trials per condition. Previous studies suggest that participants can detect dynamics in incentive value associations with far fewer trials (N=8, Ittipuripat et al., 2015) or lower reward-to-non-rewarded ratios (33:77%, Serences, 2008), and that these detections influence visual spatial priorities. In the decay reward- condition, an exponential decay function (reward value = points*(e-4*RT), RT = Response Time) was applied to each value at target onset. The monetary value of points was adjusted so that participants received the same rate of cash payments as Experiment 1 (100,000 = £1). Participants were informed at the start of the decay blocks that the value available to them would begin to run out upon appearance of the target. Procedure The procedure was the same as Experiment 1 with the follow- ing exceptions. Participants’ eyes were monitored on every trial. Calibration and validation were performed every 25 trials to minimise drift errors. If the participant’s eyes moved more than 50 pixels (1.5 ) from the fixation at cue-offset, text ap- peared to notify participants they had been “too fast”. The trial was then terminated. Terminated trials accounted for ~3% of all trials. Cue-values were increased from Experiment 1 to 5,000 versus 100 points, so that participants could gain at least 1 point when a decay was applied to the low incentive val- ue. Correct responses were rewarded on 80% of trials as in Experiment 1. Given that there were 200 trials per Atten Percept Psychophys Accuracy (a) Observed group mean RTs in ms for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels) for each reward condition. Vertical evidence for the winning model. Any values lower than 1 (dotted li support P[Alt]. The larger the BF value, the stronger the evidence for winning model. Any values lower than 1 (dotted line) support P[Alt]; values between 3 (dashed line) and 10 constitute moderate evidence Fig. 4 Response time (RT) Experiment 2. (a) Observed group mean RTs in ms for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels) for each reward condition. Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model. The winning model involved main effects of incentive value, cue validity and reward condition. (b) BFs for the probability of the winning RT model (P[Win]: v + rc + iv), relative to the five next best models (Alternative, P[Alt], models, y-axis). The larger the BF value, the stronger evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]. The larger the BF value, the stronger the evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]; BF values between 3 (dashed line) and 10 constitute moderate evidence for the winning model, values > 10 provide strong evidence. Dark bars indi- cate P[Alt] only contains an additive influence of incentive value; light bars indicate P[Alt] involves a multiplicative influence of incentive value and either spatial certainty or validity. The Alt RT models were as follows: evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]. The larger the BF value, the stronger the evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]; BF values between 3 (dashed line) and 10 constitute moderate evidence for the winning model, values > 10 provide strong evidence. Dark bars indi- cate P[Alt] only contains an additive influence of incentive value; light bars indicate P[Alt] involves a multiplicative influence of incentive value and either spatial certainty or validity. Accuracy The Alt RT models were as follows: trivial to compute (i.e., very high certainty) might best reveal non-additive effects, because these conditions should be min- imally taxing to central resources and thus be more likely to enable an influence of incentive value on visual selection. We created this condition by using very high spatial-certainty cues and then pitted incentive value and spatial certainty against each other in a spatial-orienting task, where endogenous cues signalled the likely location of upcoming letter targets. Interestingly, an additive influence of incentive value and spa- tial certainty was observed, even under conditions of very high certainty. Spatial certainty increased the size of the cueing-effect (i.e. the difference in performance between in- validly and validly cued trials), whereas incentive value had a comparable influence on both valid and invalid trial types, across all levels of spatial certainty. responses to low-value targets. Accuracy was higher for the fixed relative to the decay reward condition (.05, ± .008 (SE)), suggesting that as participants became faster in the delayed reward condition, they also produced a very modest decrease in accuracy. As in Experiment 1, the accuracy data obtained here demonstrate that the validity and incentive value results were not due to a speed-accuracy trade off, whereas the influ- ence of reward condition may indeed be due to such a trade- off (see Fig. 5). The best-fitting model to account for these data contained a cue-validity x spatial certainty interaction, and main effects of incentive value, spatial certainty and cue validity. Accuracy We followed the same data-cleaning procedures as in Experiment 1. Again, piecewise linear functions were fitted to the data to isolate the trials contaminated by spatial certainty learning effects. The number of trials removed from the start of each block were similar to Experiment 1 (mean = 14.7, SD 8.5). Differences in accuracy between valid and invalid trials grew larger as spatial certainty increased (by .15 on average, ±.07 (SE)), suggesting that as participants became slower on inva- lid trials, they also became less accurate. Furthermore, accu- racy performance was slightly higher when targets appeared in high- relative to low-incentive value placeholders (by approx- imately .0003, ± .0001 (SE)), showing that participants be- came very modestly less accurate as they slowed their We also used the same model comparison approach, with the exception that we added the reward condition term to the linear mixed effects models that were fit to the data. Atten Percept Psychophys Fig. 4 Response time (RT) Experiment 2. (a) Observed group mean RTs in ms for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels) for each reward condition. Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model. The winning model involved main effects of incentive value, cue validity and reward condition. (b) BFs for the probability of the winning RT model (P[Win]: v + rc + iv), relative to the five next best models (Alternative, P[Alt], models, y-axis). The larger the BF value, the stronger evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]. The larger the BF value, the stronger the evidence for the winning model. Any values lower than 1 (dotted line) support P[Alt]; BF values between 3 (dashed line) and 10 constitute moderate evidence for the winning model, values > 10 provide strong evidence. Dark bars indi- cate P[Alt] only contains an additive influence of incentive value; light bars indicate P[Alt] involves a multiplicative influence of incentive value and either spatial certainty or validity. The Alt RT models were as follows: Fig. 4 Response time (RT) Experiment 2. General discussion This interpretation predicts that the degree to which incentive value or spatial certainty can influence performance is dependent upon the range over which preparatory processes can be titrated and still yield acceptable performances. This would account for why, in Experiment 2, under a context with greater pressure on RT performance, we observed an influ- ence of cue validity but not spatial certainty. Presumably, it had become too costly to modulate RT performance by spatial certainty and meet the perceived demands of the task. high-incentive value/high-certainty conditions would out- weigh losses incurred by slower RTs under low-incentive value/low-certainty conditions, relative to RTs predicted by an additive model. Although the influence of spatial certainty manifested differently to Experiment 1, i.e., by modulating accuracy, rather than RT, we observed that the effect of incen- tive value remained additive to spatial certainty and to other experimental factors. Again, the findings support the Additive Hypothesis. However, this interpretation depends on the as- sumption that participants were able to infer the relationship between RT and rewards. Although participants were both explicitly informed about the decaying reward and were ex- posed to a sufficient range of the function to theoretically be able to infer the relationship between RT and rewards, an explicit measure of the participant’s understanding of the RT/reward relationship should be taken in future studies to verify this assumption. What kind of mechanism or computation could result in a robust additive influence between incentive value and spatial certainty? In concert with recent theoretical and empirical de- velopments suggesting that cognitive control processes are off- set by subjective and computational costs of effortful control (Braver, 2012; Yee & Braver, 2018), we believe the current data can be interpreted as reflecting trial-by-trial adaptations aimed at the conservation of effort. If we assume that the main- tenance of the task set, i.e., a priori preparedness to identify a data-limited target at two locations, requires energetic resources from the underlying selection mechanism, it is of benefit to the brain to predict conditions where effort can be relaxed, in order to conserve energy expenditure. For example, by learning the energetic range over which target identification mechanisms can be adjusted, to ensure good enough target detection, given the task parameters. According to this view, a cost-benefit anal- ysis could inform how much energy could be saved, given an acceptable decrement to accuracy and RT. General discussion In Experiment 2, we reasoned that if an expected value operation can bias visual selection, then a reward structure that favours a multiplicative weighting of incentive value and spatial certainty may reveal it. We applied an exponential decay function to incentive values at target onset; this ensured that if RTs were driven by multiplicative as opposed to addi- tive weighting, then reward gains accrued by faster RTs under Over two experiments we tested whether the Additive Hypothesis would outperform the expected value account, even under conditions expected to challenge the optimality of additivity. In Experiment 1, we hypothesised that if incen- tive value and spatial certainty influence a common underly- ing mechanism, then conditions wherein spatial certainty is Atten Percept Psychophys Fig. 5 Accuracy data for Experiment 2. Accuracy (Acc) for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels) for each reward condition. Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model Fig. 5 Accuracy data for Experiment 2. Accuracy (Acc) for targets appearing in high- (dark circles) or low- (light circles) value placeholders plotted as a function of spatial certainty (x-axis) for valid and invalid spatial cues (panels) for each reward condition. Vertical lines indicate ± 1 within-subject SE. Lines represent fit of the winning model steady level of task preparation favouring the high-value lo- cation, for example, by increasing the excitability of neuronal assemblies whose collective receptive fields correspond to detecting lines or edges at that location (Carrasco, 2011; Desimone & Duncan, 1995; Roelfsema et al., 2000; Schmitz & Duncan, 2018), thereby biasing the system towards a stron- ger response to the upcoming stimulus (Buschman & Kastner, 2015). Concurrently, the excitability of neuronal assemblies directed towards encoding information from the low-value location should be relaxed, as the cost of sometimes missing the target at that location, given the energy needed to detect it, should become negligible. Similarly, upon spatial cue onset, preparation of such target detection mechanisms could be fur- ther relaxed for the unlikely location, proportionally to how unlikely that location is to possess a target. Importantly, this is performed incrementally to the previous adjustment. This in- terpretation suggests that the system incrementally updates cost-efficient sensory encoding on the basis of incoming in- formation. General discussion We inspected the previous literature and observed that this is a consistent finding across other studies that varied the probabilities of explicit spatial cues in comparable tasks (Lanthier et al., 2015; Vossel et al., 2006), suggesting that this is a replicable phenomenon, rather than the consequence of a ceiling effect or something similar. In contrast, Stankevich and Geng (2014) observed the opposite: RTs decreased as target location became more likely (analogous to valid trials) but remained invariant when directed towards increasingly less likely (invalid) locations. Therefore, our results suggest that the explicit spatial cue we used resulted in preparation towards the cued location that did not vary with the certainty offered by the cue, coupled with a relaxation of preparation towards the invalid location that scaled with certainty. In con- trast, non-explicit spatial knowledge appears to cause a strengthening of preparation towards the more likely location, with no concomitant relaxation towards the unlikely location. This suggests that spatial certainty influences visual selection differently dependent on how it is learned. For example, a long history of arrows serving as useful directional cues could motivate a strong response to the directional stimulus, against which other useful behaviours can be adapted. Therefore, it appears that visual selection behaviours adapt to environmen- tal information in relation to the most contextually relevant baseline behaviour. An alternate, but not mutually exclusive, reason for the independence of incentive value and spatial certainty may be that the two are governed by different learning systems; for example, recent investigations measuring overt eye move- ments, rather than covert attention shifts, found that incentive values induce associative behaviours (Kim & Anderson, 2019; i.e. participants look at the value cue even when paid not to; Le Pelley et al., 2015), whereas spatial cues may be learned via instrumental conditioning (i.e. participants are more likely to repeat an orienting behaviour that has been reinforced; Kim & Anderson, 2019). Indeed, evidence that visual selection biases remain present when explicit spatial cues (i.e. arrows or words) are non-predictive suggests that these behaviours may be under the control of the habitual learning system (Jiang, 2018; Yin & Knowlton, 2006), which governs learned stimulus-response contingencies. Moreover, in the current experiments, we have taken a snapshot of the relationship between incentive value and spatial certainty at one time point (400 ms stimulus-onset asynchrony (SOA)), potentially when the system is still in a state of updating. General discussion The current findings shed further insights into the previous- ly proposed unitary selection mechanism that biases competi- tion between cortical representations of stimuli, in the pres- ence of both incentive and explicit spatial cues (Stănişor et al., 2013). These authors showed that overlapping clusters of ma- caque V1 neurons were sensitive to both incentive value cues and 100% valid spatial cues. The authors proposed that the explicit spatial cue served to re-weight the relative value be- tween the target and distractor, and that this re-weighting was instantiated by a unitary selection mechanism that computes Applied to the current context, after the onset of the incen- tive value cues, selection mechanisms should maintain a Atten Percept Psychophys information from each cue has undergone further processing. This is a possible avenue of further investigation in future studies. the relative expected value between targets. The current study shows that the spatial certainty derived from explicit cues does not contribute to an expected value operation with the previ- ous incentive values to re-weight the relative value between the two items. Rather, an additive influence points to the re- peated invocation of a selection mechanism on the basis of updates from separable information sources. As incentive val- ue and spatial certainty appear to have been added, rather than multiplied, to the existing output of the selection mechanism, the two sources of information may be transformed into a common representational space, or unit, prior to the enactment of their influence.3 An additive influence of incentive value on visual selection was also observed by Stankevich and Geng (2014), when value was pitted against the varying probability that a target would appear on one side versus the other, in the absence of explicit spatial cues. A visual comparison of the current data and the data from Stankevich and Geng (2014) also yields some interesting points of difference concerning the influence of spatial certainty in the presence or absence of explicit spa- tial cues. With the current explicit cues, we observe RTs that are comparable across spatial-certainty conditions for valid cues, whereas RTs on invalid trials increase as spatial certainty decreases. 3 An alternate explanation for the observed additive relationship between spa- tial certainty and incentive value on RT performance is that an expected value operation is being performed by summation on a logarithmic scale. For exam- ple, the expected utility of the scene (E(U)) could be computed as follows: General discussion It is possible that a multiplicative relationship between spatial cer- tainty and incentive value emerges later in time, once the E U ð Þ ¼ SC T ð Þ*IV T ð Þ ð Þ þ SC D ð Þ*IV D ð Þ ð Þ ð3Þ Conclusions and Over two experiments, we sought to arbitrate between com- peting theories for how learned associations pertaining to in- centive value and spatial certainty combine to influence visual selection. Specifically, we asked whether this influence was additive (Additive hypothesis), or multiplicative (Expected Value hypothesis). We tested these hypotheses by pitting in- centive values and spatial certainties against one another in a spatial cueing task under conditions expected to challenge the optimality of an additive operation. The data from two exper- iments support the notion that visual selection mechanisms show independent sensitivity to incentive value and spatial- E0 U ð Þ ¼ log E U ð Þ ð4Þ E0 U ð Þ ¼ log E U ð Þ E0 U ð Þ ¼ log E U ð Þ where SC is spatial certainty, IV is incentive value, T is the target location and D is the distractor location. In this case, if the distractor location’s priority was assigned a value of zero, E(U) would reduce to log(SC(T)) + log(IV(T)), i.e., an additive relationship between spatial certainty and incentive value. However, such an account hinges on the notion that the distractor location is somehow assigned a value of zero. This interpreta- tion seems unlikely given that the distractor location has value, and can never be fully ruled out as a potential target location. Moreover, such a computation should produce logarithmic, and not linear, relationships between spatial certainty and RTs, when the latter was observed across both Experiment 1 and Experiment 2. where SC is spatial certainty, IV is incentive value, T is the target location and D is the distractor location. In this case, if the distractor location’s priority was assigned a value of zero, E(U) would reduce to log(SC(T)) + log(IV(T)), i.e., an additive relationship between spatial certainty and incentive value. However, such an account hinges on the notion that the distractor location is somehow assigned a value of zero. This interpreta- tion seems unlikely given that the distractor location has value, and can never be fully ruled out as a potential target location. Moreover, such a computation should produce logarithmic, and not linear, relationships between spatial certainty and RTs, when the latter was observed across both Experiment 1 and Experiment 2. E U ð Þ ¼ SC T ð Þ*IV T ð Þ ð Þ þ SC D ð Þ*IV D ð Þ ð Þ Atten Percept Psychophys Button, K. S., Ioannidis, J. P. A., Mokrysz, C., Nosek, B. A., Flint, J., Robinson, E. S. J., & Munafò, M. R. (2013). Power failure: why small sample size undermines the reliability of neuroscience. Nature Reviews. Neuroscience, 14(5), 365–376. https://doi.org/10.1038/ nrn3475 certainty information, and that both information sources are converted to a common representational space, or unit, in order to influence visual selection. We also interpret our re- sults as suggesting that the mechanisms underpinning visual selection dynamically leverage distinct information sources to reflexively conserve effort within a range that allows accept- able performance given the current task parameters. Carrasco, M. (2011). Visual attention: the past 25 years. Vision Research, 51(13), 1484–1525. https://doi.org/10.1016/j.visres.2011.04.012 Carver, C. S., & White, T. L. (1994). Behavioral inhibition, behavioral activation, and affective responses to impending reward and punish- ment: The BIS/BAS Scales. Journal of Personality and Social Psychology, 67(2), 319. https://doi.org/10.1037/0022-3514.67.2. 319 Acknowledgements K.G. Garner would like to acknowledge Christopher Nolan, Andrew Clouter and Dragan Rangelov for their com- ments and insightful discussions about this work. This project was sup- ported by grant ES/L000210/1 from ESRC to J Raymond. K.G. Garner is also supported by a Marie Sklodowska Curie Global Fellowship. The authors declare no competing interests. Chelazzi, L., Eštočinová, J., Calletti, R., Lo Gerfo, E., Sani, I., Della Libera, C., & Santandrea, E. (2014). Altering spatial priority maps via reward-based learning. 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Towards quantitative root hydraulic phenotyping: novel mathematical functions to calculate plant-scale hydraulic parameters from root system functional and structural traits
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J. Math. Biol. (2017) 75:1133–1170 DOI 10.1007/s00285-017-1111-z J. Math. Biol. (2017) 75:1133–1170 DOI 10.1007/s00285-017-1111-z Mathematical Biology B F. Meunier felicien.meunier@uclouvain.be This work was funded by the “Fonds National de la recherche scientifique” (FNRS) of Belgium that supports Félicien Meunier as research fellow. Valentin Couvreur was supported by the Belgian American Educational Foundation (BAEF) as UCLouvain Fellow, by Wallonie-Bruxelles International (WBI) with a WBI.WORLD grant, and by the Fonds Spciaux de Recherche (FSR) of the Université catholique de Louvain. The following notation has been used all over the text: T, L, P and M stand for time, length, pressure and mass unit, respectively. 1 Earth and Life Institute-Environment, Université catholique de Louvain, Louvain-la-Neuve, Belgium Abbreviations RWU Root water uptake RS Root system HA Hydraulic architecture SUF Standard uptake fraction SUD Standard uptake density Towards quantitative root hydraulic phenotyping: novel mathematical functions to calculate plant-scale hydraulic parameters from root system functional and structural traits F. Meunier1 · V. Couvreur2,3 · X. Draye3 · J. Vanderborght4,5 · M. Javaux1,4 Received: 5 July 2016 / Revised: 12 February 2017 / Published online: 2 March 2017 © The Author(s) 2017. This article is published with open access at Springerlink.com Abstract Predicting root water uptake and plant transpiration is crucial for managing plant irrigation and developing drought-tolerant root system ideotypes (i.e. ideal root systems). Today, three-dimensional structural functional models exist, which allows solving the water flow equation in the soil and in the root systems under transient conditions and in heterogeneous soils. Yet, these models rely on the full represen- tation of the three-dimensional distribution of the root hydraulic properties, which is not always easy to access. Recently, new models able to represent this complex system without the full knowledge of the plant 3D hydraulic architecture and with a limited number of parameters have been developed. However, the estimation of the macroscopic parameters a priori still requires a numerical model and the knowledge of the full three-dimensional hydraulic architecture. The objective of this study is to 1 Earth and Life Institute-Environment, Université catholique de Louvain, Louvain-la-Neuve, Belgium 1 Earth and Life Institute-Environment, Université catholique de Louvain, Louvain-la-Neuve, Belgium 2 Department of Land, Air and Water Resources, University of California, Davis, CA, USA 3 Earth and Life Institute-Agronomy, Université catholique de Louvain, Louvain-la-Neuve, Belgium 4 Forschungszentrum Jülich GmbH, Agrosphere (IBG-3), Jülich, Germany 5 Division of Soil and Water Management, KU Leuven, Leuven, Belgium 123 3 1134 F. Meunier et al. provide analytical mathematical models to estimate the values of these parameters as a function of local plant general features, like the distance between laterals, the number of primaries or the ratio of radial to axial root conductances. Such functions would allow one to characterize the behaviour of a root system (as characterized by its macroscopic parameters) directly from averaged plant root traits, thereby opening new possibilities for developing quantitative ideotypes, by linking plant scale parameters to mean functional or structural properties. With its simple form, the proposed model offers the chance to perform sensitivity and optimization analyses as presented in this study. Keywords Root water uptake · Plant-scale parameters · Hydraulic architecture · Water flow equation in root Mathematics Subject Classification 92C80 · 92B05 Mathematics Subject Classification 92C80 · 92B05 1 Introduction Plant transpiration is the process by which water molecules are evaporated from leaf surfaces. When stomatas open, carbon dioxide is taken up while water vapour is lost at a certain rate function of the atmospheric evaporative demand, leaf properties and stomatal aperture (Lobet et al. 2014). Root systems (RS) play a key role by providing water from the soil, which sustains the transpiration flux (McElrone et al. 2013) and by sending hydraulic and hormonal signals to regulate stomata conductance (Tardieu et al. 2015). Location of roots within the soil, connection between root segments but also root and soil hydraulic resistance distribution determine the ability of a root system to acquire and transport water. Thus, not only the RS architecture (de Dorlodot et al. 2007) but also the RS hydraulics (Vadez 2014) influence the ability of plants to extract and transport soil water to the leaves. It has been shown that RS architecture and hydraulics are determinant under drought (Leitner et al. 2014b; Tardieu 2012). Therefore, researchers have attempted to search for ideal structural and functional characteristics of root systems, which would help plant RS to explore soil and to provide water to the plant in an optimal way (Wasson et al. 2012; Lynch 2013). High plant conductivity, steep secondary roots, long roots, high radial conductivity, low xylem have for instance been proposed as potential optimal features (Comas et al. 2013). However, these features may not be optimal under all climatic and environmental conditions (Tardieu 2012; Leitner et al. 2014b). In addition, quantitative approaches are missing to properly evaluate how these proposed features would affect the plant ability to extract water. 1135 Towards quantitative root hydraulic phenotyping... Since the first studies of Dixon and Joly (1895) more than a century ago, the understanding of the sap ascent in plant has evolved until the establishment of the cohesion-tension theory (Steudle 2001; Wheeler and Stroock 2008). The water trans- port in xylem vessels is a mainly passive process opposed to the potential hydraulic gradient between the soil and the atmosphere and the continuity of the water phase is ensured (as long as cavitation does not appear) by tension-cohesion forces. An old and widely accepted model for plant water flow uses the Ohm law analogy, where the plant system is represented as a network of connected hydraulic resistances (van den Honert 1948). 1 Introduction The Hydraulic architecture (HA) has been proposed by Zimmermann (1978) as a conceptual framework to represent roots as a connected network, whose structure (connection between root segments) and function (radial and axial hydraulic resistances) define its properties. Although this concept was initially meant to describe above ground parts of the plant and in particular the sensitivity of xylem to embolism (Tyree and Ewers 1991) later the notion was more specifically developed for RS (Root hydraulic architecture or RHA) by Doussan et al. (1998a) and by Couvreur et al. (2012), amongst others (see for example the work of Cowan 1965). Several mathematical solutions of the flow equation in a complex connected system have been developed through years. Landsberg and Fowkes (1978) developed the first analytical solution for water flow in a homogeneous single root. They also proposed a solution when regularly spaced laterals are included on a hydraulically-homogeneous root branch. However, the solution does not apply to the case of hydraulic properties varying along the root and an exact solution of the second case can be found without incorporating the laterals on the primary branch. Biondini (2008) and Roose and Schnepf (2008) also developed analytical solutions for continuous root (i.e. without segmenting the roots into small homogeneous root parts called segments) including second order roots (and higher) but they did not consider the resistance to flow along the main axis between the laterals and/or they did not include possible changes of hydraulic properties along a root branch. Recently, Couvreur et al. (2012) developed a RS-scale model for solving water flow in RHA. In this approach, RHA is characterized with only three macroscopic (i.e. plant-scale) parameters: (1) the global hydraulic conductance of the root system (Krs [L3T−1P−1]), (2) theStandardUptakeFractions (SUF[−]), or relativedistribution of water uptake under uniform soil water potential, and (3) the compensatory root water uptake conductance (Kcomp [L3T−1P−1]). The parameter Krs defines how easily water flows through a root system. The larger the conductance, the lower the pressure drop inside the root system. The SUF provides weighing factors to obtain the equivalent soil water potential sensed by the plant (called Hs,eq [P]) by averaging the distributed soil water potentials. The third plant-scale parameter Kcomp controls the redistribution of root water uptake (RWU) in non-uniform soil water potential conditions (i.e. process known as compensation, or compensatory RWU, see Jarvis 2011 or Javaux et al. 2013). 1 Introduction The advantage of this approach is that it does not rely on an explicit consideration of the RS architecture and reduces the RHA characteristric parameters to three, while keeping an exact representation of the water flow physical principles (Couvreur et al. 2012). Moreover, these macroscopic root system hydraulic parameters can be used in coupled soil–plant models (such as models of de Jong van Lier et al. 2013 or Javaux et al. 2008) to simulate the water uptake for contrasted soil and climate conditions. 12 3 3 3 1136 F. Meunier et al. Leitner et al. (2014b) indeed showed that optimal root traits depend on soil and climate properties. Leitner et al. (2014b) indeed showed that optimal root traits depend on soil and climate properties. In a context of global climate change and expected water limitations on crop yield (Cattivelli et al. 2008), identifying key factors affecting crop water productivity is essential (Volpe et al. 2013; Passioura 2006). Root systems, with their large natural variability seem to be good candidates for crop plant improvement. Developing plant- scale indices, which quantify the ability of a RS to extract water from heterogeneous soil is a key step in the search of optimal plants under dry conditions. However, no quantitativemodelexistsyettolinktheRShydraulicparameterstolocalrootfunctional and structural properties (i.e. root angle, distance between laterals, local hydraulic resistances, etc.). Our objective is thus to provide novel mathematical models linking local root structural and functional traits to plant-scale properties of RS: Krs, SUF and Kcomp. In this study, we will focus on the first two parameters after demonstrating that Kcomp is equal to Krs under certain conditions. p To achieve this goal, the following methodology will be followed: (1) to review, adapt existing or develop new mathematical solutions of the flow equations explicitly accounting for specific local root traits in simple RS with increasing degrees of com- plexity; (2) to extract the upscaled hydraulic parameters from these solutions in order to (3) investigate how root traits impact them. The structure of the paper is the following: first we develop the theory of our model with mathematical tools applied to RS with increasing complexity. We then illustrate the model functioning with two applications: the first one uses the model to derive root hydraulic parameters from experimental data; the second is theoretical and analyses the sensitivity of the macroscopic parameters of a mature maize RS to local traits. 1 Introduction Finally, we demonstrate and discuss the application of the model in quantitative phenotyping. 2.1 Root water flow model 2.1.1 Water flow equation 2.1.1 Water flow equation Two local hydraulic properties characterize any root or root zone: its hydraulic radial conductivity kr  LT−1P−1 and its axial intrinsic conductance kx  L4T−1P−1 . Here andeverywhereweassumeauniquevalueforrootradialconductivityinbothdirections (in- and outflows). Mass conservation for a segment of length dz [L] writes Mass conservation for a segment of length dz [L] writes d Jx dz = 2πrqr (1) (1) where Jx [L3T−1] is the volumetric flow rate, r [L] is the root radius and qr [LT−1] the radial flux. We call Jr [L3T−1] the radial volumetric flow given by: Jr = 2πrqrdz (2) (2) Jr = 2πrqrdz 12 123 123 Towards quantitative root hydraulic phenotyping... 1137 The axial volumetric flow rate is powered by the gradient of water xylem potential Hx [P] (defined as the sum of the gravitational and the pressure potentials): Jx = −kx d Hx dz (3) (3) The water radial flux comes from the water potential difference between the root xylem potential and the soil–root interface Hsr [P]: qr = kr (Hsr −Hx) (4) (4) Combining Eqs. (1), (3) and (4) we obtain the water flow equation in roots: d dz  −kx d Hx dz  = 2πrkr (Hsr −Hx) This equation can be solved given that two boundary conditions are provided and the root hydraulic properties are known. The solution takes the form of a continuous function of the xylem potential which leads to the axial and radial root water flows. The flow can then be used to derive the root macroscopic parameters described in the next section. 2.1.2 Macroscopic parameters Couvreur et al. (2012) model provides a solution based on three parameters to represent water flow in any complex RS. These three parameters are the root system conductance or Krs [L3P−1T−1], the Standard Uptake Fraction or SUF [−] and the compensatory water uptake conductance or Kcomp [L3T−1P−1]. This physically-based model of the water flow in root systems can be summarized with two main equations. The first one is the segment water uptake which is given by the sum of two processes: the water uptake in homogeneous soil conditions and the compensatory water uptake that occurs in heterogeneous conditions: Jr = Tact SU F + Kcomp  Hsr(z) −Hs,eq  SU F (5) (5) with Jr [L3T−1] the radial flow, Tact [L3T−1] the actual transpiration, Hsr(z) [P] the soil–root interface potential and Hs,eq [P] the soil equivalent potential felt by the plant defined as the soil–root interface potential weighted by the standard uptake fraction. The second equation describes the relation between the actual transpiration and the collar potential Hcollar [P]: Tact = Krs  Hs,eq −Hcollar  (6) (6) What we learn from such a model is that we can simulate accurately the water uptake in any condition of any root system once we know the macroscopic parameters. As What we learn from such a model is that we can simulate accurately the water uptake in any condition of any root system once we know the macroscopic parameters. As 12 3 1138 F. Meunier et al. shown by the authors, these plant-scale variables can be calculated numerically. Let us first detail what they exactly mean and how we can calculate them. shown by the authors, these plant-scale variables can be calculated numerically. Let us first detail what they exactly mean and how we can calculate them. The first plant-scale macroscopic parameter is the global conductance of the RS Krs. It represents the ability of a root system to uptake water to sustain the transpiratio 3 1 The first plant-scale macroscopic parameter is the global conductance of the RS, Krs. It represents the ability of a root system to uptake water to sustain the transpiration Tact [L3T−1] under a specific difference between the soil water status and the xylem plant potential and can be calculated in homogeneous soil conditions. We fix the collar potential and impose a uniform soil–root interface. 2.1.2 Macroscopic parameters In such a case, Hsr(z) = Hs,eq and the conductance can be obtained calculating the actual transpiration that the root system can deliver: Eq. (6) yields: Krs = Tact  Hs,eq −Hcollar  (7) (7) Note that the root system conductance can also be calculated using Thevenin the- orem (1883). Note that the root system conductance can also be calculated using Thevenin the- orem (1883). The second macroscopic root system parameter is the Standard Uptake Fraction SUF or the normalized water uptake under uniform conditions. By definition, SUF is the radial flow entering each root segment divided by the collar root flow under homo- geneous soil conditions. Again it can be calculated solving the water flow problem in uniform soil conditions, Eq. (5) yields: SU F = Jr Tact SU F = Jr Tact In continuous domains, we can define a new parameter characterizing the distri- bution of water uptake rate densities under uniform soil water potential distribution: SUD [L−1]) for Standard Uptake Density. SUD is directly related to SUF through the segment length lseg [L]: SU D = SU F lseg SU D = SU F lseg By definition, the integration of SUD over the total root length yields 1. Mathemat- ically, SUD is defined as: SU D = 2πrqr Tact SU D = 2πrqr Tact The third parameter is Kcomp, the compensatory conductance. It characterizes the ability of a plant to compensate, i.e. taking up more water in regions where it is more available (i.e. under heterogeneous soil conditions). It must be calculated under heterogeneous soil conditions. If the collar flow is null, the root water uptake/release is due to the heterogeneous soil–root potential only and controlled by Kcomp. In this case, Eq. (5) yields (Tact = 0): Jr = Kcomp  Hsr(z) −Hs,eq  SU F 123 123 1139 Towards quantitative root hydraulic phenotyping... The definition of the equivalent soil water potential slightly changes in continuous domains: Hs,eq =  lroot 0 Hsr(z)SUD(z, lroot)dz. The equivalent potential sensed by the plant is now obtained integrating the soil–root interface potential Hsr(z) [P] weighted by the Standard Uptake Density. The integration domain in the case of a single root is between 0 and the root length lroot [L] as shown here or in case of complex root systems it is performed on the whole root system length. 2.2 Single homogeneous root Let us consider a simple RS made of a single root branch with uniform hydraulic properties. The RS has a radius r and a length lroot [L] and is oriented along the z- axis (a list of the principal symbols can be found in “Appendix 1”). z is defined as the position along the root, zero at the root tip, positive towards the plant collar by convention. Let us assume that the water potential at the soil–root interface Hsr(z) [P] is uniform and equal to Hsoil [P]. The xylem water potential in the root collar is called Hcollar [P]. This situation is shown in Fig. 1 (left). The analytical solution for water flow in a uniform root was proposed by Landserg and Fowkes (1978) when a constant collar potential and no-flux at the tip are imposed: Hx(z,lroot) = Hsoil + (Hcollar −Hsoil) cosh (τz) cosh (τlroot) Jx(z,lroot) = kxτ (Hsoil −Hcollar) sinh (τz) cosh (τlroot) Jr(z,lroot) = dzkxτ 2 (Hsoil −Hcollar) cosh (τz) cosh (τlroot) where cosh, sinh and tanh are the hyperbolic cosine, sine and tangent functions, respec- tively. We call τ = 2πrkr kx [L−1]. This parameter is similar to α introduced by Alm et al. (1992) and Landsberg and Fowkes (1978). See “Appendix 2” for mathematical details (adapted from Landsberg and Fowkes 1978). Since the equivalent potential in case of uniform soil water potential yields: Hs,eq = Hsoil we obtain for the root conductance Krs(lroot) using its definition (7) (Tact is the axial flow at the root collar, i.e. in z = lroot): we obtain for the root conductance Krs(lroot) using its definition (7) (Tact is the axial flow at the root collar, i.e. in z = lroot): Krs(lroot) Δ= Jx(z = lroot,lroot) Hsoil −Hcollar = kxτ sinh (τlroot) cosh (τlroot) = κtanh(τlroot) (8) (8) with with κ = τkx = 2πrkrkx [L3P−1T−1] (9) κ = τkx = 2πrkrkx [L3P−1T−1] (9) (9) Interestingly in case of non-limiting xylem conductance and/or small roots Interestingly in case of non-limiting xylem conductance and/or small roots: τlroot << 1 ⇐⇒tanh(τlroot) ≃τlroot 3 1140 F. Meunier et al. Fig. 1 Continuous model for a homogeneous root: Layout of the uniform root simulated (left). The second (center) and third (right) panels illustrate the sensitivity of the Krs macroscopic parameter decreasing the axial intrinsic conductance and radial conductivity, respectively. For details, see text Fig. 123 2.2 Single homogeneous root 1 Continuous model for a homogeneous root: Layout of the uniform root simulated (left). The second (center) and third (right) panels illustrate the sensitivity of the Krs macroscopic parameter decreasing the axial intrinsic conductance and radial conductivity, respectively. For details, see text the global conductance can be approximated by a linear relation: Krs(lroot) ≃2πrkrlroot (10) (10) Inthiscasetheglobalconductanceoftherootisproportionaltothetotalrootsurface: 2πrlroot. In case of very long roots and/or xylem conductance limiting conditions (τlroot >> 1), an asymptotic value κ is reached: Krs(lroot) →κ (11) (11) This asymptotic value of conductance implies that for given local hydraulic prop- erties and water potential difference between the soil and the root collar, there is a maximal flow rate. This suggests that there is an optimal length beyond which the root conductance does not increase significantly. At that point plant marginal interest in investing structural carbon in the root for water acquisition is not significant. Centre and right panels of Fig. 1 illustrate the sensitivity of the global conductance of the uniform root to changes in axial and radial conductivities. In these subplots Eqs. (8), (10) and (11) are plotted as blue solid lines, dark/grey linear relations and dark/grey dashed-dotted horizontal lines, respectively. The dark arrows indicate the decreasing trend of Krs curves when decreasing root hydraulic conductivities (resp. axial and radial conductivities in central and right subplots). Note that for this figure and all other figures in this study we used maize root con- ductivities values obtained by Doussan et al. (1998b) and architectural traits (radius, internodal distances) from RootTyp (Pags et al. 2004) parametrized for maize (Cou- vreur et al. 2012). In particular young segments have been considered for the first figures. Since a maximal flow rate exists for a given water potential difference between the soil and the collar we can define the fraction α [−] of the actual flow to the maximal one. By definition it is the root conductance divided by its asymptotic value. Inversely, 123 Towards quantitative root hydraulic phenotyping... 1141 Fig. 2 l∗(α): Root length necessary to reach two different α fractions of κ as a function of the root local conductivities Fig. 2 l∗(α): Root length necessary to reach two different α fractions of κ as a function of the root loc conductivities we derive the root length l∗needed to reach a specific ratio of the maximal flow. 2.2 Single homogeneous root α(l∗) Δ= κtanh(τl∗) κ = tanh(τl∗) ⇐⇒l∗(α) = atanh(α) τ with atanh the inverse hyperbolic tangent function. This solution allows us to calculate the length necessary to reach a specific conductance such as α = 0.5, or α = 0.99. This length depends on the root radial and axial conductivity, as illustrated in Fig. 2. This equation puts the bases of allometric relations for roots allowing to relate optimal root branch length to their local properties. The second root system parameter is the Standard Uptake Density or SUD. Using its definition we obtain: SU D(z,lroot) Δ= 2πrqr(z,lroot) Krs (Hsoil −Hcollar) = τ cosh (τz) sinh(τlroot) (12) (12) We can also approximate the Standard Uptake Density by a Taylor series (τlroot << 1 ⇒τz << 1, ∀z): We can also approximate the Standard Uptake Density by a Taylor series (τlroot << 1 ⇒τz << 1, ∀z): SU D(z,lroot) ≃ 1 lroot 1 + (τz)2 2 (13) (13) In this case the Taylor approximation of the the distribution of water uptake rate densities is a parabola meaning that the maximal uptake occurs at the proximal part of the uniform root (z = lroot). Under non-limiting xylem conductance conditions though, the SUD is almost uni- form along the root and equals to 1 lroot . We obtain the latter result by neglecting the quadratic dependence of SUD in z: SU D(z,lroot) ≃ 1 lroot 1 + (τz)2 2 ≃ 1 lroot 12 3 1142 F. Meunier et al. Fig. 3 Continuous model for a homogeneous root: sensitivity analysis of the SUD macroscopic parameter according to changes in lroot and τ parameters. For details, see text Fig. 3 Continuous model for a homogeneous root: sensitivity analysis of the SUD macroscopic parameter according to changes in lroot and τ parameters. For details, see text Figure 3 illustrates the uptake rate relative density in standard conditions and its sensitivity to lroot and τ, respectively. In the left panel, the lighter blues solid line are smaller roots. The gray dashed lines are the harmonic approximations predicted from Eq. (13). The smaller the τl product the better the approximation. In the right subplot τ intrinsic property of the root is gradually increased. The smaller the radial to axial conductivity ratio the more homogeneous the water uptake. 2.2 Single homogeneous root Indeed the axial resistance is less and less limiting so that the radial barrier becomes progressively the highest resistance to root water flow. The area below the curve always remains equal to 1. To derive the third macroscopic parameter (Kcomp), new boundary conditions have to be imposed. Indeed this parameter only plays a role in non-uniform soil water potential conditions. We impose now non-uniform soil–root interface potential, no flux at the tip and at the collar, which write: ⎧ ⎨ ⎩ Hsr(z) = αexp (βz) Jx(z = 0,lroot) = 0 Jx(z = lroot,lroot) = 0 ⎧ ⎨ ⎩ Hsr(z) = αexp (βz) Jx(z = 0,lroot) = 0 Jx(z = lroot,lroot) = 0 In this case the solution for the xylem water potential distribution becomes: Hx(z,lroot) = τ 2 τ 2 −β2 αexp (βz) + c1exp (τz) + c2exp (−τz) with c1 and c2 that can be obtained using previously described root boundary conditions:  c1 c2  =  τ −τ τexp(τlroot) −τexp(−τlroot) −1 ⎡ ⎣ −αβτ 2 τ 2−β2 −−αβτ 2 τ 2−β2 exp(βlroot) ⎤ ⎦ 123 12 1143 Towards quantitative root hydraulic phenotyping... Interestingly, we find: Jr SU F τlroot→0 ≃ 2πrkrlroot  Hsr −Hs,eq + c.O (τlroot)  with c a constant depending on the root properties and soil conditions. So that Kcomp is equal to Krs (see Eq. 10) as long as the xylem conductance is non-limiting, as shown numerically by Couvreur et al. (2012). When xylem conductance is limiting, Kcomp cannot be defined analytically in a simple way, but in tested maize and wheat HA a value close to Krs predicts compensatory uptake satisfyingly well (Couvreur et al. 2014). In the following sections we only consider Krs = Kcomp for sake of simplicity but we always can use the previous equation to evaluate this approximation’s accuracy. y p q pp y To summarize this first theoretical section, from the physical equations of water flow in a root cylinder with uniform hydraulic properties (solutions of Landsberg and Fowkes 1978), we derived analytical expressions for the macroscopic root hydraulic parameters of Couvreur et al. (2012) model in terms of local hydraulic properties. These plant-scale parameters depend on both hydraulic (axial and radial conductivi- ties) and geometrical traits (length, radius). 2.2 Single homogeneous root Particularly, two local hydraulic properties determine the macroscopic parameters: κ provides the asymptotic root conductance, and τ determines how quickly a growing root approaches its asymptotic conductance, and how uniform is the water uptake profile along the root. These local properties do not depend on the boundary conditions. They are intrinsic root properties, just like the macroscopic parameters. 2.3 Root with heterogeneous properties We consider now a root cylinder made of several homogeneous root zones. Each zone is characterized by its own hydraulic radial conductivity kr,i and axial conductance kx,i with i varying from one to N the total zone number. To calculate the macroscopic parameters of the entire root we first focus on one of the root zones called generically hereafter i. For all zones except the apical one, the upstream root part can be seen as an initial conductance attached to the considered zone. The root zone under focus has a root length lroot,i and the position zi [L] along the root portion varies between 0 and lroot,i. We define the upstream or distal end of the root zone as the position closest to the root tip (z = 0) and the downstream or proximal end as the end of the root zone (z = lroot,i). , We can define an asymptotic conductance κi and a convergence speed τi for each root zone. Therefore we apply the same methodology than in the previous sections except for the boundary conditions that are different than no flux at the lower end of the root cylinder to solve the water flow equation. For the ith zone, the boundary conditions write:  Hx(zi = lroot,i,lroot,i) = Hx,i Jx(zi = 0,lroot,i) = Ji−1 12 3 3 1144 F. Meunier et al. where the first variable under parentheses represents the location within the ith zone, and the second one, the length of this zone). Hx,i [P] is the proximal pressure head and Ji−1 [L3T−1] is the distal flow. The latter condition is used to simulate the presence of an upstream root cylinder below the current one (distal end). Using the methodology developed in the “Appendix 2” to solve the flow equation, we obtain after simplifica- tion: Hx(zi,lroot,i) = Hsoil +  Hx,i −Hsoil  cosh(τiz) cosh(τilroot,i) + Ji−1 κi sinh(τilroot,i −τiz) cosh(τilroot,i) (14) and Jx(zi,lroot,i) = −kx,i d Hx dzi = ΔHiκi sinh(τizi) cosh(τilroot,i) + Ji−1 cosh(τilroot,i −τizi) cosh(τilroot,i) with Hi = Hsoil −Hx,i [P]. It can be seen that these two equations verify the boundary conditions Jx(zi,lroot,i) = −kx,i d Hx dzi = ΔHiκi sinh(τizi) cosh(τilroot,i) + Ji−1 cosh(τilroot,i −τizi) cosh(τilroot,i) with Hi = Hsoil −Hx,i [P]. It can be seen that these two equations verify the boundary conditions. 2.3 Root with heterogeneous properties Using the macroscopic parameters definitions given in a previous section (see 2.1.2) we obtain for the root conductance Krs(lroot,i) defined as the conductance of the entire root system including the distal part and the root segment with length lroot,i: Krs,i(lroot,i) Δ= Jx(lroot,i,lroot,i) ΔHi = κitanh(τilroot,i) + Ji ΔHcosh(τilroot,i) (15) (15) The conductance of the distal root zone in zi = 0 is given by: The conductance of the distal root zone in zi = 0 is given by: Krs,i−1 Δ= Ji Hsoil −Hx(zi = 0,lroot,i) The conductance of the zone i–1 is indeed by definition the flow Ji which arrives to segment i divided by the potential difference between the soil and the xylem at the connection between the root cylinders, zi = 0 in this case. When Krs,i−1 with Hx(zi, lroot,i) estimated by Eq. (14) is inserted in Eq. (15), it yields: κi  tanh(τilroot,i) + Krs,i−1 cosh(τilroot,i)  Krs,i−1sinh(τilroot,i) + κicosh(τilroot,i)   (16) Krs,i(lroot,i) = κi  tanh(τilroot,i) Krs,i(lroot,i) = κi  tanh(τilroot,i)  (16) Let us point that, if the zone length we are focusing on is zero, the root conductance is simply Krs,i−1, the conductance of the upstream zone. Let us point that, if the zone length we are focusing on is zero, the root conductance is simply Krs,i−1, the conductance of the upstream zone. 123 Towards quantitative root hydraulic phenotyping... 1145 Similarly a new solution is obtained for the SUD. The standard uptake density along the considered root fraction is independent of the soil water potential: SU D(zi,lroot,i) = τi  κicosh(τizi) + Krs,i−1sinh(τizi) κisinh(τilroot,i) + Krs,i−1cosh(τilroot,i)  (17) (17) These solutions (Eqs. 16, 17) are a generalisation of the solutions of the homoge- neous root Eqs. (8) and (12) to roots with several differentiated zones (straightforward with Krs,0 = 0). These solutions (Eqs. 16, 17) are a generalisation of the solutions of the homoge- neous root Eqs. (8) and (12) to roots with several differentiated zones (straightforward with Krs,0 = 0). To summarize, we just have presented a method that can be used to assemble the elementary building blocks (i.e. the zones) into complex hydraulic systems. We can now generalize for a compartmented root of total length lroot made of N differentiated zones. The locations of the connections between zones are called li, which corresponds to the cumulative sums of the different zone lengths lroot,i. 2.3 Root with heterogeneous properties When continuity of both axial fluxes and potentials is imposed at the junction between distinct zones as ’inter- mediary’ conditions and when no flux at the root tip, i.e. Jx(z = 0, lroot) = 0 and a potential top boundary condition Hx(z = lroot, lroot) = Hcollar are imposed just as in the first section, a unique solution can be found. Mathematically we impose from the tip to the collar: ⎧ ⎪⎪⎨ ⎪⎪⎩ Jx(z = 0,lroot) = 0 Hx(z = li,lroot)|i = Hx(z = li,lroot)|i+1 ∀i ∈[1, N −1] Jx(z = li,lroot)|i = Jx(z = li,lroot)|i+1 ∀i ∈[1, N −1] Hx(z = lroot,lroot) = Hcollar (18) (18) where . . . |i means that the function is evaluated in the ith zone. where . . . |i means that the function is evaluated in the ith zone. The solution of the flow equation inside each root zone is given by the general solution (19) applied to the boundary conditions (18). Hx(z,lroot) = ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ Hsoil + c1exp (τ1z) + c2exp (−τ1z) , z ∈[0,l1] Hsoil + c3exp (τ2(z −l1)) + c4exp (−τ2(z −l1)) , z ∈[l1,l2] ... Hsoil + c2N−1exp (τN(z −lN−1)) + c2Nexp (−τN(z −lN−1)) , z ∈  lN−1,lN  (19) (19) where c is the vector of coefficients that can be determined by the boundary conditions (18). For instance, if we consider two zones equations (18) become: ⎧ ⎪⎪⎨ ⎪⎪⎩ Jx(z = 0,lroot) = 0 Hx(z = l1,lroot)|1 = Hx(z = l1,lroot)|2 Jx(z = l1,lroot)|1 = Jx(z = l1,lroot)|2 Hx(z = lroot,lroot) = Hcollar ⎧ ⎪⎪⎨ ⎪⎪⎩ Jx(z = 0,lroot) = 0 Hx(z = l1,lroot)|1 = Hx(z = l1,lroot)|2 Jx(z = l1,lroot)|1 = Jx(z = l1,lroot)|2 Hx(z = lroot,lroot) = Hcollar 123 1146 F. Meunier et al. 123 2.3 Root with heterogeneous properties Or Or Or ⎧ ⎪⎪⎨ ⎪⎪⎩ −kx1 (c1τ1 −c2τ1) = 0 c1exp(τ1l1) + c2exp(−τ1l1) = c3 + c4 −kx1τ1 (c1exp(τ1l1) −c2exp(−τ1l1)) = −kx2τ2 (c3 −c4) Hsoil + c3exp(τ2(l2 −l1) + c4exp(−τ2(l2 −l1) = Hcollar ⎧ ⎪⎪⎨ ⎪⎪⎩ −kx1 (c1τ1 −c2τ1) = 0 c1exp(τ1l1) + c2exp(−τ1l1) = c3 + c4 −kx1τ1 (c1exp(τ1l1) −c2exp(−τ1l1)) = −kx2τ2 (c3 −c4) Hsoil + c3exp(τ2(l2 −l1) + c4exp(−τ2(l2 −l1) = Hcollar This set of four boundary conditions can be rewritten in matrix notations factorizing the coefficients ci: R ∗c = H with: c = ⎡ ⎢⎢⎣ c1 c2 c3 c4 ⎤ ⎥⎥⎦ R = ⎡ ⎢⎢⎣ τ1 −τ1 0 0 exp (τ1l1) exp (−τ1l1) −1 −1 κ1exp (τ1l1) −κ1exp (−τ1l1) −κ2 κ2 0 0 exp (τ2(l2 −l1)) exp (−τ2(l2 −l1)) ⎤ ⎥⎥⎦ H = ⎡ ⎢⎢⎣ 0 0 0 Hcollar −Hsoil ⎤ ⎥⎥⎦ R ∗c = H with: This mathematical development allows the splitting of any root in an arbitrary number of differentiated homogeneous zones by calculating the vector c using: c = R−1H It allows solving the water flow equation in a single root branch with hetereoge- neous properties. By inserting the values of vector c in Eq. (19), we obtain the xylem potential everywhere inside the root branch and consequently the axial and radial fluxes by applying Eqs. (29) and (30), respectively. The macroscopic parameters are then obtained using their definition (8) and (12). g Figure 4(top) shows a RS made of three zones, each of them having homogeneous hydraulic properties. In the left subplot the root global conductance is shown as a function of the total root length (dark blue solid line). The Krs can be calculated at each location z between the tip and lroot, and will depend on the hydraulic properties of therootsegmentandzonesbetween0andthelocationz.HowtheKrs willchangewithz will depend on the values of the root properties along the root branch. If we change each of the radial or axial hydraulic conductivities separately, Krs will change accordingly in a different way, as showed in Fig. 4. The sensitivity of this macroscopic parameter to the different radial conductivities when increasing the local hydraulic traits by 25% 123 1147 Towards quantitative root hydraulic phenotyping... Fig. 4 Root with heterogeneous properties: the top-drawn root is a layout of a three-compartmented root. 2.3 Root with heterogeneous properties The two subplots represent the root global conductance as a function of different local properties (dark blue solid line) and its sensitivity to the local radial (lighter blue lines, left subplot) or axial (lighter blue lines, right subplot) conductivities Fig. 4 Root with heterogeneous properties: the top-drawn root is a layout of a three-compartmented root. The two subplots represent the root global conductance as a function of different local properties (dark blue solid line) and its sensitivity to the local radial (lighter blue lines, left subplot) or axial (lighter blue lines, right subplot) conductivities are plotted (light blue solid lines). In the right subplot we present the same sensitivity analysis of this root conductance to each local hydraulic axial conductances. The reference values are the different hydraulic conductivity plateau obtained by Doussan et al. (1998b) for a maize lateral root. The sensitivity of this macroscopic parameter to the different radial conductivities when increasing the local hydraulic traits by 25% are also plotted (light blue solid lines). In the right subplot we present the same sensitivity analysis of this root con- ductance to each local hydraulic axial conductances. Increasing by 25% the radial or axial conductivity of one of the zones affects in different ways the global conductance according to the root length. Figure 5 shows SUD in the case of heterogeneous root properties and its sensitivity. Here again we consider a three-zoned root. In subplots b and c, the uptake fraction density is plotted as a function of the position along the root (blue solid line). We clearly see the locations of the transition between zones. By definition the integral remains equal to unity. The sensitivity of the normalized uptake is plotted when local radial conductivity of each zone is respectively increased by 25% as compared to the generic hydraulic properties obtained by Doussan et al. (1998b) (light blue dashed, dotted and dash-dotted lines, from tip to collar zones). In the right subplot the impact of an increase of the axial conductances of each zone is shown (light blue dashed, dotted and dash-dotted lines from tip to collar zones). 2.4 Root system with laterals Unlike the uniform root or the root with homogeneous zones, no solution of the water flow equation could be found for a root bearing regularly spaced laterals. Landsberg 12 3 1148 F. Meunier et al. Fig. 5 Root with heterogeneous properties: SUD along a three-zones root (dark solid line) and sensitivity of the normalized uptake to the local radial (left subplot) and axial (right subplot) hydraulic conductivities (lighter blue lines) Fig. 5 Root with heterogeneous properties: SUD along a three-zones root (dark solid line) and sensitivity of the normalized uptake to the local radial (left subplot) and axial (right subplot) hydraulic conductivities (lighter blue lines) and Fowkes (1978) suggested an approximation when incorporating these laterals all over the principal root length by increasing the radial conductivity of the principal root. Yet it is possible to find the conductance of this idealized root system using a discrete approach. The mathematical solution of this problem is not straightforward and we will introduce it progressively. In a first step, we present such a discrete model, its parameters and equations (Sect. 2.4.1). We then apply and solve it for the simplest case: the uniform root (Sect. 2.4.2). We show that the discrete root model converges to the exact continuous model for sufficiently small root segments. Finally we repeat the methodology for the more complex case of a root bearing laterals (Sect. 2.4.3). 2.4.1 Discrete model In the discrete approach, we consider that the basic element is a 1D root segment of length lseg [L] in which local hydraulic properties are considered as uniform. Each single segment (numbered n) has a radial (Kr,n [L3P−1T−1]) and an axial (Kx,n [L3P−1T−1]) hydraulic conductance that depends on the hydraulic and geo- metric root segment properties as:  Kr = 2πrlsegkr Kx = kx lseg The segment radial conductance is thus defined as the radial conductivity multi- plied by the segment surface while the root segment axial conductance is obtained by dividing the intrinsic axial conductance by the segment length. Note that we now use the subscript n instead of i to clearly distinguish root zones (that exhibit contrasted root hydraulic properties, see previous section) and root segments (that may have identical root hydraulic properties, as it will be shortly developed). Using the segment conductances, the flow in the segmented root systems is approximated for the segment numbered n (supposed here not linked to lateral roots) by: 123 Towards quantitative root hydraulic phenotyping... 1149 sed of t  Jr,n = Kr,n  Hsr,n −Hx,n  Jx,n = Kx,n  Hx,n−1 −Hx,n   Jr,n = Kr,n  Hsr,n −Hx,n  Jx,n = Kx,n  Hx,n−1 −Hx,n  with Hsr,n the water potential at the soil root interface, Hx,n and Hx,n−1 the water xylem potential inside the segment n and n–1 respectively. Jr,n and Jx,n are the radial and axial flows in the nth segment. The potential as well as the radial and axial flow are considered as uniform inside the root segment of finite length. Fig. 6 Conceptual models used for the discrete model of the homogeneous root: overview of the simple homogeneous root made of n segments (a) with details on the recurrence relationship (b) and the first recurrence (c) 2.4.2 Discretized uniform root branch A root branch can be conceptualized as a recurrence of the diagram represented in Fig. 6b whose initial value is illustrated in Fig. 6c. Mathematically, the equivalent conductance for n segments Krs,n is: Krs,n = ⎧ ⎪⎨ ⎪⎩  1 Kr,n + 1 Kx,n −1 , n = 1  1 Kx,n + 1 Kr,n+Krs,n−1 −1 , elsewhere The derivation of the discrete problem from continuous approach is given in the “Appendix 3”. 3 1150 F. Meunier et al. 1150 F. Meunier et al. Note that in the specific case of homogeneous root: Kr,n = Kr, ∀n (and the same for all Kx,n. It can be demonstrated that the root conductance for n segments is given by: Krs,n =  κ+ κ− 2n −1 1 κ−  κ+ κ− 2n −1 κ+ n→∞ −−−−−→κ+ Kx>>Kr −−−−−→√Kr Kx = √2πrkrkx (20) (20) where κ−[L3P−1T−1] and κ+ [L3P−1T−1] are function of the radial and axial con- ductances: ⎧ where κ−[L3P−1T−1] and κ+ [L3P−1T−1] are function of the radial and axial con ductances: ⎧ √ ⎧ ⎨ ⎩ κ−= −Kr−√ K 2r +4Kr Kx 2 < 0 κ+ = −Kr+√ K 2r +4Kr Kx 2 > 0 (21) (21) Demonstration is given in “Appendix 4”. κ+ is the asymptotic value of conductance: for a homogeneous root, Eq. (20) shows that the apparent conductance tends to κ+, when the number of segments (or the root branch length) tends to infinity, as previously shown in Fig. 1 and Eq. (9). For the first limit of Eq. (20) to be verified, κ+ must have a smaller absolute value than κ−. But this is always the case since, in absolute values, according to their definitions, the former is the difference of two positive terms and the latter the sum of the same terms. Note that for lseg →0, κ+ goes to the exact asymptotic conductance given in Eq. (9) which is the second limit of Eq. (20). If we define χ = κ+ κ− 2 (22) (22) Eq. (20) simply becomes: Krs,n = χn −1 1 κ−χn −1 κ+ . Krs,n = χn −1 1 κ−χn −1 κ+ . χ determines the rate of convergence of the root conductance towards its asymptotic value κ+. It is worth mentioning that for lseg →0, κ+ →κ and Krs,n →Krs(nlseg). This implies that the size of the segments that lead to a certain accuracy of the discrete solution can be derived. 2.4.2 Discretized uniform root branch A finite difference solution of SUF for a homogeneous root can be found in function of the kr kx ratio (and on the considered segment length lseg). It leads to an equation equivalent to the one found in the continuous solution section when the segment length is small enough. Details of the mathematical development are given in “Appendix 5”. 2.4.3 Discretized root branch with laterals 2.4.3 Discretized root branch with laterals For RS with lateral roots, no continuous solution of Hx and Jr was found but a recur- rence series can still be used to obtain the macroscopic hydraulic parameters. In this 123 Towards quantitative root hydraulic phenotyping... 1151 Fig. 7 Root system with laterals: the left panel illustrate the conceptual root system. The two other subplots show κ+,lat as a function of Klat, dinter with increasing kr (left) and increasing kx (right) (from light to dark blue in both figures). The asymptotic conductance of an unbranched root system is given by the horizontal dashed lines Fig. 7 Root system with laterals: the left panel illustrate the conceptual root system. The two other subplots show κ+,lat as a function of Klat, dinter with increasing kr (left) and increasing kx (right) (from light to dark blue in both figures). The asymptotic conductance of an unbranched root system is given by the horizontal dashed lines series radial conductivity and axial conductance of the principal root are assumed to be uniform as well as the conductance of lateral roots. Lateral roots can be reduced to their effective total conductance Klat [L3T−1P−1], each connected in parallel to the principal root. Note that the lateral conductance Klat is assumed to be constant. This assumption makes sense because it was demonstrated in the two previous sections that beyond a certain length the conductance of a uniform or heterogeneous root branch does not change dramatically. We call dinter [L] the distance between two successive laterals. The link with the number of internodal segments (Ninter) is: dinter = Ninterlseg dinter = Ninterlseg Figure 7(left) shows the recurrence that must be solved to obtain the asymptotic conductance of our system Krs,nlat after addition of nlat laterals. kr and kx are the radial conductivity and intrinsic axial conductance of the main root, dinter and Klat are the branching distance and the lateral root conductance, respectively. Using Eq. (16), we obtain the following recurrent series after simplification: Krs,nlat = κ  Klatcosh(τdinter) + sinh(τdinter) + Krs,nlat−1cosh(τdinter)  Klatsinh(τdinter) + κcosh(τdinter) + Krs,nlat−1sinh(τdinter) here Krs,nlat is the conductance of the whole system after addition of nlat laterals. where Krs,nlat is the conductance of the whole system after addition of nlat laterals. Applying the method developed for the homogeneous single discrete root (see “Appendix 4”), we obtain the convergence values of the recurrent series for the whole root system: ⎧ ⎪⎪⎨ ⎪⎪⎩ κ+,lat = −Klat+  K 2 lat+4  κ2+ Klat κ tanh(τdinter )  2 > 0 κ−,lat = −Klat−  K 2 lat+4  κ2+ Klat κ tanh(τdinter )  2 < 0 (23) (23) 12 3 1152 F. Meunier et al. κ+,lat is the asymptotic conductance of the system. This is the root system conduc- tance including the effect of laterals. Interestingly, it yields: κ+,lat ⎧ ⎪⎪⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎪⎪⎩ Klat≫κ −−−−−→ κ tanh(τdinter) Klat≪κ −−−−−→κ dinter→0 −−−−−→ Klatkx dinter dinter→∞ −−−−−→κ In others words, the asymptotic conductance of a RS with laterals depends on four parameters: the radial and axial conductivities of the principal root segments, the con- ductance of the laterals and the distance between two laterals. When this distance becomes infinite—i.e. there is no more laterals—we come back to the conductance we found in Sect. 2.2 (continuous solution of the single homogeneous root). Same observation if the laterals conductance becomes negligible. Other possible simplifica- tion: if the Klat is much larger than κ, the root system conductance is the principal root conductance κ increased by a factor 1 tanh(τdinter). If the internodal distance becomes very short the asymptotic conductance of the RS depends only on the principal axial conduc- tance, the distance between the successive laterals and their conductance. The fact that a maximal conductance for a root system with laterals is progressively reached as the principal root branch grows puts the basis of allometric relations (links functional— structural properties) for root systems. 123 dinter = Ninterlseg The RS conductance has the same shape than the discrete solution for a homoge- neous root: ( )nl 1 Krs,nlat = (χlat)nlat −1 1 κ−,lat (χlat)nlat − 1 κ+,lat (24) Krs,nlat = (χlat)nlat −1 1 κ−,lat (χlat)nlat − 1 κ+,lat (24) with: χlat =  κ κcosh(τdinter) −κ−,latsinh(τdinter) 2 (25) Krs,nlat = (χlat)nlat −1 1 κ−,lat (χlat)nlat − 1 κ+,lat (24) with: χlat =  κ κcosh(τdinter) −κ−,latsinh(τdinter) 2 (25) (24) with: with: χlat =  κ κcosh(τdinter) −κ−,latsinh(τdinter) 2 (25) (25) Let us note that in their original paper Landsberg and Fowkes found a similar relation for the total conductance of a root bearing laterals but they considered the following simplification: the laterals were not accounted directly for but their presence was simulated increasing the radial conductivity of the main root everywhere. This assumption leads to overestimate the root system conductance especially when the density of the laterals is low or when their conductance is high. Moreover it does not predict correctly the water uptake location (which is ’diluted’ along the root branch). In addition this model can be coupled with the previous one (root with heteroge- neous properties) to take into account the differentiated zones close to the root tip or at the root basis. From this solution we can derive how many segments are necessary to reach a certain fraction of the maximal conductance κlat,+. We derive the fraction αlat of the 123 Towards quantitative root hydraulic phenotyping... 1153 actual system conductance to the asymptotic one and its inverse function: actual system conductance to the asymptotic one and its inverse function: αlat(nlat) = χnlat lat −1 κ+,lat κ−,lat χnlat lat −1 ⇐⇒nlat(αlat) = log αlat−1 αlat κ+,lat κ−,lat −1 log (χlat) (26) (26) Figure 7 shows the asymptotic conductance κ+,lat of the root with laterals, clarifying the effect of kx, kr, Klat, and dinter. The horizontal dashed lines give the asymptotic conductance κ for the principal root without laterals, for different ratio of radial to axial conductances (Eq. 24), increasing the radial conductivity (left, from light to dark blue lines) or axial conductivity (right, from light to dark blue lines). Besides it prevents the accurate location of the root water uptake along the root since the effect of laterals is diluted along the whole root length. dinter = Ninterlseg The continuous lines give the conductance of a branched root when Klat increases and dinter is 1 cm (open triangles) or 5 cm (open circles). The larger the distance between branches, the more the asymptotic conductance is controlled by the principal root properties. The equivalent conductance starts to be affected by the conductance of the lateral roots after a certain Klat value that varies with kr. When Klat tends to zero, the solution becomes equivalent to a single unbranched root system and reaches the dashed lines asymptotically. Let us note that if the internodal distance between successive laterals becomes very short, the root conductance, as suggested by Landsberg and Fowkes (1978), is given by: Krs = κnewtanh(τnewlroot) Krs = κnewtanh(τnewlroot) with the same notation as before and: with the same notation as before and: kr,new = kr + Klat 2πrdinter kx,new = kx 3 Applications In this section, we present possible applications of these models. We first use exper- imental data published by Zwieniecki et al. (2002) to estimate local root hydraulic properties from multiple global conductance measurements. Then we perform a sen- sitivity analysis of a maize root system to understand the key factors of its hydraulics. Finally we develop applications regarding quantitative ideotypes. 3.1 Application to experimental data In a first experiment, Zwieniecki et al. (2002) cut the proximal end of primary maize root branches and measured the conductance of the remaining part. Repeating this 12 3 1154 F. Meunier et al. Fig. 8 Application of the model to retrieve root local properties (experiments from Zwieniecki et al. 2002): experimental results (symbols, each colour stand for one particular root) as compared to model best fit (solid lines, with the corresponding colours) for both experiments (left panel proximal end cut, right panel distal end cut) Fig. 8 Application of the model to retrieve root local properties (experiments from Zwieniecki et al. 2002): experimental results (symbols, each colour stand for one particular root) as compared to model best fit (solid lines, with the corresponding colours) for both experiments (left panel proximal end cut, right panel distal end cut) procedure several times and for several root axes, they obtained data of water flow under a given potential difference as a function of the distance to the root tip. The experimental results are shown in Fig. 8 (left panel, symbols). Each colour stands for a different root branch analysed by Zwieniecki et al. (2002). A second experiment was performed on other RS in which the distal part of the roots that was cut (right panel, symbols). This time they measured the conductance of the proximal root branch region. Adding some microscopic cross sections to this framework, they observed two distinct zones in the root branches: the axially nonconductive tip and a second radially active zone whose cortex was composed by early metaxylem vessels. Although the root axis is composed of two parts, we can consider that only the second part takes up water and treat the RS as a homogeneous root made only by the oldest segments. Equations from single homogeneous root section are consequently valid. The second equality of Eq. (8) relates the root branch conductance Krs to the root segment length lroot. It can be used directly for the first experiment consequently. This relation was fitted to the dataset in order to optimize local axial and radial conductivity for each root using the reference potential differences mentioned in the study of 0.38MPa. For the second experiment the measured flow are given as the difference between the total uncut root flow and the flow of the proximal end. 3.1 Application to experimental data So we present the results in a similar form: Krs(lroot) −Krs,prox with Krs,prox = κtanh(τz). p p Only considering one root zone allows an extremely good fit to the observations represented by the colour symbols. We obtain a very strong correlation between the modelled (solid lines) and the measured conductances (symbols): r2 = 0.99 as shown in the left panel representing the first experiment. The second experiment (right panel) provides results almost as good as the previous ones: r2 = 0.92. In this plot, the results are presented in a form similar to the the original one of Zwieniecki et al. (2002): as the difference of the total root conductance minus the conductance of the proximal zone. As it can be seen easily from the left panel, as the root grows (but this is valid also for the second experiment), the conductance reaches a plateau just as the Eq. (8) predicts it. It means that the xylem conductance becomes limiting the water flow in these roots. 12 3.2 Application example: a maize root system The functions developed in the theory are of potential use for developing quantitative ideotypes, i.e., quantitative estimatse of optimal root hydraulic or architectural traits for drought stress tolerance. We consider as an example a maize RS generated by RootTyp (Pags et al. 2004). This RS is made of several primary roots attached to the basis of a stem. On these parallel primaries are attached regularly spaced laterals. We used the relationships of Doussan et al. (1998b) (see Figure 4) to represent the evolution of root segment radial and axial hydraulic properties with root segment age as a succession of plateau for both primaries and laterals. The conductance of a mature maize lateral root (whose oldest segments have the hydraulic properties of the last plateau) can be predicted using the equations of the root branch with heteregeneous properties section: we used an equation similar to Eq. (16) with three successive plateau’s. To derive the total RS conductance we assume that the stem is not taking up water (its radial conductivity is zero) and that its xylem conductance is hydraulically non-limiting (its axial conductivity is very high). Consequently the RS conductance is simply the sum of the primary conductances. As illustrated in the top panel of Fig. 9 the majority of the root primary surface for a mature maize RS is made by the oldest zone so that we can consider with good approximation the primary root hydraulic properties as uniform when analysing a mature root system. Around 70% of the root surface of the primaries is made of segments older than 23 days, 18% have between 10 and 25 days, the remaining 12% is made of young segments. (younger than 10 days) Similar contribution is observed for the root surface of the laterals (14% younger, 16% intermediate and 66% older, respectively, the rest is the non-conductive tips). We can use the model including laterals to calculate the primary conductance and finally the RS conductance. The dinter parameter is provided by the mean value of the inter-branches distance of RootTyp parametrized for maize (Couvreur et al. 2014). In the bottom panels of Fig. 9 we consider a change of ±50% for the local conduc- tivities of both primary (left) and lateral (right) roots, everything else kept constant. In these panels the dashed lines stand for the radial conductivity while the solid lines represent the axial intrinsic conductances. 123 1155 Towards quantitative root hydraulic phenotyping... 3.2 Application example: a maize root system The colour blue indicate the primary roots and the red colour, the laterals. The darker the lines, the younger the zones. The interest of such an example is to evaluate the potential benefit of increasing locally the root conductivity on RS conductance.Performing a sensitivity analysis on mature roots, we investigate which zone is the most resistive part of complex root systems. This sensitivity analysis is based on both hydraulic (obtained by Doussan et al. 1998b) and architectural parameters (growth and shape parameters of the maize plant for RootTyp (Pags et al. 2004). We can first notice that hydraulic property relative changes of primaries would not have a big impact on the total RS conductance. Indeed the axial conductivity of mature primaries are high enough to conduct the total amount of water until the collar while their radial conductivity is so low that even if the pressure head drops until the collar would be negligible increasing them the consequent change in conductance would be tiny. The radial conductivity of young segments of lateral roots will have an important (the biggest) impact on global conductance even if they are not the majority of lateral 12 123 1156 F. Meunier et al. Fig. 9 Sensitivity analysis of a maize root system: the top panels represent a maize root system architecture highlighting the different root zones: young (left), intermediate (center) and old (right) root zones for both primaries (blue) and lateral (red) roots. Bottom subplots show the effect of modifying local radial (dashed) or axial (solid) conductivities of the primary (left) or lateral (right) roots on the RS conductance in a one- by-one sensitivity analysis. The curves are coloured as a function of the segment ages and consequently their hydraulic conductivities: the darker, the younger Fig. 9 Sensitivity analysis of a maize root system: the top panels represent a maize root system architecture highlighting the different root zones: young (left), intermediate (center) and old (right) root zones for both primaries (blue) and lateral (red) roots. Bottom subplots show the effect of modifying local radial (dashed) or axial (solid) conductivities of the primary (left) or lateral (right) roots on the RS conductance in a one- by-one sensitivity analysis. 123 3.2 Application example: a maize root system The curves are coloured as a function of the segment ages and consequently their hydraulic conductivities: the darker, the younger segments as it can be seen from the left top panel (they constitue only 14% of the lateral root surfaces). Javot and Maurel (2002) reviewed the aquaporin expression in roots and its effect on the root hydraulic conductivity. They listed a set of examples demonstrating the possible regulation of the root conductance according to outer stimuli. Using the present model and this sensitivity analysis we can predict the magnitude of a change in local radial conductivity needed to induce a desired modification of the water uptake in a given soil (so far only in homogeneous soil conditions but the present model could and will be coupled in the future with a soil model to simulate transient flow). Another application would be the effect of drought period and its consequent root cells shrinkage leading to a decrease of the soil–root contact and thus of the root radial conductance on macroscopic behaviour (North and Nobel 1991). Finally, the benefits of increasing local xylem vessels (Vercambre et al. 2002) or rise the radial conductivity of certain zones (Blum 2010) can be predicted using both this approach 123 Towards quantitative root hydraulic phenotyping... 1157 and/or complete simulations (Leitner et al. 2014b). For example from the picture (Fig. 9) it can readily be seen that increasing axial conductances of both young and intermediate lateral segments would allow raising the RS conductance significantly. Other architectural parameters can also be easily tested with this model. For example increasing the root density by reducing the value of dinter. and/or complete simulations (Leitner et al. 2014b). For example from the picture (Fig. 9) it can readily be seen that increasing axial conductances of both young and intermediate lateral segments would allow raising the RS conductance significantly. Other architectural parameters can also be easily tested with this model. For example increasing the root density by reducing the value of dinter. If we performed here a one-by-one sensitivity analysis, combination of traits may also be considered. RS ideotypes as suggested by Lynch (2013) for maize may be eval- uated through the here-developed models if proposed under the form of quantitative traits. 3.3 Allometric relations as a function of hydraulic properties The new root branch length is always smaller when increasing the radial conductivity as compared to increasing the axial conductivity. Moreover the new branch length is always larger than 0.9 in this range when raising the axial hydraulic properties while it decreases until 0.1 when focusing on the radial hydraulic properties. Th d l h i hi i d l i hRSi l di l l E i Fig. 10 Quantitative phenotyping illustration: ratio of new to old root branch length needed to reach half the original asymptotic conductance when increasing the radial conductivity by a factor a (solid line) or the axial conductivity when increasing the axial conductivity by a factor b (dashed line). The horizontal axis represents the factor a or b factor a (solid line) or b are in the range [1 10]. The new root branch length is always smaller when increasing the radial conductivity as compared to increasing the axial conductivity. Moreover the new branch length is always larger than 0.9 in this range when raising the axial hydraulic properties while it decreases until 0.1 when focusing on the radial hydraulic properties. ThesecondexampleshowninthissectiondealswithRSincludinglaterals.Equation (23) gives the maximal conductance of a branched root system as a function of lateral conductance, local conductivities of the main root and the internodal distance. If the internodal distance increases or the lateral conductance decreases, the asymptotic RS conductance will progressively converge towards κ the asymptotic conductance of an unbranched root branch. Beyond a maximal branching length or a minimal lateral conductance, the lateral roots have no more effects on the RS global conductance. Thus for a set of three paramaters (kr, kx and dinter or Klat) a fourth one can be derived that enables increasing the unbranched conductance by a fraction ϵ [−]. Mathematically we are looking for the minimal K∗ lat or maximal d∗ inter such as: (1 + ϵ) κ = κ+,lat (28) (28) (1 + ϵ) κ = κ+,lat with ϵ > 0. Here again an analytical solution of the problem can be found. In Fig. 11, we plot these relations as a function of kr to kx ratio. In the left subplot, five different levels of Klat are considered while in the right subplot we draw the results for four distinct internodal distances. In other words, if we consider a plant root whose kr kx ratio is fixed as well as Klat, then Fig. 3.3 Allometric relations as a function of hydraulic properties Allometry is the study of the relationship of body size to shape, anatomy, physi- ology and finally behaviour (Damuth 2001). For root systems allemoteric relations have already been proposed by Biondini (2008) linking structural functions to local functional properties. p p It has been demonstrated in the section theory (see single homogeneous root or root system with laterals) that a maximal value of Krs is progressively approached as the root length increases. This means that an optimal root length must exist in terms of carbon cost as compared to root conductance gain. This optimal length allows defining allometric relations between structural and functional root properties. Here below we define these relations for 2 cases. First we consider a single homogeneous branch and ask the following question: If we increase the radial (resp. axial) conductivity of a homogeneous root by a factor a (resp. by a factor of b), how much may we reduce the root length and keep the same global conductance value? Mathematically, this question can be addressed by finding the length l∗ root that keeps the same global conductance while its local hydraulic properties are altered. l∗ root such as Krs(lroot) = ⎧ ⎨ ⎩ √2πrakrkxtanh  2πrakr kx l∗root  √2πrkrbkxtanh  2πrkr bkx l∗root If we take as reference global conductance value half of the maximal original conductance Krs(lroot) = κ 2. Using Eq. (8) we obtain ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ l∗ root lroot = 1 √a atanh  1 2 1 √a  atanh  1 2  l∗root lroot = √ b atanh  1 2 1 √ b  atanh  1 2  (27) (27) Interestingly Eqs. (27) are independent on the root hydraulics or geometric prop- erties. Increasing the radial (resp. axial) root branch conductivity, Eq. (27) a (resp. b) provides the new length necessary to reach half of the original asymptotic conduc- tance normalized by the previous length. Figure 10 shows these functions when the 12 3 1158 F. Meunier et al. Fig. 10 Quantitative phenotyping illustration: ratio of new to old root branch length needed to reach half the original asymptotic conductance when increasing the radial conductivity by a factor a (solid line) or the axial conductivity when increasing the axial conductivity by a factor b (dashed line). The horizontal axis represents the factor a or b factor a (solid line) or b are in the range [1 10]. 3.3 Allometric relations as a function of hydraulic properties 11a gives the maximal internodal distance that makes sense in a hydraulic framework that increases significantly the unbranched conductance). Beyond this maximal internodal distance the primary axial intrinsic conductance avoids a significant contribution of the laterals on the RS conductance. The same explanation is valid for the second subplot: if kr kx and dinter are fixed then the five lines represent the minimal conductance of laterals compatible with a significant increase of the global conductance. ϵ was fixed to 5%. If the lateral conductance 123 Towards quantitative root hydraulic phenotyping... 1159 Fig. 11 Example of relationship between architecture and hydraulics: maximal internodal distance d∗ inter (left) and minimal Klat∗(right) that allows increasing the RS asymptotic conductance by 5% as compared to an unbranched root branch as a function of kr kx ratio of the main root Fig. 11 Example of relationship between architecture and hydraulics: maximal internodal distance d∗ inter (left) and minimal Klat∗(right) that allows increasing the RS asymptotic conductance by 5% as compared to an unbranched root branch as a function of kr kx ratio of the main root is lower, it is too tiny compared to the primary radial conductivity to significantly contribute to the RS conductance. is lower, it is too tiny compared to the primary radial conductivity to significantly contribute to the RS conductance. 3.4 Future model applications The performance of root systems when simulated in contrasted environment will be indeed highly dependent on the macroscopic parameters (that represent the ability to take up water and the location of the root water uptake) as well as the pedoclimatic situations. Next studies will consequently focus on plant–environment interactions in order to look for best associations and to decipher key traits for an optimal root water uptake in contrasted and heterogeneous conditions. Some of the major roles of root systems are water and nutrient uptake from soils. In this paper, we considered the hydraulic macroscopic parameters of the root system and their dependence on the hydraulic properties of root segments and the distribution of lateral roots. However, the search for an optimized root system is a multi-objective optimization problem that should consider optimal water but also nutrient uptake under minimal carbon costs. This optimum might furthermore depend on the soil hydraulic and chemical properties, the nutrient and water distribution and the climate. As far as the nutrient uptake is considered, further work is needed to link the distribution of nutrientuptaketorootpropertiesandnutrientdistributions.But,thecurrentworkpartly contributes to this issue since water uptake distributions influence nutrient transport to the root surfaces directly by advective flow and indirectly by changing water content and consequently the diffusion coefficient. Next studies simulating simultaneous water and nutrient uptakes may help identifying key properties for the capture of these resources in each type of environments. Further studies will consequently focus on the use of such equations to decipher the soil–plant relation in a complete Root–Soil model solving the water flow in soil and root systems in three dimensions and in particular on the relations between the macroscopic parameters and the cumulative transpiration in a range of environments. 3.4 Future model applications The emergence of common database collecting root structural, topological and hydraulic properties in a unique format (Lobet et al. 2015) makes possible the use of the present model to calculate and to collect macroscopic parameters of any listed species. A coupling can also be made between any software analysing root architecture from experiments (a non-exhaustive list is referenced by Lobet et al. 2013) and such a model enabling an automatized analysis of global properties of genotypes if local conductivity functions are available a priori. Otherwise an additional work of deter- mining these functions will be needed but the equations presented in this paper can also be useful to retrieve shapes and values of these conductivity relationships from some global conductance measurements added to any technique delivering a complete root architecture (topology, age and order distribution such as in Leitner et al. 2014a) Finally this model could be coupled with the novel macroscopic root water uptake model developed by Couvreur et al. (2012) to investigate the effect of modifying any trait first on plant parameters and afterwards on plant ability to maintain or to increase its yield (through transpiration) under drought condition when the environ- ment is provided. In further studies, breeding proposals of Comas et al. (2013), Lynch 12 3 3 1160 F. Meunier et al. (2013) and Wasson et al. (2012) will be tested in silico in various contexts defined as a combination of soil and climate. Using a model solving water flow in soil and plant will also allow us to retrieve the impact of given traits on soil moisture and poten- tial distribution (Teuling et al. 2006). The new developments presented here allow us to calculate plant-scale parameters of idealized root systems. These parameters (cal- culated in homogeneous conditions) can be used for simulating root water uptake in non-homogeneous conditions as demonstrated by Couvreur et al. (2012). The method- ology presented here constitutes thus the first step in a larger study to predict the plant performance in contrasted (and heterogeneous) environments. Our results are tools to accurately and efficiently calculate plant-scale parameters that can be used in turn in soil–root system model simulating the water flow to assess plant performance. They consequently need to be coupled with soil model (de Jong van Lier et al. 2008). 4 Conclusion We present novel mathematical functions that predict plant scale hydraulic properties from measurable local structural and functional properties. The first parameter is the root global conductance Krs; the second parameter describes the potential relative uptake in uniform soil and is called the Standard Uptake Density SUD. These new root models were developed based on analytical solutions (and their approximations) of root water flow equations. Such functions may allow breeders to quantitatively a 12 Towards quantitative root hydraulic phenotyping... 1161 priori assess how a local root trait affects the global plant hydraulic properties, which opens new avenues for optimizing plant drougth tolerance for instance. priori assess how a local root trait affects the global plant hydraulic properties, which opens new avenues for optimizing plant drougth tolerance for instance. Three types of simplistic root systems were considered: a homogeneous single root branch, a single root branch split into several homogeneous zones or a single root branch with regularly spaced laterals. For these three root systems, macroscopic parameters can always be predicted as a function of local functional and architectural root traits. The relevant architectural traits were the total root length, the length of the homogeneous root zone, the distance between laterals. The functional parameters were the axial conductance and the radial conductivity of the homogeneous zones, of the primary and of the laterals. For a homogeneous root or a root with laterals, it was demonstrated that a maximum conductance is reached after a certain length for given set of kr and kx values, which mean that optimal root lengths must exist in terms of hydraulics. These models were successfully applied to laboratory data or used in sensitivity analyses of RHA to investigate the impact of local traits on global plant hydraulic behaviour. Finally they can be seen as a tool for deriving quantitative links between hydraulic and architectural traits of root systems. Based on these simple RS functions, models for more realistic RS architectures can be built, paving the way for quantitative estimation of the impact of changes in root traits on plant uptake efficiency. It is expected that other architectural traits like root angles will play a role in the estimate of SUD with more complex systems. Applications of these functions may allow one to quantitatively assess in silico ideotypes, i.e. root systems with ideal traits for drought tolerance as proposed by Comas et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional 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. 4 Conclusion (2013), Lynch (2013) and Wasson et al. (2012). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional 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. Appendix 1 Symbol Dimension Description α − Fraction of the asymptotic conductance reached by root length l∗ αlat L3P−1T −1 Fraction of the asymptotic conductance reached with nlat χ − Eq. (22) χlat L3P−1T −1 Eq. (25) dinter L Inter-branching distance Hcollar P Collar water potential Hs,eq P Equivalent soil water potential Hsoil P Soil water potential Hsr P Soil–root water potential Hx P Xylem water potential i − Root zone number Jr L3T −1 Radial volumetric flow rate Jx L3T −1 Axial volumetric flow rate κ L3P−1T −1 Asymptotic root conductance κ+ L3P−1T −1 Eq. (21) κ− L3P−1T −1 Eq. (21) κ+,lat L3P−1T −1 Eq. (23) κ−,lat L3P−1T −1 Eq. (23) Kcomp L3P−1T −1 Compensatory water uptake conductance Krs L3P−1T −1 Global conductance of the root system Krs,nlat L3P−1T −1 Root system conductance after addition of nlat laterals kr L P−1T −1 Root radial conductivity kx L4 P−1T −1 Root axial conductivity Kr L3P−1T −1 Segment radial conductance Kx L3P−1T −1 Segment axial conductance l∗ L Root length for a specific for a faction α of the asymptotic conductance lroot L Root length lseg L Segment length Ninter − Number of root segments between two successive branches n − Root segment number nlat − Number of laterals qr LT −1 Root radial flux r L Root radius SU D L−1 Standard uptake density SU F − Standard uptake fraction τ T −1 Root intrinsic property Tact L3T −1 Actual transpiration of the root system z L Distance to root tip Appendix 2 123 123 123 1162 F. Meunier et al. Appendix 1 Appendix 2 The axial flow Jx  L3T−1 inside a homogeneous root is given by: Jx(z,lroot) = −kx d Hx(z,lroot) dz  (29) (29) 123 123 12 1163 Towards quantitative root hydraulic phenotyping... This flow is a function of the position inside the root z [L] and the root length. In Eq. (29), Hx [P] is the total water potential (the sum of gravitational and pressure potentials). Mass conservation for a segment of length dz [L] (see Fig. 1a) leads to: −2πrkr (Hx(z,lroot) −Hsoil) = d Jx(z,lroot) dz (30) (30) The left hand side of the previous equation gives the radial flow between the soil– root interface and the inner root, [L3T−1] divided by a root layer dz [L]. Combining Eqs. (29) and (30) we obtain a differential equation: −2πrkr (Hx(z,lroot) −Hsoil) = −kx d2Hx(z,lroot) dz2 The general solution of this partial derivative equation has the shape: Hx(z,lroot) = Hsoil + c1exp (τz) + c2exp (−τz) with: τ = 2πrkr kx [L−1]. with: τ = 2πrkr kx [L−1]. If we impose the following boundary conditions Jx (z = 0, lroot) = 0 and Hx  z = lroot, lroot  = Hcollar, we obtain the unique solution for the water potential inside the root: h ( ) Hx(z,lroot) = Hsoil + (Hcollar −Hsoil) cosh (τz) cosh (τl) (31) (31) Using Eqs. (29) and (30) and the specific solution (31) we may find the axial and radial flows: Using Eqs. (29) and (30) and the specific solution (31) we may find the axial and radial flows: Jx(z,lroot) = kxτ (Hsoil −Hcollar) sinh (τz) cosh (τl) Jr(z,lroot) = dzkxτ 2 (Hsoil −Hcollar) cosh (τz) cosh (τl) Jx(z,lroot) = kxτ (Hsoil −Hcollar) sinh (τz) cosh (τl) J (z l t) = dzk τ 2 (H il −H ll ) cosh (τz) ( ) Jr(z,lroot) = dzkxτ 2 (Hsoil −Hcollar) cosh (τz) cosh (τl) Appendix 3 To derive the discrete recursion formula from discretising the continuum equations we consider a homogeneous root of length lroot and made of n segments of length lseg in a homogeneous of water potential Hsoil. We start from the conductance definition: the root branch conductance is the axial flow divided by the difference of water potential 1 Krs,n = Hsoil −Hx,n Jx,n It is equivalent to: 1 Krs,n = Hsoil −Hx,n−1 + Hx,n−1 −Hx,n Jx,n 1 Krs,n = Hsoil −Hx,n Jx,n It is equivalent to: 1 Krs,n = Hsoil −Hx,n−1 + Hx,n−1 −Hx,n Jx,n 12 3 3 1164 F. Meunier et al. Splitting into two terms the right hand side, it yields: Splitting into two terms the right hand side, it yields: 1 Krs,n = Hsoil −Hx,n−1 Jx,n + Hx,n−1 −Hx,n Jx,n The first term can be expressed as the root conductance when one segment is removed. The second term is simply the inverse of the segment axial conductance: The first term can be expressed as the root conductance when one segment is removed. The second term is simply the inverse of the segment axial conductance: 1 Krs,n = 1 Krs,n−1 Jx,n−1 Jx + lseg kx (32) (32) On the other hand, the water flow entering the root inside the last root segment is: Jx,n −Jx,n−1 = 2πrkr  Hsoil −Hx,n−1  lseg Dividing by Jx,n−1: Jx,n Jx,n−1 −1 = 2πrkr  Hsoil −Hx,n−1  lseg Jx,n−1 We obtain Jx,n Jx,n−1 = 2πrkrlseg Krs,n−1 + 1 (33) Finally combing Eqs. (32) and (33), it yields: 1 Krs,n = lseg kx + 1 2πrkrlseg + Krs,n−1 Which is equivalent to: 1 Krs,n = 1 Kx + 1 Kr + Krs,n−1 Appendix 4 We obtain Jx,n Jx,n−1 = 2πrkrlseg Krs,n−1 + 1 (33) Jx,n Jx,n−1 = 2πrkrlseg Krs,n−1 + 1 (33) Finally combing Eqs. (32) and (33), it yields: 1 Krs,n = lseg kx + 1 2πrkrlseg + Krs,n−1 Which is equivalent to: Which is equivalent to: Appendix 4 We want to solve the recurrent series shown in Fig. 6 to derive the root conductance Krs,n after addition of n identical segments. It can be demonstrated that this sequence converges and the possible values of convergence (κ−and κ+) can be expressed as follows: 1 Krs = 1 Kx + 1 Kr + Krs ⇐⇒κ−= −Kr−√ K 2r +4Kr Kx 2 < 0 κ+ = −Kr+√ K 2r +4Kr Kx 2 > 0 (34) (34) 123 Towards quantitative root hydraulic phenotyping... 1165 These solutions are found assuming Krs,n = Krs,n−1. As conductances are always larger than zero, κ+ is the only possible solution. We want to discover now how fast the addition of new resistances will make the model converge to this value. To do so we can rewrite equation Eq. (34) as the following sequence whose Krs,n is the nth element: Krs,n+1 = f (Krs,n) = 1 1 Kx + 1 Kr+Krs,n = Kx Krs,n + Kr Kx Krs,n + (Kr + Kx) = aKrs,n + b cKrs,n + d with: ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ f (x) =  1 Kx + 1 Kr+x −1 a = Kx b = Kr Kx c = 1 d = Kr + Kx ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ f (x) =  1 Kx + 1 Kr+x −1 a = Kx b = Kr Kx c = 1 d = Kr + Kx Formulated now as an homographic sequence we can evaluate the rate of conver- gence. This convergence speed indeed is defined by: | Krs,n+1 −x∗| | Krs,n −x∗| where x∗is the value of convergence (= κ+). But in the case of an homographic sequence, this can be simplified and tends to absolute value of the derivative of the function f, | f ′(x∗) | : where x∗is the value of convergence (= κ+). Appendix 4 But in the case of an homographic sequence, this can be simplified and tends to absolute value of the derivative of the function f, | f ′(x∗) | : | Krs,n+1 −x∗| | Krs,n −x∗| = | f (Krs,n) −f (x∗) | | Krs,n −x∗| →| f ′(x∗) | The derivative in this case yields: The derivative in this case yields: The derivative in this case yields: | f ′(x) |= 1 1 Kx + 1 Kr+x ′ = K 2 x (x + Kr + Kx)2 This function evaluated in x∗= κ+ yields: | f ′(x∗) |= κ+ κ− 2 The latter equality can be obtained with mathematical manipulations (not shown here). Now we know the speed of convergence, the homographic sequence can be The latter equality can be obtained with mathematical manipulations (not shown here). Now we know the speed of convergence, the homographic sequence can be 12 3 1166 F. Meunier et al. transformed into a geometrical one called hereafter vn: transformed into a geometrical one called hereafter vn: transformed into a geometrical one called hereafter vn: transformed into a geometrical one called hereafter vn: vn+1 = κ+ κ− 2 vn vn+1 = κ+ κ− 2 vn Link between both series is: Krs,n = vnκ−−κ+ vn −1 (35) (35) The previous steps allow us to predict the global conductance of a root with its number n of identical segments writing first the following equality The previous steps allow us to predict the global conductance of a root with its number n of identical segments writing first the following equality vn = v1 κ+ κ− 2n = K0 −κ+ K0 −κ− κ+ κ− 2n (36) (36) with K0 [L3T−1T−1] an initial conductance connected the considered root branch. Because the v sequence tends to zero the equivalent resistance will tend towards the positive fixed point, i.e. solution of Eq. (34) when the number of segments is high enough. Finally combining Eqs. (35) and (36) we obtain: Krs,n = K0−κ+ K0−κ−  κ+ κ− 2n κ−−κ+ K0−κ+ K0−κ−  κ+ κ− 2n −1 And if K0 = 0 (i.e. no initial conductance attached to the root branch), we obtain: And if K0 = 0 (i.e. no initial conductance attached to the root branch), we obtain: Krs,n =  κ+ κ− 2n −1 1 κ−  κ+ κ− 2n −1 κ+ Appendix 5 To calculate the Standard Uptake Fraction of an homogeneous root, we proceed as follow: the key variable in the SUF determination is the xylem potential of each segment since, in homogeneous condition, the difference between this value and the outside (uniform) potential gives the product of the radial conductance and the lateral flux. Kirchoff and Ohm laws give us two equations (we define now the first segment at the top of the root rather than at the distal end of the root and we consider a saturated soil: Hsoil = 0 for sake of simplicity):  Kx,n  Hx,n −Hx,n−1  = Jx,n Jx,n−1 = Jx,n −Kr,n−1Hx,n−1 123 12 Towards quantitative root hydraulic phenotyping... Towards quantitative root hydraulic phenotyping... 1167 These equations can be summarised as a system of two recurrent equations:  Hx,n Jx,n  = 1 + Kr,n Kx,n 1 Kx,n Kr,n 1 .  Hx,n−1 Jx,n−1  At the nth segment of an homogeneous zone (Kr,n = Kr and Kx,n = Kx) we can state: At the nth segment of an homogeneous zone (Kr,n = Kr and Kx,n = Kx) we can state:  Hx,n Jx,n  = 1 + Kr Kx 1 Kx Kr 1 n−1 .  Hx,1 Jx,1  Since we have Krs,n = κ+α and Tact = −Hcollarκ+α for the global root conductance Krs,n and for the collar flow Tact, respectively. Since we have Krs,n = κ+α and Tact = −Hcollarκ+α for the global root conductance Krs,n and for the collar flow Tact, respectively.  Hx,n Jx,n  =  1 + Kr Kx 1 Kx Kr 1 n−1 . the exact solution we found in the continuous model. the exact solution we found in the continuous model. Appendix 5  Hcollar(1 + κ+α Kx ) −Hcollarκ+α  (37) (37) If we define matrix A = 1 + Kr Kx 1 Kx Kr 1  we can, finding the eigenvalues and eigen- vectors of A, develop the following relations: If we define matrix A = 1 + Kr Kx 1 Kx Kr 1  we can, finding the eigenvalues and eigen vectors of A, develop the following relations: If we define matrix A = 1 + Kr Kx 1 Kx Kr 1  we can, finding the eigenvalues and eigen- vectors of A, develop the following relations:   vectors of A, develop the following relations: An−1 = P.Λn−1.P−1 An−1 = P.Λn−1.P−1 with: P = − κ− Kr Kx − κ+ Kr Kx 1 1  Λ = 1 −κ− Kx 0 0 1 −κ+ Kx  and P−1 =  Kr Kx κ+−κ− κ+ κ+−κ− −Kr Kx κ+−κ−− κ− κ−−κ+  with: and And combining Eq. (37) and the previous matrix definitions we find an exact solu- tion of SUF for an homogeneous root, depending only on the kr kx ratio. Indeed, by definition: SU Fn = Jr,n Tact = Kr Hx,n −κ+Hcollarα 12 3 1168 F. Meunier et al. We obtain: We obtain: We obtain: SU Fn = Kr 2κ+  1 −κ+ Kx n −  1 + κ+ Kx n + Kr 2κ+αKx  1 −κ+ Kx n +  1 + κ+ Kx n Let us note that if the segment length is small enough, we can simplify the matrices: Let us note that if the segment length is small enough, we can simplify the matrices: P →  1 κ −1 κ 1 1  Λ → 1 + κ Kx 0 0 1 − κ Kx  P−1 →  κ 2 1 2 −κ 2 1 2  And the SUF simply becomes: And the SUF simply becomes: And the SUF simply becomes: And the SUF simply becomes: SU Fn →−(n + 1)  τlseg 2 + τlseg tanh(τlroot) Consequently: SU D → τ tanh(τlroot) ≃τcosh(τz) sinh(τlroot) the exact solution we found in the continuous model. SU Fn →−(n + 1)  τlseg 2 + τlseg tanh(τlroot) : SU D → τ tanh(τlroot) ≃τcosh(τz) sinh(τlroot) Consequently: References Vadose Zone J 12(3):0 de Jong van Lier Q, van Dam JC, Metselaar K, de Jong R, Duijnisveld WHM (2008) Macroscopic root water uptake distribution using a matric flux potential approach. 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An Improved Adaptive Exponential Reaching Sliding Mode Control Strategy for Intelligent Connected Vehicle System under Complex Communication Environment and Different Types of Interference Mode Control Strategy for Intelligent Connected Vehicle System under Complex Communication Environment and Different Types of Interference Tao Song  Shandong University School of Control Science and Engineering WENXING ZHU  (  zhuwenxing@sdu.edu.cn ) Shandong University School of Control Science and Engineering https://orcid.org/0000-0001-7061- 5359 Shibin Su  Qingdao Hisense Network Technology Co.,Ltd Wenwen Wang  Qingdao Hisense Network Technology Co.,Ltd Research Article Keywords: Adaptive slide mode control, Intelligent connected vehicle, Performance analysis, Communication environment change, Input interference Posted Date: January 18th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2299180/v1 Tao Song  Shandong University School of Control Science and Engineering WENXING ZHU  (  zhuwenxing@sdu.edu.cn ) Shandong University School of Control Science and Engineering https://orcid.org/0000-0001-7061- 5359 Shibin Su  Qingdao Hisense Network Technology Co.,Ltd Wenwen Wang  Qingdao Hisense Network Technology Co.,Ltd Tao Song  Shandong University School of Control Science and Engineering WENXING ZHU  (  zhuwenxing@sdu.edu.cn ) Shandong University School of Control Science and Engineering https://orcid.org/0000-0001-7061- 5359 Shibin Su  Qingdao Hisense Network Technology Co.,Ltd Wenwen Wang  Qingdao Hisense Network Technology Co.,Ltd * Corresponding author. zhuwenxing@sdu.edu.cn. Tel. 86-531-88395289. Research Article Keywords: Adaptive slide mode control, Intelligent connected vehicle, Performance analysis, Communication environment change, Input interference Posted Date: January 18th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2299180/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: January 18th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. An Improved Adaptive Exponential Reaching Sliding Mode Control Strategy for Intelligent Connected Vehicle System under Complex Communication Environment and Different Types of Interference Tao Song a, Wen-Xing Zhu a, *, Shi-Bin Su b, Wen-Wen Wang b a School of Control Science and Engineering, Shandong University, Jinan 250061, China b Qingdao Hisense Network Technology Co., Ltd, Qingdao 266075, China analysis; Communication environment change; Input interference analysis; Communication environment change; Input interference An Improved Adaptive Exponential Reaching Sliding Mode Control Strategy for Intelligent Connected Vehicle System under Complex Communication Environment and Different Types of Interference Tao Song a, Wen-Xing Zhu a, *, Shi-Bin Su b, Wen-Wen Wang b a School of Control Science and Engineering, Shandong University, Jinan 250061, China b Qingdao Hisense Network Technology Co., Ltd, Qingdao 266075, China Abstract: This paper studies the performance of a nonlinear sliding mode controller in the cooperative control of the intelligent connected vehicles system. The study begins by proposing a V2V car-following model that takes into account the impacts of disturbances both external and within in the system. Subsequently, an improved adaptive exponential reaching sliding mode controller (IAERSMC) based on a non- singular terminal sliding mode variable is proposed. It is shown through theoretical analysis that with this controller, the system can converge in finite time and enhance dynamic performance. To verify the effectiveness of the controller proposed in this study, two simulation experiments are designed and performed. The first experiment tests the change of communication environment by transforming the communication topology, and the second experiment tests response to three different types of input interference: continuous regular interference, continuous irregular interference, and episodic disturbance. Simulation results show that the controller proposed in this study can maintain a stable operating state of the intelligent connected vehicles system in the face of the complex variable communication environments and different types of interference. The theoretical analysis agrees with the results of the simulated experiments. Keywords: Adaptive slide mode control; Intelligent connected vehicle; Performance Keywords: Adaptive slide mode control; Intelligent connected vehicle; Performance 1. Introduction Intelligent transportation system (ITS) integrates advanced detection, computing, control, communication and management technologies into the transportation management system, providing comprehensive and timely information for the main traffic and travelers, which can improve travel efficiency and reduce environmental pollution and traffic accidents. In recent years, intelligent transportation systems have entered a new stage of development, and one of the main features is the introduction of intelligent connected vehicles to build a vehicle-road collaboration system to improve traffic capacity and travel rates. Intelligent connected vehicles integrate the intelligence and networked technology of vehicles, which can form multi-vehicle systems through vehicle-vehicle communication to realize the collaboration of sensing, decision-making and control. The intelligent vehicle-road system based on connected vehicles and vehicle-road cooperative control is the future development goal and direction of intelligent transportation system [1]. Connected Vehicle Platoon Control is one of the most promising research directions in the field of intelligent connected vehicle control technology. The vehicle platoon utilizes wireless communication technology to connect multiple vehicles, and enables these connected vehicles to transmit and share information within a certain distance. It greatly expands the range of vehicular information acquisition and makes Vehicle-to-Vehicle (V2V) and Vehicle-to-Infrastructure (V2I) collaboration possible, improving the overall performance of vehicular and traffic systems. This introduction provides a brief overview of key research developments concerning three key aspects of vehicle platoon control systems: spacing policy, communication topology and cooperative controller. Vehicle spacing policy is also known as the “geometric configuration” of the vehicle platoon, there are two common spacing policies, which are Constant Spacing (CS) and Constant Time Headway (CTH). In terms of Constant Spacing, Silva et al. [2] proposes an integral controller, which can ensure string stability and restrain disturbance in the vehicle platoon system with disturbance. Darbha et al. [3] proposed a control controller based on constant spacing policy for vehicle platooning system using multiple vehicles look ahead information. Ge and Orosz [4] proposed acceleration-based connected cruise control (CCC)to increase roadway traffic mobility, and the results show that adding a few appropriate CCC vehicles to the traffic flow can stabilize otherwise string unstable vehicle platoons. Lin et al. [5] proposed optimal localized feedback gains control for one-dimensional formations in which vehicles only use information from their immediate neighbors. In terms of Constant Time Headway, Bian et al. [6] study a multiple-predecessor following strategy based on CTH to reduce time headway via vehicle-to-vehicle (V2V) communication. 1. Introduction Besselink and Johansson [7] proposed a novel delay-based spacing policy for the control of vehicle platoons together with a notion of disturbance string stability. In addition, Li [8] proposed a new variable time headway (VTH) policy-based platoon controller by considering motion coupling interactions to eliminate the adverse impacts of external disturbances and time delays. The communication topology of the vehicles platoon refers to the feedback information flow used for vehicle control, which has an important impact on the stability of the vehicles platoon. The communication topology determines the information interaction between the member vehicles within the platoon and extends the environmental perception range of the self-vehicle, and different communication topologies correspond to different vehicle platoon performances. Li et al. [9,10] studied the impact of switching communication topology on the dynamic performance of connected vehicles based on car-following model. Li et al. [11] extend existing studies on distributed platoon control to more generic topologies with complex eigenvalues. Konduri et al. [12] study the effects of multiple vehicles looking ahead in a vehicle platoon that employs a constant spacing policy-based controller in the presence of parasitic lags. Oncu et al. [13] presented an NCS framework for analyzing the effects of wireless communication between vehicles on CACC string stability performance. Salvi et al. [14] presented a decentralized control protocol able to guarantee achievement of a platooning configuration in a vehicular network affected by time- varying delays. Tang et al. [15,16] propose a new car-following model to describe the communication between vehicles, which is used to study the driving behavior in the event of an accident. The goal of controller design makes to eliminate the influence of disturbing factors such that the controlled object can operate according to the desired rules. Vehicle controllers are usually used in distributed control systems. Distributed control is a decomposition of the connected vehicle platoon into individual subsystems to design their respective controllers, which calculate the amount of control based only on limited information about neighboring vehicles. It is characterized by low computational effort and light communication load. Di Bernardo et al. [17] proposed a distributed double- integrator controller to achieve platooning of vehicles in the presence of heterogeneous time-varying delays. Dunbar and Caveney [18] consider the problem of distributed control of a platoon of vehicles with nonlinear dynamics and present distributed receding horizon control algorithms. Lin et al. 1. Introduction [19] consider the optimal control of one- dimensional formations with nearest-neighbor interactions between the vehicles and formulate a structured optimal control problem in which local information exchange of relative positions between immediate neighbors imposes structural constraints on the feedback gains. Zhu et al. [20-22] introduced a series of methods including classical compensators and discrete controllers into Autonomous traffic flow control. Based on the sliding mode control principle, Yan [23,24] designed feed-forward compensation- based finite-time traffic flow controller and improved super-twisting compensator, Gao [25], Guo [26] and Kown [27] designed adaptive sliding mode controllers. The sliding mode control method can suppress disturbances and make the system converge in a finite time. The principle of sliding mode control involves a discontinuous switching control to dynamically drive the state of the system to the lower order sliding mode surface, after which the system will move to the origin according to the equation of the sliding mode surface to reach the steady state. After years of development, sliding mode control theory has become an important research direction of nonlinear control theory, with widespread concerned and applied in a variety of fields. The sliding mode control strategy is introduced in this study for cooperative control of intelligent connected vehicles, and an improved adaptive exponential reaching sliding mode controller is proposed based on the non-singular terminal sliding mode variables. Through theoretical analysis, it is found that the improved controller proposed in this study can make the system converge in finite time and improve the dynamic performance of the system. Further, simulation experiments are designed to analyze the improved controller and demonstrate that the improved system can maintain stable operation in the face of complex communication environments and different types of external disturbances. The rest of this paper is structured as follows. The connected vehicle system is modeled in the second part, and the V2V car-following model is proposed based on the FVD model, with the effect of system perturbation is considered in the modeling process. The third section proposes IAEASMC based on the non-singular terminal sliding mode. In the fourth part, the theoretical analysis of the improved controller is carried out. The fifth part is simulation experiments to verify the effect of the improved controller. The sixth part is the conclusion. 2. 1. Introduction Model 2.1 V2V System Fig.1 Illustration of a connected and automated vehicles traffic flow in the V2V environment 2.1 V2V System ig.1 Illustration of a connected and automated vehicles traffic flow in the V2V environment The illustration of the intelligent connected vehicle system is shown in Fig. 1. It is assumed that all vehicles have hardware and software systems that enable autonomous driving and communication through the on-board communication unit as well as the roadside unit to achieve intelligent network connectivity. This means that each vehicle can receive information from all vehicles in the platoon and send information about its vehicle, which includes the vehicle's position, velocity and acceleration, etc. There are two ways for vehicles to transfer information, the first is through on-board sensors that gather information from nearby vehicles, as shown by the blue solid line in Fig. 1; the other way is to use the onboard V2V communication unit to obtain information from other vehicles in the platoon, as shown by the red and green dashed lines in Fig. 1. In particular, the green dashed line in the figure indicates the information interaction between the vehicles in the platoon and the lead vehicle, and the red dashed line refers to the information interaction between the vehicles in the platoon excluding the lead vehicle. Next, we combine traffic flow theory to model the intelligent connected vehicles in a V2V environment. It is well known that the FVD model proposed by Jiang et al. [28] has widely used to explore the traffic flow theory, which can accurately describe many characteristics of the traffic flow, such as "walking and stopping", etc. The FVD model considering the positive and negative velocity difference between the front vehicle and the rear vehicle is as follows. 𝑎𝑖(𝑡) = 𝑝(𝐹(ℎ𝑖(𝑡)) −𝑣𝑖(𝑡)) + 𝜆∆𝑣𝑖(𝑡) (1) (1) Where 𝑥𝑖(𝑡), 𝑣𝑖(𝑡) and 𝑎𝑖(𝑡) denote the position, velocity and acceleration of the ith vehicle at time t, respectively. ℎ𝑖(𝑡) = 𝑥𝑖−1(𝑡) −𝑥𝑖(𝑡) and ∆𝑣𝑖(𝑡) = 𝑣𝑖−1(𝑡) −𝑣𝑖(𝑡) denote the headway and velocity difference between the ith vehicle and the preceding vehicle, respectively. In autonomous vehicles, 𝑝> 0 and 𝜆> 0 denote the sensitivity coefficients of the sensors built into the vehicle. 𝐹(ℎ𝑖(𝑡)) is the optimal velocity function (OVF). 1. Introduction 𝐹(ℎ𝑖(𝑡)) = 𝑣𝑚𝑎𝑥 2 ∙[tanh(ℎ𝑖(𝑡) −ℎ𝑐) + tanh(ℎ𝑐)] (2) (2) where 𝑣𝑚𝑎𝑥 denotes the maximum speed of the vehicle traveling, ℎ𝑐 denotes the safe headway, and tanh(∙) is the hyperbolic tangent function. where 𝑣𝑚𝑎𝑥 denotes the maximum speed of the vehicle traveling, ℎ𝑐 denotes the safe headway, and tanh(∙) is the hyperbolic tangent function. Based on the FVD model, a new V2V car-following model is established by considering the communication between vehicles, 𝑎𝑖(𝑡) = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝐹(ℎ𝑖,𝑙(𝑡)) 𝑛 𝑙=0 −𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙∆𝑣𝑖,𝑙(𝑡) 𝑛 𝑙=0 (3) (3) Assume that the vehicles platoon consists of n+1 vehicles including one leader (i.e., vehicle 0) and 𝑛 followers (i.e., vehicle 1 to vehicle 𝑛 ). ℎ𝑖,𝑙(𝑡) = (𝑥𝑙(𝑡) −𝑥𝑖(𝑡) −(𝑖−𝑙) ℎ𝑐 ⁄ ) (𝑖−𝑙) ⁄ denotes the average headway between the ith vehicle and the lth vehicle. 𝑟𝑖,𝑙 denotes the communication connection between vehicle i and vehicle l. If 𝑟𝑖,𝑙= 1, it means that the two vehicles establish the connection, and if 𝑟𝑖,𝑙= 0, it means that the vehicle communication is disconnected; 𝜇𝑖,𝑙 is defined as the communication weight coefficient between vehicle i and vehicle l [9], which is defined as follows, 𝜇𝑖,𝑙= { 1 (𝑖+1)𝑖−1 𝑓𝑜𝑟 𝑙= 0 𝑖 (𝑖+1)𝑖−𝑙 𝑓𝑜𝑟 𝑙∈(0, 𝑖−1) 1 − 1 (𝑖+1)𝑖−1 −∑ 𝑖 (𝑖+1)𝑖−𝜓 𝑓𝑜𝑟 𝑙= 𝑖−1 𝑖−2 𝜓=1 0 𝑓𝑜𝑟 𝑙∈[𝑖, 𝑛] (4) 𝜇𝑖,𝑙= { 1 (𝑖+1)𝑖−1 𝑓𝑜𝑟 𝑙= 0 𝑖 (𝑖+1)𝑖−𝑙 𝑓𝑜𝑟 𝑙∈(0, 𝑖−1) 1 − 1 (𝑖+1)𝑖−1 −∑ 𝑖 (𝑖+1)𝑖−𝜓 𝑓𝑜𝑟 𝑙= 𝑖−1 𝑖−2 𝜓=1 0 𝑓𝑜𝑟 𝑙∈[𝑖, 𝑛] (4) (4) Based on the above analysis, in this study, when 𝑙∈[𝑖, 𝑛], 𝜇𝑖,𝑙= 0 , that is, the vehicle mainly receives the operation information from the previous vehicle. Based on this, the dynamic equation of the intelligent connected vehicle is as follows, { 𝑑𝑥𝑖(𝑡) 𝑑𝑡 = 𝑣𝑖(𝑡) 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 (5) { 𝑑𝑥𝑖(𝑡) 𝑑𝑡 = 𝑣𝑖(𝑡) (5) 2.2 Complex Disturbance of V2V System First, complex perturbations of the system are defined [23,24]. Define 𝜔𝑣𝑖 as the velocity measurement error of vehicle i and 𝜔𝑥𝑖 as the position measurement error of vehicle i. 𝜔𝑣𝑖,𝑗 and 𝜔ℎ𝑖,𝑗, 𝑗= 0,1,2, ⋯, 𝑛 denote the velocity as well as the headway error caused by the communication between vehicle i and other vehicles, respectively. The main reasons for this error include packet loss, communication delay caused by channel congestion and communication interference caused by the external natural environment, etc. 1. Introduction Additionally, 𝑑𝑜𝑖 is defined as the disruption caused by the natural environment, such as wind gusts, rain, and snow to the ith vehicle. Bringing all the above perturbations into the intelligent connected vehicle dynamic Eq. (5), we can obtain, { 𝑑(𝑥𝑖(𝑡)+𝜔𝑥𝑖(𝑡)) 𝑑𝑡 = 𝑣𝑖(𝑡) + 𝜔𝑣𝑖 𝑑(𝑣𝑖(𝑡)+𝜔𝑣𝑖(𝑡)) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝐹(ℎ𝑖,𝑙(𝑡) + 𝜔ℎ𝑖,𝑙) 𝑖−1 𝑙=0 −(𝑣𝑖(𝑡) −𝜔𝑣𝑖)) +𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙((𝑣𝑙(𝑡) −𝜔𝑣𝑙) −(𝑣𝑖(𝑡) −𝜔𝑣𝑖)) 𝑖−1 𝑙=0 + 𝑑𝑜𝑖 (6) (6) For convenience, it is reasonable to define 𝑑𝐹(𝜔ℎ𝑖,𝑙) as the perturbation deviation of the standard desired velocity 𝐹(ℎ𝑖,𝑙(𝑡)), then, 𝐹(ℎ𝑖,𝑙(𝑡) + 𝜔ℎ𝑖,𝑙) = 𝐹(ℎ𝑖,𝑙(𝑡)) + 𝑑𝐹(𝜔ℎ𝑖,𝑙). Further, define 𝑑𝑥𝑖 and 𝑑𝑣𝑖 of the following form. 𝑑𝐹(𝜔ℎ𝑖,𝑙). Further, define 𝑑𝑥𝑖 and 𝑑𝑣𝑖 of the following form. { 𝑑𝑥𝑖= 𝜔𝑣𝑖− 𝑑𝜔𝑥𝑖(𝑡) 𝑑𝑡 = 0 𝑑𝑣𝑖= 𝑝∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙 𝑖−1 𝑙=0 𝑑𝐹(𝜔ℎ𝑖,𝑙) + 𝑝𝜔𝑣𝑖+ 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙(𝜔𝑣𝑙−𝜔𝑣𝑖) 𝑖−1 𝑙=0 + 𝑑𝑜𝑖− 𝑑𝜔𝑣𝑖(𝑡) 𝑑𝑡 (7) (7) Based on the above form of perturbation, Eq. (6) can be rewritten as follows. { 𝑑𝑥𝑖(𝑡) 𝑑𝑡 = 𝑣𝑖(𝑡) 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 + 𝑑𝑣𝑖 (8) (8) {𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 + 𝑑𝑣𝑖 (8) Assume that the leading vehicle of the connected vehicle platoon maintains a desired steady state and the entire vehicle platoon also runs in steady state [𝑣∗, ℎ∗]𝑇. Define the various errors in the deviation from the steady state of the connected vehicle system as 𝛿𝑥𝑖(𝑡) = 𝑥𝑖(𝑡) −𝑥0(𝑡) + 𝑖∙ℎ∗ , 𝛿𝑣𝑖(𝑡) = 𝑣𝑖(𝑡) −𝑣∗ and 𝛿𝐹(ℎ𝑖,𝑙) = 𝐹(ℎ𝑖,𝑙) −𝐹(ℎ𝑖,𝑙 ∗) = 𝛿𝑥𝑙(𝑡)−𝛿𝑥𝑖(𝑡) 𝑖−𝑙 𝐹′(ℎ𝑖,𝑙 ∗) , respectively. Further introducing the controller 𝑢𝑖(𝑡) in system (8) to improve the state of the system, system (8) can be rewritten as, { 𝑑𝛿𝑥𝑖(𝑡) 𝑑𝑡 = 𝛿𝑣𝑖(𝑡) 𝑑𝛿𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝛿𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 + 𝑑𝑣𝑖+ 𝑢𝑖(𝑡) { 𝑑𝛿𝑥𝑖(𝑡) 𝑑𝑡 = 𝛿𝑣𝑖(𝑡) { 𝑑𝑡 𝑖( ) 𝑑𝛿𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝛿𝑣𝑖(𝑡)) + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 + 𝑑𝑣𝑖+ 𝑢𝑖(𝑡) ( 9 ) ( 9 ) 3. Improved Adaptive Exponential Reaching Sliding Mode Controller Design 3. Improved Adaptive Exponential Reaching Sliding Mode Controller Design 3. Improved Adaptive Exponential Reaching Sliding Mode Controller Design Design This section discusses the design of an improved adaptive exponential reaching sliding mode controller based on non-singular terminal sliding variables to optimize the intelligent connected vehicle system for excellent steady-state and dynamic performance [29,30]. 1. Introduction First, choose the non-singular terminal sliding mode variable as, 𝑠𝑖(𝑡) = 𝛿𝑥𝑖(𝑡) + 𝛽|𝛿𝑣𝑖(𝑡)|𝛼∙sign(𝛿𝑣𝑖(𝑡)) (10) where, 1 < 𝛼< 2, 𝛽> 0, sign(∙) is the signum function. 𝑠𝑖(𝑡) = 𝛿𝑥𝑖(𝑡) + 𝛽|𝛿𝑣𝑖(𝑡)|𝛼∙sign(𝛿𝑣𝑖(𝑡)) (10) where, 1 < 𝛼< 2, 𝛽> 0, sign(∙) is the signum function. (10) Taking the first order derivative of the sliding variable 𝑠𝑖(𝑡) yields, 𝑠̇𝑖(𝑡) = 𝑑𝛿𝑥𝑖(𝑡) 𝑑𝑡 + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙ 𝑑𝛿𝑣𝑖(𝑡) 𝑑𝑡 (11) Bringing system (9) into 𝑠̇𝑖(𝑡), (11) 𝑠̇𝑖(𝑡) = 𝛿𝑣𝑖(𝑡) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑝(∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−1 𝑙=0 −𝛿𝑣𝑖(𝑡)) + ∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿∆𝑣𝑖,𝑙(𝑡) 𝑖−1 𝑙=0 ) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑑𝑣𝑖+ 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑢𝑖(𝑡) (12) The error generated by perturbation, the measurement error and the error generated by V2V communication are defined as the input interference 𝑑𝐼𝑁𝐸𝑖, i.e. The error generated by perturbation, the measurement error and the error generated by V2V communication are defined as the input interference 𝑑𝐼𝑁𝐸𝑖, i.e. 𝑑𝐼𝑁𝐸𝑖= 𝑝∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿𝐹(ℎ𝑖,𝑙(𝑡)) 𝑖−2 𝑙=0 + 𝜆∑ 𝑟𝑖,𝑙𝜇𝑖,𝑙𝛿∆𝑣𝑖,𝑙(𝑡) 𝑖−2 𝑙=0 + 𝑑𝑣𝑖 (13) (13) Thus, 𝑠̇𝑖(𝑡) can be further rewritten as, 𝑠̇𝑖(𝑡) = 𝛿𝑣𝑖(𝑡) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑝𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑝𝛿𝑣𝑖(𝑡) + 𝑠̇𝑖(𝑡) = 𝛿𝑣𝑖(𝑡) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑝𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑝𝛿𝑣𝑖(𝑡) + 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡)) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑑𝐼𝑁𝐸𝑖+ 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑢𝑖(𝑡) 𝑠𝑖(𝑡) = 𝛿𝑣𝑖(𝑡) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)| (𝑝𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑝𝛿𝑣𝑖(𝑡) + 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡)) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑑𝐼𝑁𝐸𝑖+ 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑢𝑖(𝑡) (14) 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡)) + 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑑𝐼𝑁𝐸𝑖+ 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝑢𝑖(𝑡) (14) (14) For the system (9), convergence in finite-time (i.e., lim 𝑡→𝑡1 𝛿𝑥𝑖(𝑡) = 0 ) can be achieved if the general constant speed reaching controller 𝑢𝑖1(𝑡), is chosen as follows. 1. Introduction 𝑢𝑖1(𝑡) = − 1 𝛼𝛽|𝛿𝑣𝑖(𝑡)|2−𝛼∙sign(𝛿𝑣𝑖(𝑡)) + 𝑞𝛿𝑣𝑖(𝑡) −𝑞𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) − 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −𝑘∙sign(𝑠𝑖(𝑡)) (15) (15) At this time, the first order derivative of the sliding mode variable 𝑠̇𝑖(𝑡) is as follows At this time, the first order derivative of the sliding mode variable 𝑠̇𝑖(𝑡) is as follows 𝑠̇𝑖(𝑡) = 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑑𝐼𝑁𝐸𝑖−𝑘∙sign(𝑠𝑖(𝑡))) (16) (16) In this study, in order to obtain better dynamic performance, we improve the above In this study, in order to obtain better dynamic performance, we improve the above general constant speed reaching controller (15) and propose the following improved adaptive exponential reaching sliding mode controller (17), which can guarantee the system to obtain better dynamic performance while converging in finite time, and the new controller 𝑢𝑖(𝑡) is designed as follows, 𝑢𝑖(𝑡) = − 1 𝛼𝛽|𝛿𝑣𝑖(𝑡)|2−𝛼∙sign(𝛿𝑣𝑖(𝑡)) + 𝑞𝛿𝑣𝑖(𝑡) −𝑞𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) − 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −( 𝑘 𝑔(𝑠𝑖) sign(𝑠𝑖(𝑡)) + 𝜑𝑠𝑖(𝑡)) (17) 𝑢𝑖(𝑡) = − 1 𝛼𝛽|𝛿𝑣𝑖(𝑡)|2−𝛼∙sign(𝛿𝑣𝑖(𝑡)) + 𝑞𝛿𝑣𝑖(𝑡) −𝑞𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) − 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −( 𝑘 𝑔(𝑠𝑖) sign(𝑠𝑖(𝑡)) + 𝜑𝑠𝑖(𝑡)) (17) (17) At this time, the first order derivative of the sliding mode variable 𝑠̇𝑖(𝑡) is as follows At this time, the first order derivative of the sliding mode variable 𝑠̇𝑖(𝑡) is as follows 𝑠̇𝑖(𝑡) = 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑑𝐼𝑁𝐸𝑖− 𝑘 𝑔(𝑠𝑖) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡)) (18) where 𝑔(𝑠𝑖) = 𝜀+ 𝜃(1 −tanh2(𝜔|𝑠𝑖(𝑡)|)), 0 < 𝜀< 1. 𝑠̇𝑖(𝑡) = 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 ∙(𝑑𝐼𝑁𝐸𝑖− 𝑘 𝑔(𝑠𝑖) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡)) (18) (18) It is seen that the improved controller (17) adds two components 𝜑𝑠𝑖(𝑡) and 𝑔(𝑠𝑖) compared to the general constant speed reaching controller (15). For 𝜑𝑠𝑖(𝑡), it can accelerate the reaching speed when the system trajectory is far away from the sliding surface, and close to zero when it is near the surface. For 𝑔(𝑠𝑖) , if the value of |𝑠𝑖(𝑡)| increases, 𝑔(𝑠𝑖) approximates 𝜀 and 𝑘 𝜀> 𝑘. Whereas when the value of |𝑠𝑖(𝑡)| decreases close to zero, 𝑔(𝑠𝑖) approximates 𝜀+ 𝜃. This means that a faster reaching time can be obtained when the system trajectory is far away from the sliding surface, whereas when the system trajectory is near the sliding surface, if 𝜀+ 𝜃> 1, it reduces the gain of the signal function to serve to suppress chattering. This analysis of the specific dynamic process will be shown in section 4.2. 4. Performance Analysis 4.1 Convergence Analysis Lemma 1: If Lyapunov function 𝑉(𝑠) is defined on a neighborhood of the origin and 𝑉̇ (𝑥) + 𝜆0𝑉𝑟(𝑥) ≤0 ,𝜆0 > 0 ,0 < 𝑟< 1 , there exists an area, which can reach within finite time 𝑇𝑟𝑒𝑎𝑐ℎ≤𝑉1−𝑟(0) [𝜆0(1 −𝑟)] ⁄ with the initial value of 𝑉(0). When 𝛿𝑣𝑖(𝑡) ≠0, choose Lyapunov function 𝑉1 as 1 = 1 2 𝑠𝑖 2(𝑠) (19) 𝑉1 = 1 2 𝑠𝑖 2(𝑠) (19) 𝑉1 = 1 2 𝑠𝑖 2(𝑠) (19) aking the derivative of 𝑉1 gives, Taking the derivative of 𝑉1 gives, 𝑉̇1 = 𝑠𝑖(𝑡)𝑠̇𝑖(𝑡) = 𝑠𝑖(𝑡) ∙𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 (𝑑𝐼𝑁𝐸𝑖− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) − 𝜑𝑠𝑖(𝑡)) = 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 (− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) |𝑠𝑖(𝑡)| + 𝑑𝐼𝑁𝐸𝑖𝑠𝑖(𝑡) −𝜑𝑠𝑖 2(𝑡)) ≤ 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 (− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) |𝑠𝑖(𝑡)| + 𝑑𝐼𝑁𝐸𝑖𝑠𝑖(𝑡)) ≤ 𝑠𝑖(𝑡)) = 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 (− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) |𝑠𝑖(𝑡)| + 𝑑𝐼𝑁𝐸𝑖𝑠𝑖(𝑡) −𝜑𝑠𝑖 2(𝑡)) ≤ 𝑘 tanh2(𝜔|𝑠𝑖(𝑡)|)) −𝑑1) |𝑠𝑖(𝑡)| ∙𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 (20) (20) where 𝑑1 is a positive number, and it is assumed that the input interference 𝑑𝐼𝑁𝐸𝑖 satisfies |𝑑𝐼𝑁𝐸𝑖| ≤𝑑1. where 𝑑1 is a positive number, and it is assumed that the input interference 𝑑𝐼𝑁𝐸𝑖 satisfies |𝑑𝐼𝑁𝐸𝑖| ≤𝑑1. s |𝑑𝐼𝑁𝐸𝑖| ≤𝑑1. The following relationship exists, The following relationship exists, ( 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|))) min = 𝑘 𝜀+𝜃 (21) (21) If appropriate values of 𝑘, 𝜀, 𝜃 are chosen such that 𝑘 𝜀+𝜃≥𝑑1 + 𝜉, where 𝜉> 0. here, If appropriate values of 𝑘, 𝜀, 𝜃 are chosen such that 𝑘 𝜀+𝜃≥𝑑1 + 𝜉, where 𝜉> 0. here, 𝑉̇1 = 𝑠𝑖(𝑡)𝑠̇𝑖(𝑡) ≤𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝜉|𝑠𝑖(𝑡)| = −√2𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝜉𝑉1 1 2 (22) (22) According to (22) and Lemma 1, the system trajectory converges to origin within 1 finite time such that lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ1 𝑠𝑖(𝑡) = 0, 𝑇𝑟𝑒𝑎𝑐ℎ1 ≤ √2𝑉1 1 2(0) 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝜉, where is the settling time and 𝑉1(0) is the initial value of 𝑉1. e such that lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ1 𝑠𝑖(𝑡) = 0, 𝑇𝑟𝑒𝑎𝑐ℎ1 ≤ √2𝑉1 1 2(0) 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝜉, where is the settling finite time such that lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ1 𝑠𝑖(𝑡) = 0, 𝑇𝑟𝑒𝑎𝑐ℎ1 ≤ √2𝑉1 2(0) 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1𝜉, where is the settling time and 𝑉1(0) is the initial value of 𝑉1. time and 𝑉1(0) is the initial value of 𝑉1. time and 𝑉1(0) is the initial value of 𝑉1. time and 𝑉1(0) is the initial value of 𝑉1. 𝑉1(0) is the initial value of 𝑉1. 4.1 Convergence Analysis Let 𝜌≥2 1+𝛼 2𝛼( 1 𝛽) 1 𝛼>0, (26) 𝑉̇2 ≤−𝜌𝑉2 1+𝛼 2𝛼 𝑉̇2 ≤−𝜌𝑉2 1+𝛼 2𝛼 (27) 𝑉̇2 ≤−𝜌𝑉2 1+𝛼 2𝛼 (27) (27) According to (27) and Lemma 1, after the system trajectory arrives on the sliding According to (27) and Lemma 1, after the system trajectory arrives on the sliding surface 𝑠𝑖(𝑡) = 0, in finite time 𝑇𝑟𝑒𝑎𝑐ℎ2 ≤ 𝑉2 1−1+𝛼 2𝛼(0) 𝜌(1−1+𝛼 2𝛼), we can obtain lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ2 𝜎𝑖(𝑡) = surface 𝑠𝑖(𝑡) = 0, in finite time 𝑇𝑟𝑒𝑎𝑐ℎ2 ≤ 𝑉2 1−1+𝛼 2𝛼(0) 𝜌(1−1+𝛼 2𝛼), we can obtain lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ2 𝜎𝑖(𝑡) = lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ2 𝛿𝑥𝑖(𝑡) = 0 . And for the whole process of intelligent vehicles platoon ℎ2 𝛿𝑥𝑖(𝑡) = 0. tion, there exists lim 𝑡→𝑇𝑟𝑒𝑎𝑐ℎ1+𝑇𝑟𝑒𝑎𝑐ℎ2 𝛿𝑥𝑖(𝑡) = 0. 4.2 Dynamic Analysis 4.1 Convergence Analysis When 𝛿𝑣𝑖(𝑡) = 0, bringing the controller (17) into the system (9) yields that, 𝑑𝛿𝑣𝑖 𝑑𝑡= − 1 𝛼𝛽|𝛿𝑣𝑖|2−𝛼− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) + 𝑑𝐼𝑁𝐸𝑖= − 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) + 𝑑𝐼𝑁𝐸𝑖 (23) 𝑑𝛿𝑣𝑖 𝑑𝑡= − 1 𝛼𝛽|𝛿𝑣𝑖|2−𝛼− 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) + 𝑑𝐼𝑁𝐸𝑖= 𝑘 i ( ( )) ( ) 𝑑 (23 − 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) + 𝑑𝐼𝑁𝐸𝑖 (23 (23) When 𝛿𝑣̇𝑖(𝑡) < 0 , 𝑠𝑖(𝑡) > 0 ; conversely, when 𝛿𝑣̇𝑖(𝑡) > 0 , 𝑠𝑖(𝑡) < 0 . This indicates that 𝛿𝑣𝑖(𝑡) = 0 is not an attractor, i.e., the trajectory of the system will not stop on 𝛿𝑣𝑖(𝑡) = 0, but will continue to move to the sliding surface 𝑠𝑖(𝑡) = 0 on it. And, after the system state arrives on the sliding surface 𝑠𝑖(𝑡) = 0, the system state will continue to move along the sliding surface to the origin, i.e., 𝛿𝑣𝑖(𝑡) = 0, 𝛿𝑥𝑖(𝑡) = 0. will continue to move along the sliding surface to the origin, i.e., 𝛿𝑣𝑖(𝑡) = 0, 𝛿𝑥𝑖(𝑡) = 0. 0. When 𝑠𝑖(𝑡) = 0, the following equation can be derived from the sliding mode variable (10), 𝛿𝑣𝑖(𝑡) = −( 1 𝛽) 1 𝛼|𝛿𝑥𝑖(𝑡)| 1 𝛼sign(𝛿𝑥𝑖(𝑡)) (24) 𝛿𝑣𝑖(𝑡) = −( 1 𝛽) 1 𝛼|𝛿𝑥𝑖(𝑡)| 1 𝛼sign(𝛿𝑥𝑖(𝑡)) (24) Let 𝜎𝑖(𝑡) = 𝛿𝑥𝑖(𝑡), choose Lyapunov function 𝑉2 as (24) Let 𝜎𝑖(𝑡) = 𝛿𝑥𝑖(𝑡), choose Lyapunov function 𝑉2 as 𝑉2 = 1 2 𝜎𝑖 2(𝑡) (25) (25) Taking the derivative of 𝑉2 gives, Taking the derivative of 𝑉2 gives, 𝑉̇2 = 𝜎𝑖(𝑡)𝜎̇𝑖(𝑡) = 𝛿𝑥𝑖(𝑡) ∙(−( 1 𝛽) 1 𝛼|𝛿𝑥𝑖(𝑡)| 1 𝛼sign(𝛿𝑥𝑖(𝑡))) = 𝑉̇2 = 𝜎𝑖(𝑡)𝜎̇𝑖(𝑡) = 𝛿𝑥𝑖(𝑡) ∙(−( 1 𝛽) 𝛼|𝛿𝑥𝑖(𝑡)| 1 𝛼sign(𝛿𝑥𝑖(𝑡))) = −( 1 𝛽) 1 𝛼|𝛿𝑥𝑖(𝑡)|1+1 𝛼sign(𝛿𝑥𝑖(𝑡)) = −2 1+𝛼 2𝛼( 1 𝛽) 1 𝛼𝑉2 1+𝛼 2𝛼 (26) Since 1 < 𝛼< 2, 1+𝛼 2𝛼< 1. Let 𝜌≥2 1+𝛼 2𝛼( 1 𝛽) 1 𝛼>0, −( 1 𝛽) 1 𝛼|𝛿𝑥𝑖(𝑡)|1+1 𝛼sign(𝛿𝑥𝑖(𝑡)) = −2 1+𝛼 2𝛼( 1 𝛽) 1 𝛼𝑉2 1+𝛼 2𝛼 (26) Since 1 < 𝛼< 2, 1+𝛼 2𝛼< 1. 4.2 Dynamic Analysis In this subsection, the improvement of the system dynamic performance by the improved controller (IAERSMC) is discussed. First, comparing the general constant speed reaching controller (15) with the improved controller (17) in this study, the −𝜑𝑠𝑖(𝑡) term of the exponential reaching law is introduced. Due to the introduction of the −𝜑𝑠𝑖(𝑡) term, when the value of the sliding mode variable 𝑠𝑖(𝑡) is larger, its first-order derivative 𝑠̇𝑖(𝑡) is also larger, i.e., the reaching speed is accelerated, and when 𝑠𝑖(𝑡) reaches near the sliding mode surface and close to zero, the −𝜑𝑠𝑖(𝑡) term will also converge to zero. Therefore, the introduction of the −𝜑𝑠𝑖(𝑡) term can accelerate the reaching speed without changing the amplitude of the chattering around 𝑠𝑖(𝑡) = 0 after the sliding mode variable reaches the sliding mode surface, so it can improve the dynamic performance of the system. Next, let's discuss the control effect of another improvement on the system. For convenience, let 𝑑𝐼𝑁𝐸𝑖= 0,𝜑= 0,𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 = 1, here, onvenience, let 𝑑𝐼𝑁𝐸𝑖= 0,𝜑= 0,𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 = 1, here, 𝑠̇𝑖(𝑡) = − 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) (28) (28) 𝑠̇𝑖(𝑡) = − 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) (28) Simple treatment of the above formula, Simple treatment of the above formula, 𝑑𝑠 𝑑𝑡= − 𝑘 𝜀+𝜃(1−tanh2(𝜔|𝑠𝑖(𝑡)|)) sign(𝑠𝑖(𝑡)) (29) (29) Shift items on both sides of the equals sign, Shift items on both sides of the equals sign, 1 sign(𝑠𝑖(𝑡)) (𝜀+ 𝜃(1 −tanh2(𝜔|𝑠𝑖(𝑡)|))) 𝑑𝑠= −𝑘𝑑𝑡 (30) (30) Assume that 𝑡1 is the time required for the sliding mode variable 𝑠𝑖(𝑡) of the system under the action of the improved controller (17) to reach the sliding mode surface 𝑠𝑖(𝑡) = 0 for the first time. Integrating both sides of the equal sign of equation (30). ∫ 1 sign(𝑠𝑖(𝑡)) (𝜀+ 𝜃(1 −tanh2(𝜔|𝑠𝑖(𝑡)|))) 𝑑𝑠 𝑠𝑡1 𝑠0 = ∫ −𝑘𝑑𝑡 𝑡1 0 (31) (31) Where, 𝑠0 denotes the initial value of the sliding mode variable and 𝑠𝑡1 = 0. 4.2 Dynamic Analysis When 𝑠0 > 0, When 𝑠0 > 0, ∫ 1 sign(𝑠𝑖(𝑡)) (𝜀+ 𝜃(1 −tanh2(𝜔𝑠𝑖(𝑡)))) 𝑑𝑠 𝑠𝑡1 𝑠0 = ∫ −𝑘𝑑𝑡 𝑡1 0 (32) (32) sign(𝑠𝑖(𝑡)) 0 Here, Here, 𝑡1 = 1 𝑘(𝜀𝑠0 + 𝜃 𝜔tanh(𝜔𝑠0)) (33) (33) 𝑡1 = 𝑘(𝜀𝑠0 + 𝜔tanh(𝜔𝑠0)) (33) When 𝑠0 < 0, When 𝑠0 < 0, ∫ 1 sign(𝑠𝑖(𝑡)) (𝜀+ 𝜃(1 −tanh2(−𝜔𝑠𝑖(𝑡)))) 𝑑𝑠 𝑠𝑡1 𝑠0 = ∫ −𝑘𝑑𝑡 𝑡1 0 (34) (34) Here, Here, 𝑡1 = 1 𝑘(−𝜀𝑠0 + 𝜃 𝜔tanh(𝜔𝑠0)) (35) 𝑡1 = 1 𝑘(−𝜀𝑠0 + 𝜃 𝜔tanh(𝜔𝑠0)) (35) And thus, And thus, And thus, 𝑡1 = 1 𝑘(−𝜀|𝑠0| + 𝜃 𝜔tanh(𝜔|𝑠0|)) (36) 𝑡1 = 1 𝑘(−𝜀|𝑠0| + 𝜃 𝜔tanh(𝜔|𝑠0|)) (36) (36) Given that 0 < tanh(𝜔|𝑠0|) < 1, the following inequality holds, 𝑡1 < 1 𝑘(−𝜀|𝑠0| + 𝜃 𝜔) (37) 𝑡1 < 1 𝑘(−𝜀|𝑠0| + 𝜃 𝜔) (37) (37) If the values of 𝜃, 𝜔, 𝑘 satisfy 𝑘𝜔≫𝜃, then, 𝑡1 < 𝜀 𝑘|𝑠0| (38) 𝑡1 < 𝜀 𝑘|𝑠0| (38) 𝑡1 < 𝜀 𝑘|𝑠0| (38) And for controller (15), again let 𝑑𝐼𝑁𝐸𝑖= 0 and 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 = 1, then, And for controller (15), again let 𝑑𝐼𝑁𝐸𝑖= 0 and 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 = 1, then, And for controller (15), again let 𝑑𝐼𝑁𝐸𝑖= 0 and 𝛼𝛽|𝛿𝑣𝑖(𝑡)|𝛼−1 = 1, then, 𝑠̇𝑖(𝑡) = −𝑘∙sign(𝑠𝑖(𝑡)) (39) (39) Similarly, expand and shift items, 1 sign(𝑠𝑖(𝑡)) 𝑑𝑠= −𝑘𝑑𝑡 (40) (40) Integrating both sides of the equal sign, we can obtain that Integrating both sides of the equal sign, we can obtain that 𝑡0 = 1 𝑘|𝑠0| (41) 𝑡0 = 1 𝑘|𝑠0| (41) Here, 𝑡0 denotes the time required for the sliding mode variable 𝑠𝑖(𝑡) of the system to reach the sliding mode surface 𝑠𝑖(𝑡) = 0 for the first time under the action of the general constant speed reaching controller (15), Since the value 0 < 𝜀< 1 taken in the improved controller (17), then 𝑡< 𝜀|𝑠| < 1 |𝑠| = 𝑡 Since the value 0 < 𝜀< 1 taken in the improved controller (17), then Since the value 0 < 𝜀< 1 taken in the improved controller (17), then 𝑡1 < 𝜀 𝑘|𝑠0| < 1 𝑘|𝑠0| = 𝑡0 (42) 𝑡1 < 𝜀 𝑘|𝑠0| < 1 𝑘|𝑠0| = 𝑡0 (42) 𝑡1 < 𝜀 𝑘|𝑠0| < 1 𝑘|𝑠0| = 𝑡0 (42) That is, the improved controller in this study can effectively reduce the time for the sliding mode variables to reach the sliding surface. 4.2 Dynamic Analysis On the other hand, let us discuss the effect of the improved controller (17) on the chattering of the sliding mode variables near the sliding mode surface. On the other hand, let us discuss the effect of the improved controller (17) on the chattering of the sliding mode variables near the sliding mode surface. For the reaching speed (28) of the sliding mode variable, the dynamic characteristics of the sliding mode switching function 𝑠𝑖(𝑡) near the sliding mode surface 𝑠𝑖(𝑡) = 0 can be expressed as, 𝑠𝑖−1(𝑡)−𝑠𝑖(𝑡) 𝑇 = − 𝑘 𝜀+𝜃 sign(𝑠𝑖(𝑡)) (43 (43) where 𝑇 denotes the system sampling period. When the sliding mode variable 𝑠𝑖(𝑡) is close enough to the sliding mode surface, it can be considered that there are two cases of 𝑠𝑖(𝑡) →0+ or 𝑠𝑖(𝑡) →0−, and then, where 𝑇 denotes the system sampling period. 5. Simulation Analysis 5.1 Different Communication Environment 4.2 Dynamic Analysis When the sliding mode variable 𝑠𝑖(𝑡) is close enough to the sliding mode surface, it can be considered that there are two cases of 𝑠𝑖(𝑡) →0+ or 𝑠𝑖(𝑡) →0−, and then, { 𝑠𝑖−1(𝑡) = − 𝑘𝑇 𝜀+𝜃, 𝑠𝑖(𝑡) →0+ 𝑠𝑖−1(𝑡) = 𝑘𝑇 𝜀+𝜃, 𝑠𝑖(𝑡) →0− (44) (44) Define the chattering width of the sliding mode variable under the action of the improved controller (17) to be ∆1 in the vicinity of the sliding mode surface, then, Define the chattering width of the sliding mode variable under the action of the improved controller (17) to be ∆1 in the vicinity of the sliding mode surface, then, ∆1= 2𝑘𝑇 𝜀+𝜃 (45 ∆1= 2𝑘𝑇 𝜀+𝜃 (45) And for the reaching speed (39) of the sliding mode variable of the general constant speed reaching controller (15), the dynamic characteristics of the sliding mode variable 𝑠𝑖(𝑡) near the sliding mode surface 𝑠𝑖(𝑡) = 0 can be expressed as, 𝑠𝑖−1(𝑡)−𝑠𝑖(𝑡) 𝑇 = −𝑘∙sign(𝑠𝑖(𝑡)) (46) (46) When 𝑠𝑖(𝑡) is close enough to the sliding surface, {𝑠𝑖−1(𝑡) = −𝑘𝑇, 𝑠𝑖(𝑡) →0+ 𝑠𝑖−1(𝑡) = 𝑘𝑇, 𝑠𝑖(𝑡) →0− (47) (47) Define the chattering width of the sliding mode variable under the action of the controller (15) to be ∆0 in the vicinity of the sliding mode surface, then, ∆0= 2𝑘𝑇 (48) ∆0= 2𝑘𝑇 (48) If the parameters of the controller are taken to satisfy 𝜀+ 𝜃> 1, then ∆1= 2𝑘𝑇 𝜀+𝜃< 2𝑘𝑇= ∆0 (49) ∆1= 2𝑘𝑇 𝜀+𝜃< 2𝑘𝑇= ∆0 (49 (49) That is, the improved controller proposed in this study can effectively reduce the chattering width of the sliding mode variable near the sliding mode surface. In summary, the parameter values of the improved controller (17) designed in this study need to satisfy 𝑘𝜔≫𝜃 and 𝜀+ 𝜃> 1 . As a result, the controller can effectively shorten the reaching speed and reduce the chattering amplitude, which effectively improves the dynamic performance of the system of the intelligent connected vehicle. 5. Simulation Analysis 5.1 Different Communication Environment Communication is affected by many factors, such as the blockage of buildings, more closed underground tunnels, rain and snow and other natural weather changes. Since the external environment would disrupt the vehicle-vehicle communication network during the platooning process, accordingly, the communication connection between vehicles cannot guarantee absolute reliability. There are various communication topologies for intelligent connected vehicles, including Predecessor Following (PF), Predecessor-Leader Following (PLF), Two-Predecessor Following (TPF) and Two-Predecessor-Leader Following (TPLF). Several typical communication topologies illustrate in Fig. 2, and the V2V dynamics models corresponding to various communication topologies are Eqs. (50)-(53) shown respectively. In order to operate more steadily, the intelligent connected vehicle will modify the communication topology of the system in accordance with the various operational environments. Communication topology switching could interfere with the operation of the connected vehicle platoon, and different communication environments also produce different effects on the operation of the platoon. PF topology: PF topology: PF topology: PF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑣𝑖(𝑡)) + 𝜆∆𝑣𝑖,𝑖−1(𝑡) (50) PLF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑣𝑖(𝑡)) + 𝜆∆𝑣𝑖,𝑖−1(𝑡) (50) (50) 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,0𝐹(ℎ𝑖,0(𝑡)) + 𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,0∆𝑣𝑖,0(𝑡) + 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,0𝐹(ℎ𝑖,0(𝑡)) + 𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,0∆𝑣𝑖,0(𝑡) + 𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡)) (51) (51) TPF topology: TPF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡)) (52 TPF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇 ∆𝑣 (𝑡) + 𝜇 ∆𝑣 (𝑡)) 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡)) (52) (52) 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡)) (52) TPLF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) + 𝜇𝑖,0𝐹(ℎ𝑖,0(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡) + 𝜇𝑖,𝑖−0∆𝑣𝑖,𝑖−0(𝑡)) (53) TPLF topology: 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) + 𝜇𝑖,0𝐹(ℎ𝑖,0(𝑡)) −𝑣𝑖(𝑡)) + 𝑑𝑣𝑖(𝑡) 𝑑𝑡 = 𝑝(𝜇𝑖,𝑖−1𝐹(ℎ𝑖,𝑖−1(𝑡)) + 𝜇𝑖,𝑖−2𝐹(ℎ𝑖,𝑖−2(𝑡)) + 𝜇𝑖,0𝐹(ℎ𝑖,0(𝑡)) −𝑣𝑖(𝑡)) + 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡) + 𝜇𝑖,𝑖−0∆𝑣𝑖,𝑖−0(𝑡)) (53) 𝑑𝑡 𝜆(𝜇𝑖,𝑖−1∆𝑣𝑖,𝑖−1(𝑡) + 𝜇𝑖,𝑖−2∆𝑣𝑖,𝑖−2(𝑡) + 𝜇𝑖,𝑖−0∆𝑣𝑖,𝑖−0(𝑡)) (53) (53) Fig.2 Illustration of typically connected vehicle communication topology Fig.2 Illustration of typically connected vehicle communication topology In this section, we discuss the operation of the intelligent vehicles platoon in different communication environments and the effect of the improved controller (IAEASMC) proposed in this study. Set up the following simulation environment, a platoon of 10 intelligent connected vehicles driving on an infinitely long road, no lane change or overtaking occurs, and the platoon is uniformly arranged at a headway of ℎ∗= 10𝑚. 5.1 Different Communication Environment The initial velocity of all vehicles is 𝑣0 = 𝐹(ℎ∗), each simulation step is 0.1 seconds, and the total simulation step is set to 1200 steps. Assuming the initial communication topology of the connected vehicle platoon is TPLF, as the communication environment changes, the communication topology of the platoon changes to PLF, TPF and PF at the 300th, 600th and 900th steps, respectively, in that order. To increase the accuracy and reasonableness of the experiment, a disturbance of size 2 𝑚𝑠 ⁄ is added to the velocity of the pilot car at the 50th step before the communication topology change to represent the change in the communication environment. In this case, the velocity of the leading vehicle changes as 𝑣0(𝑡) = 𝐹(ℎ∗) + 2𝑒−(𝑡−250)2 2×62 −2𝑒−(𝑡−550)2 2×62 + 2𝑒−(𝑡−850)2 2×62 . The four controllers are set up as follows: no control; the general constant speed reaching controller, as shown in Eq. (15), takes the parameter value of 𝑘= 2.0; the general exponential reaching controller, as shown in Eq. (51), the parameters are 𝑘= 2.1, 𝜑= 0.8 ; The improved controller proposed in this study, as shown in Eq. (17), takes the values of 𝑘= 2.1, 𝜀= 0.7, 𝜃= 1.2, 𝜔= 6.5, 𝜑= 0.8 and the other parameters take the values of 𝛼= 1.8, 𝛽= 𝐹(ℎ∗) + 2𝑒−(𝑡−250)2 2×62 −2𝑒−(𝑡−550)2 2×62 + 2𝑒−(𝑡−850)2 2×62 . The four controllers are set up as follows: no control; the general constant speed reaching controller, as shown in Eq. (15), takes the parameter value of 𝑘= 2.0; the general exponential reaching controller, as shown in Eq. (51), the parameters are 𝑘= 2.1, 𝜑= 0.8 ; The improved controller proposed in this study, as shown in Eq. (17), takes the values of 𝑘= 2.1, 𝜀= 0.7, 𝜃= 1.2, 𝜔= 6.5, 𝜑= 0.8 and the other parameters take the values of 𝛼= 1.8, 𝛽= 1.2, 𝑝= 2.0, 𝜆= 0.5. 1.2, 𝑝= 2.0, 𝜆= 0.5. 𝑢𝑖2(𝑡) = − 1 𝛼𝛽|𝛿𝑣𝑖(𝑡)|2−𝛼∙sign(𝛿𝑣𝑖(𝑡)) + 𝑞𝛿𝑣𝑖(𝑡) −𝑞𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) − 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −𝑘∙sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) (54) 𝑢𝑖2(𝑡) = − 1 𝛼𝛽|𝛿𝑣𝑖(𝑡)|2−𝛼∙sign(𝛿𝑣𝑖(𝑡)) + 𝑞𝛿𝑣𝑖(𝑡) −𝑞𝜇𝑖,𝑖−1𝛿𝐹(ℎ𝑖,𝑖−1(𝑡)) − 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −𝑘∙sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) (54) 𝜆𝜇𝑖,𝑖−1𝛿∆𝑣𝑖,𝑖−1(𝑡) −𝑘∙sign(𝑠𝑖(𝑡)) −𝜑𝑠𝑖(𝑡) (54) (54) Fig. 3-6 illustrate that the improved controller proposed in this study can better overcome the negative impact of the change in the communication environment on the operation of the intelligent connected vehicle platoon, enabling the better operational performance of the platoon. The specific results are summarized as follows. 5.1 Different Communication Environment Fig.3 The position error profiles under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.3 The position error profiles under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.4 The velocity profiles under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.4 The velocity profiles under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.5 The acceleration profiles under different communication environments: (a) no control; (b) Fig.5 The acceleration profiles under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.6 Sliding Variable Response Curve of the Fifth Vehicle under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.6 Sliding Variable Response Curve of the Fifth Vehicle under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.6 Sliding Variable Response Curve of the Fifth Vehicle under different communication environments: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig. 3 - Fig. 6 show the position, velocity, and acceleration of the vehicles in the platoon and the variation of the sliding mode variable of the fifth vehicle in the platoon over time for the case of a change in the communication environment, respectively. The (a) - (d) modules in the figure represent the situation under the action of no controller, general constant speed reaching controller, general exponential reaching controller and IAERSMC respectively. First, comparing the operation of (a) with (b)-(c) in Fig. 3-Fig. 6, it can be observed that the performance of the system for the case without controller is much lower than the remaining three cases using controller. And for the performance of the remaining three controller control effects, it can be found that the improved controller represented by (d) diagram has the most effective results. 5.1 Different Communication Environment From the variation of vehicle position information over time represented in Fig. 3, it can be found that the curves of (c)(d) are smoother than (b), while at the communication topology switching, (d) is smoother compared to (c), indicating that the vehicles in the platoon follow each other better and the vehicles operate more stably under the action of the improved controller. The same conclusion can be obtained in Fig. 4-Fig. 6. Under the improved controller, the system vehicle operates more stably and with less fluctuation and smoother operation during the communication topology switching. In addition, due to the use of the sliding-mode controller, chattering occurs near the steady state for the velocity and acceleration of the system, and with the improved controller proposed in this study, the chattering of the system is significantly reduced compared to other controllers, keeping it within an acceptable small range. In summary, the improved controller proposed in this study can effectively improve the operation of the system and increase the stability of the system operation in the case of a change in the communication environment that leads to the need to change the communication structure of the intelligent connected vehicle, which can largely overcome the negative impact caused by the change in the communication environment. 5.2 Different Disturbance The improved controller provided in this study can effectively suppress disturbances and enable the system to obtain excellent performance, as was shown in the previous analysis, and in this section, we will design simulation experiments to prove this matter. The simulation environment is set up as follows, with a platoon of 20 vehicles driving on an infinitely long road with no lane changes or overtaking occurring. The same basic conditions as in the simulation experiment in the previous section, the vehicle platoon is arranged uniformly at a headway of ℎ∗= 10𝑚. The initial velocity of all vehicles is 𝑣0 = 𝐹(ℎ∗) , each simulation step is 0.1 seconds, and the total simulation step is set to 1200 steps. The communication topology is chosen as TPLF. In this section, the communication topology is selected as TPLF and three different interferences (𝑑𝑣𝑖 defined in Eq. (7)) are selected as follows. { 𝑑𝑣𝑖1 = 1 + 0.8 ∙sin(𝑡) 𝑑𝑣𝑖2 = 0.6 ∙rand(𝑡) 𝑑𝑣𝑖3 = 0.5𝑒−(𝑡−120)2 2×62 −0.5𝑒−(𝑡−240)2 2×62 (55) (55) Interference is defined in Eq. (7), where 𝑑𝑣𝑖1 , 𝑑𝑣𝑖2 and 𝑑𝑣𝑖3 represent three Interference is defined in Eq. (7), where 𝑑𝑣𝑖1 , 𝑑𝑣𝑖2 and 𝑑𝑣𝑖3 represent three different kinds of interference respectively. 𝑑𝑣𝑖1 is a sinusoidal interference, which indicates that the system is suffering from a continuous regular interference; 𝑑𝑣𝑖2 is a random interference, which indicates that the system is subject to a continuous irregular interference, where rand(𝑡) is a random function that generates a random real number greater than zero and less than 1; 𝑑𝑣𝑖3 indicates that a interference of size 0.5 is generated at the 120th and 240th simulation steps, and this case is an episodic interference. The same four conditions as in the previous section are no control, the general constant speed reaching controller, the general exponential reaching controller and the improved controller, respectively, with the same parameters as in the previous section. The following is an analysis of the specific control effects. This system consists of a total of 20 vehicles, and under the premise of ensuring consistent conclusions, in order to make the results more concise and clearer, only some of the vehicles have been selected for operation in the simulation results. Fig. 6-Fig. 8, Fig. 9-Fig. 11, and Fig. 12-Fig. 5.2 Different Disturbance 14 show the variation of headway, position error, and velocity of the system in the cases of encountering continuous regular interference 𝑑𝑣𝑖1, continuous irregular interference 𝑑𝑣𝑖2 , and episodic interference 𝑑𝑣𝑖3 , respectively. And the modules (a)-(d) in Fig. 6-Fig. 14 represent the operating situation of the system under no control, the general constant speed reaching controller, the general exponential reaching controller and the improved controller actions, respectively. First, by comparing module (a) with modules (b)-(d) in Fig. 6-Fig. 14, it is observed that without the controller, the system has more fluctuations in headway, position and velocity under the influence of the three disturbances. And after using the controller, the performance of the system has been significantly improved in all cases. Next, we compare the effect of the three controllers on different disturbances. For the effect of the continuous regular This system consists of a total of 20 vehicles, and under the premise of ensuring consistent conclusions, in order to make the results more concise and clearer, only some of the vehicles have been selected for operation in the simulation results. Fig. 6-Fig. 8, Fig. 9-Fig. 11, and Fig. 12-Fig. 14 show the variation of headway, position error, and velocity of the system in the cases of encountering continuous regular interference 𝑑𝑣𝑖1, continuous irregular interference 𝑑𝑣𝑖2 , and episodic interference 𝑑𝑣𝑖3 , respectively. interference 𝑑𝑣𝑖1 on the system, comparing the modules (b)-(d) in Figs. 6-fig. 8, respectively, it is observed that the effect of the general exponential reaching controller is slightly better than the general constant speed reaching controller, while the effect of the improved controller proposed in this study is significantly better than the other controllers. With the improved controller, the headway, position and velocity fluctuate less and run more stably. For the effect of the continuous irregular interference 𝑑𝑣𝑖2 on the system, comparing modules (b)-(d) in Fig. 9-Fig. 11 respectively, it is observed that the general exponential reaching controller has almost the same effect as the general constant speed reaching controller, while the improved controller proposed in this study has a significantly better effect on this kind of disturbance than other controllers. For the episodic interference 𝑑𝑣𝑖3 , comparing modules (b)-(d) in Fig. 12-Fig. 5.2 Different Disturbance 14 respectively, it is observed that the effect of the general exponential reaching controller is better than the general constant speed reaching controller, while the improved controller proposed in this study is better than the general exponential reaching controller, and the change of the effect between different controllers is more noticeable. In summary, the improved controller proposed in this study has demonstrated excellent performance in maintaining the vehicle in the system in a favorable operating condition regardless of the effect of any kind of disturbance. This indicates that the improved controller proposed in this study has superior performance and stronger anti- interference capability. Fig.6 The headway profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) Fig.6 The headway profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC IAERSMC Fig.7 The position error profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.7 The position error profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.8 The velocity profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.8 The velocity profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) Fig.8 The velocity profiles under continuous regular interference 𝑑𝑣𝑖1: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC IAERSMC IAERSMC Fig.10 The position error profiles under continuous irregular interference 𝑑𝑣𝑖2: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) Fig.10 The position error profiles under continuous irregular interference 𝑑𝑣𝑖2: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC IAERSMC Fig.9 The headway profiles under continuous irregular interference 𝑑𝑣𝑖2: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC IAERSMC Fig.11 The velocity profiles under continuous irregular interference 𝑑𝑣𝑖2: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.11 The velocity profiles under continuous irregular interference 𝑑𝑣𝑖2: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC IAERSMC Fig.12 The headway profiles under episodic interference 𝑑𝐼𝑁𝐸𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.12 The headway profiles under episodic interference 𝑑𝐼𝑁𝐸𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.13 The position error profiles under episodic interference 𝑑𝑣𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.13 The position error profiles under episodic interference 𝑑𝑣𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.13 The position error profiles under episodic interference 𝑑𝑣𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC Fig.14 The velocity profiles under episodic interference 𝑑𝑣𝑖3: (a) no control; (b) the general constant speed reaching controller; (c) the general exponential reaching controller; (d) IAERSMC 6. Conclusion In this study, a V2V car-following model describing inter-vehicle communication is proposed based on the FVD model. Based on this, the interference existing in the intelligent connected vehicles system is further considered and these interferences are defined in the model and the concept of input interference is defined. Then, the improved controller is proposed based on the non-singular terminal sliding mode in the intelligent connected vehicle system by utilizing the sliding mode control method. The convergence and dynamic performance of the system with the improved controller are analyzed, and it is proved that the improved controller can make the system converge in finite time and obtain excellent dynamic performance. Finally, two simulation experiments are designed in this study. The first experiment sets up the system to switch the communication topology due to the change of communication environment during operation and applies a perturbation before the change of communication topology. The results show that the system is less affected by the communication topology change and operates stably with the improved controller. The second experiment is to introduce three different types of interferences to the system, namely, continuous regular interference, continuous irregular interference and episodic interference, and the results show that the improved controller achieves excellent effect on the control of these three different types of disturbances. These two simulation experiments show that the improved controller proposed in this study is able to maintain a stable operation of the intelligent connected vehicles system in the face of complex and variable communication environments and different types of interference. In our future research, we will continue to focus on the application of nonlinear sliding mode control strategies in the control of intelligent connected vehicle systems, and introduce second-order, or even high-order sliding mode control methods into our research. Further take into account more traffic situations in practice, and further develop the theoretical system of intelligent connected vehicle system control. Acknowledgements This work is supported by the industry university research innovation fund of science and technology development center of the Ministry of Education (Grant No. 2021JQR007), Major Technology Innovation Projects of Shandong Province (Grant No. 2019TSLH0203) and the National Key Research and Development Program of China (Grant No. 2020YFB1600501). Reference 1. J. A. G. Lbanez, S. Zeadally, J. Contreras-Castillo: Integration challenges of intelligent transportation system with connected vehicle, cloud computing, and internet of things technologies. 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Control. 62, 4376- 4391(2017) 8. Y. F. Li, Q. X. Lv, H. Zhu, et al.: Variable time headway policy based platoon control for heterogeneous connected vehicles with external disturbances. IEEE Trans. Intell. 8. Y. F. Li, Q. X. Lv, H. Zhu, et al.: Variable time headway policy based platoon control for heterogeneous connected vehicles with external disturbances. IEEE Trans. Intell. Transp. Syst. (2022). https://doi.org/10.1007/s11071-022-07922-5 Transp. Syst. (2022). https://doi.org/10.1007/s11071-022-07922-5 9. Y. F. Li, B. J. Chen, H. Zhao, et al.: A car-following model for connected and automated vehicles with heterogeneous time delays under fixed and switching communication topologies. IEEE Trans. Intell. Transp. Syst. 23,14846-14858(2022) 10. H. Zhao, Y. F. Li, W. Hao, et al.: Evaluating the effects of switching period of communication topologies and delays on electric connected vehicles stream with car-following theory. IEEE Trans. Intell. Transp. Syst. 22,7631-7641(2021) 11. S. E. Li, X. H. Qin, Y. Zheng, K. Q. Li, et al.: Distributed platoon control under topologies with complex eigenvalues: stability analysis and controller synthesis. IEEE Trans. Intell. Transp. Syst. 27,206-220(2019) 12. S. Konduri, P. R. Pagilla, S. Darbha: Vehicle platooning with multiple vehicle looking-ahead information. IFAC PAPERSONLINE. 50,5768-5773(2018) 13. S. Oncu, J. Ploeg, et al.: Cooperative adaptive cruise control: network-aware analysis of string stability. IEEE Trans. Intell. Transp. Syst. 15,1527-1537(2014) 14. A. Salvi, S. Santini, A. S. Reference Valente: Design, analysis and performance evaluation of a third order distributed protocol for platooning in the presence of time varying delays and switching topologies. IEEE Trans. Intell. Transp. Syst. 80,360-383(2017) 15. T. Q. Tang, W. F. Shi, H. Y. Shang, Y. P. Wang: A new car-following model with consideration of inter-vehicle communication. Nonlinear Dynam. 76,2017- 2023(2014) 16. T. Q. Tang, W. F. Shi, H. Y. Shang, Y. P. Wang: An extend car-following model with consideration of the reliability of inter-vehicle communication. Measurement. 58,286-283(2014) 17. M. di Bernardo, A. Salvi, S. Santini: Distributed consensus strategy for platooning of vehicles in the presence of time-varying heterogeneous communication delays. IEEE Trans. Intell. Transp. Syst. 16,102-112(2015) 18. W. B. Dunbar, D. S. Caveney: Distributed receding horizon control of vehicle platoons: stability and string stability: IEEE Trans. Autom. Control. 57,620- 633(2012) 19. F. Lin, M. Fardad, M. R. Jovanovic: Optimal control of vehicular formations with nearest neighbor interactions. IEEE Trans. Autom. Control. 57,2203-2218(2012) 20. L. D. Zhang, W. X. Zhu, J. L. Liu: Proportional-differential effects in traffic car- following model system. Physica A. 406,89-99(2014) following model system. Physica A. 406,89-99(2014) following model system. Physica A. 406,89-99(2014) 21. W. X. Zhu, J. Du, L. D. Zhang:A compound compensation method for car-following model. Commun. Nonlinear Sci. Numer. Simul. 39,427-441(2016) 22. W. X. Zhu, H. M. Zhang: Analysis of feedback control scheme on discrete car- following system. Physica A. 503,322-330(2018) 23. R. D. Yan, D. G. Yang, B. Wijaya, C. L. Yu: Feedforward compensation-based finite-time traffic flow controller for intelligent connected vehicle subject to sudden velocity changes of leading vehicle. IEEE Trans. Intell. Transp. Syst. 21,3357- 3356(2020) 24. R. D. Yan, D. G. Yang, J. Huang, et al.: Distributed car-following control for intelligent connected vehicle using improving super-twisting compensator subject to sudden velocity changes of leading vehicle. IEEE Trans. Intell. Transp. Syst. 23,6689-6693(2022) 25. F. Gao, X. S. Hu, S. E. Li, et al.: Distributed adaptive sliding mode control of vehicle platoon with uncertain interaction topology. IEEE Trans. Veh. Technol. 65,6352- 6361(2018) 26. X. G. Guo, J. L. Wang, F. Liao, et al.: Distributed adaptive integrated-sliding-mode controller synthesis for string stability of vehicle platoons. IEEE Trans. Intell. Transp. Syst. 17,2419-2429(2016) 27. J. W. Kwon, D. Chwa: Adaptive bidirectional platoon control using a coupled sliding mode control method. IEEE Trans. Intell. Transp. Syst. 15,2040-2048(2014) 28. R. Jiang, Q. S. Wu, Z. J. Reference Zhu: A new continuum model for traffic flow and numerical 27. J. W. Kwon, D. Chwa: Adaptive bidirectional platoon control using a coupled sliding mode control method. IEEE Trans. Intell. Transp. Syst. 15,2040-2048(2014) 28. R. Jiang, Q. S. Wu, Z. J. Zhu: A new continuum model for traffic flow and numerical tests. Transp. Res. C,36,405-419(2002) 29. L. Liu, W. X. Zheng, S. H. Ding: An adaptive SOSM controller design by using a sliding-mode-based filter and its application to buck converter. IEEEE Trans. Circuits Syst. I, Reg. Papers. 66,4840-4849(2019) 30. S. Y. Lin, W. D. Zhang, H. Wang: Controller design via an adaptive reaching law for DSMC systems. IEEEE Trans. Circuits Syst. II, Exp. Briefs. 67,330-334(2020)
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https://essopenarchive.org/doi/pdf/10.1002/essoar.10508406.1
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Phenotypic Parameters Prediction of Lettuce based on Computer Vision and Regression
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2,022
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Phenotypic Parameters Prediction of Lettuce based on Computer Vision and Regression Minjuan Wang1 , Maowei Li1 , Wanwan Li1 , Jinsong Li1 , Ying Wang1 , Yu Zhang1 , Xiyue Guo1 , and Minjuan Wang1 1 China Agricultural University November 23, 2022 Abstract Posted on 23 Nov 2022 — CC-BY 4.0 — https://doi.org/10.1002/essoar.10510410.1 — This a preprint and has not been peer reviewed. Data may be preliminary. To obtain phenotypic parameters by means of lossy measurement, we proposed a comprehensive and integrated approach to predict different parameters of four varieties of lettuces. By building different prediction models, we required predicted value of five phenotypic parameters of lettuce. Test results indicate that prediction models we have constructed are reliable and feasible. In addition,our methods can be better transferred to the research of other crops, and producers can adjust the growing environment of crops in time, so as to obtain higher yield. 1 Phenotypic Parameters Prediction of Lettuce based on Computer Vision and Regression Maowei Lia , Wanwan Lia , Jinsong Lia , Ying Wanga , Yu Zhanga , Xiyue Guoa , and Minjuan Wanga a Key Laboratory of Modern Precision Agriculture System Integration Research, China Agricultural University, Beijing, China ABSTRACT To obtain phenotypic parameters by means of lossy measurement, we proposed a comprehensive and integrated approach to predict different parameters of four varieties of lettuces. By building different prediction models, we required predicted value of five phenotypic parameters of lettuce. Test results indicate that prediction models we have constructed are reliable and feasible. In addition,our methods can be better transferred to the research of other crops, and producers can adjust the growing environment of crops in time, so as to obtain higher yield. Keywords: Lettuce, Computer Vision, Regression, Phenotyping Parameters 1. INTRODUCTION The traditional phenotypic parameters are usually measured by manual method, which is difficult to meet the requirements of automation and high precision1, 2 . Meanwhile, it is time-wasting and labor-consuming. More importantly, it is unacceptable to obtain phenotypic parameters by means of lossy measurement in practical agricultural production. Therefore, we proposed a nondestructive method to measure phenotypic parameters of lettuce in this research, aiming to monitor the growth and development of lettuce dynamically and efficiently. The predicted phenotypic parameters are plant height(PH), plant diameter(PD), leaf area(LA), fresh weight(FW) and dry weight(DW), respectively. The five types of phenotypic parameters obtained by this method will help producers adjust the growing environment of crops in time, which have count for much meaning to increase yield. 2. PROPOSED METHOD Figure 1. The overall research flowchart. Further author information: (Send correspondence to Minjuan Wang) Minjuan Wang: E-mail: minjuan@cau.edu.cn, Telephone: +86 136 0111 7404 Maowei Li: E-mail: sy20203081578@cau.edu.cn, Telephone: +86 178 5331 7947 The dataset in this experiment includes four types of lettuce(Lugano, Aphylion, Salanova and Satine) based on the 3rd Greenhouse Autonomous Challenge organized by Wageningen University and Tencent Lab, which were taken by Intel depth camera, containing 391 sets of depth images and color images. Among them, fifty sets of data are used to test the performance of the model. The overall research flowchart is shown in Figure. 1. Before building prediction models, we define some evaluation indexes, including R2 , Adjusted R2 (AR2 ), RMSE, RMSE Radio(RR), NMSE, MAPE and Total Error(TE). Finally, R2 , NMSE, MAPE and TE are used to evaluate verification results on test data. AR2 is corrected determination coefficient, which offsets the influence of sample size and the number of independent variables in regression on R2 . RR is the proportion of RMSE in actual average. The calculation formula of the above indicators is as follows. Pn (yi − fi )2 2 (1) R = 1 − Pni=1 2 i=1 (yi − fi ) Adjusted R2 = 1 − (1 − R2 )(n − 1) n−p−1 r Pn i=1 (yi RM SE = − fi )2 (3) n RM SE Pn i=1 yi (4) Pn (y − f )2 i=1 Pn i 2 i i=1 yi (5) RM SE Ratio = N M SE = (2) 1 n n M AP E = 1 X yi − fi | | × 100% n i=1 yi T otal Error = m X i=1 Pn j=1 (yij − fij ) Pn 2 j=1 (yij ) (6) 2 (7) Our approach is divided into the following two parts. The goal of the first step is predict PH and PD, as is shown in Table 1. Firstly, U-net network3 is used to perform color image segmentation to obtain binary image, and then it is projected to depth image. Through projection results, the pixel point with the smallest depth is obtained, which corresponds to the highest point of lettuce. The relative plant height (PH1) can be defined by making the difference between mininum of depth and the camera height. Secondly, the number of pixels in binary image is counted as relative projection area (RPA). Based on segmentation results, we apply contour detection function of OpenCV library to obtain all contour pixels, among which point pair with farthest distance are found to calculate the relative diameter (PD1). Finally, the linear regression model is used to fit the relationship between predicted value and actual value of PH1 and PD1 to get predict PD and PD, where AR2 of model is 94.4% and 91.8%, respectively. When average PH is 13cm and average PD is 23cm, RMSE is as low as 0.816cm and 0.980cm, respectively. Table 1. Optimal model of PH and PD. Parameters model AR2 RR RMSE PH PD 0.974PH1+0.778 0.0536PD1+2.1697 4.59 4.47 7.6 9.33 16.83 18.49 The second step is predict LA, FW, and DW of different types of lettuce. Due to large difference of lettuce shape among different species and growing period(GP), we build the model of fresh weight and dry weight separately. The solution of each parameter is divided into five parts, and they are all varieties at seedling stage, Aphylion, Lugano, Satine and Salanova at mature stage. According to data of different stages of lettuce, we use PH1, GP and RPA to fit the prediction model. In terms of LA, we find that all varieties at seedling stage and Salanova at mature stage have a significant linear relationship with GP, RPA, PH and the product of two, respectively. Lugano and Satine have a significant linear relationship with the power function of GP, PH and RPA. While Aphylion has a significant linear relationship with GP and RPA. The average AR2 of LA is 91.8% and the average RMSE is 192.58, accounting for 14.92% of actual average LA, as is shown in Table 2. In terms of FW, we find that there is a significant relationship between the power function of all varieties and LA at seedling stage, while Lugano at mature stage has an obvious linear relationship with GP, RPA and their product. FW of Aphylion, Salanova and Satine at mature stage is significantly related to exponential function of GP and power function of LA. The average AR2 of FW is 92.08% and the average RMSE is 11.63, accounting for 16.75% of actual average FW, as is shown in Table 3. In terms of DW, we find that all varieties at seedling stage and mature stage have a significant relationship with exponential function of GP and power function of LA. The average AR2 of DW is 93.42% and the average RMSE is 0.50, accounting for 15.44% of actual DW, as is shown in Table 4. Table 2. Optimal model of LA. GP variety model AR2 RR RMSE seedling mature mature mature mature All Aphylion Lugano Salanova Satine -16.54GP+0.000294LA+3.329PH+0.00317GP∗LA+32.99 395.83GP+0.0154RPA-449.93 1.1 86.68GP +0.000053RPA1.47 +1.66PH2.32 -45.02 398.3GP-0.00437RPA-93.03PH+0.00159RPA∗PH +451.64 111.28GP0.979 +0.000315RPA1.3281 +0.946PH1.902 -30.547 0.876 0.965 0.906 0.911 0.932 11.36% 13.03% 18.42% 17.86% 13.95% 11.69 217.2 217.2 328.59 148.85 Table 3. Optimal model of FW. GP variety model AR2 RR RMSE seedling mature mature mature mature All Aphylion Lugano Salanova Satine 0.0143RPA1.19 -0.0444 3.47e0.62GP +0.0153RPA1.15 -1.003 0.254GP-0.00526RPA+0.0179GP∗RPA+23.7 0.018e1.56GP +0.0076RPA1.2 +5.88 5.5e0.5GP +0.0042RPA1.32 -0.102 0.809 0.942 0.958 0.975 0.920 18.45% 19.83% 15.33% 11.03% 19.10% 0.659 18.02 16.48 8.86 14.11 Table 4. Optimal model of DW. GP variety model AR2 RR RMSE seedling mature mature mature mature All Aphylion Lugano Salanova Satine 0.0133e1.37GP +3.02E-05RPA1.84 -0.0251 0.452e0.52GP +0.0026RPA0.96 -0.0954 0.686e0.44GP +0.00183RPA0.98 -0.179 0.307e0.5GP +0.00169RPA1.002 -0.183 0.448e0.45GP +0.00052RPA1.16 -0.01 0.884 0.954 0.947 0.968 0.918 18.34% 15.59% 14.52% 11.14% 17.60% 0.0495 0.7138 0.662 0.4578 0.6048 3. RESULT AND DISCUSSION According to optimal model we have selected, verification result based on test data is shown in Table 5. It can be clearly seen that MAPE is below 20% basically. The correlation analysis of actual value and predicted value is shown in Figure.2−6, NMSE of PH, PD, LA, FW and DW are 0.00773, 0.00763, 0.01688, 0.02370 and 0.02123, respectively, whose sum is the value of TE. More importantly, when our model proposed in this paper is used to predict five phenotypic parameters of test data, final TE can reach 0.07717. While the first place’s TE was 0.08128 in the 3rd Greenhouse Independent Challenge based on this dataset, it is obvious that we have exceeded the first place in the above competition, which further proves that our model has good applicability. 4. CONCLUSION In this research, we propose a series of a comprehensive and integrated approach to predict different parameters of four varieties of lettuces. By building different prediction models, AR2 of predicted value and actual value have reached more than 90%. Test results indicate that prediction models we have constructed in this study are reliable and feasible. Although it is specific to different varieties, the early treatment methods can be better transferred to the research of other crops, and the later parameter prediction models can be better analogized to other similar crops. According to the five phenotypic parameters obtained by this method, producers can adjust the growing environment of crops in time, so as to obtain higher yield. Figure 2. Correlation analysis of PH. Figure 3. Correlation analysis of PD. Figure 4. Correlation analysis of LA. Figure 5. Correlation analysis of FW. Figure 6. Correlation analysis of FW. Table 5. Test results of different models. parameters GP variety R2 MAPE RMSE RR NMSE PH PD all all seedling all all all Aphylion Lugano Salanova Satine all Aphylion Lugano Salanova Satine all Aphylion Lugano Salanova Satine 0.948 0.888 0.918 0.897 0.895 0.963 0.847 0.697 0.876 0.936 0.951 0.861 0.846 0.896 0.954 0.950 0.910 7.92% 7.20% 8.43% 13.86% 10.07% 11.51% 10.33% 15.48% 15.96% 13.17% 9.85% 10.65% 15.69% 23.30% 10.67% 13.18% 9.70% 1.209 2.084 17.656 545.828 312.733 269.897 269.140 1.271 51.234 26.398 20.303 20.502 0.097 1.968 1.490 0.968 0.719 9.04% 7.45% 11.26% 18.92% 12.75% 11.93% 10.38% 23.22% 24.90% 16.26% 12.11% 11.25% 20.50% 20.83% 19.64% 12.35% 8.53% 0.007735 0.00763 LA mature seedling FW mature seedling FW mature 0.01688 0.02370 0.02123 REFERENCES [1] Rahaman, M., et al., Advanced phenotyping and phenotype data analysis for the study of plant growth and development. Frontiers in plant science, 2015. 6: p. 619. [2] Watt, M., et al., Phenotyping: new windows into the plant for breeders. Annual review of plant biology, 2020. 71: p. 689-712. [3] Ronneberger, O., P. Fischer and T. Brox. U-net: Convolutional networks for biomedical image segmentation. in International Conference on Medical image computing and computer-assisted intervention. 2015: Springer.
https://openalex.org/W2324343575
https://zenodo.org/records/2414695/files/article.pdf
English
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THE DISSOCIATION PRESSURES OF MERCUROUS CHLORIDE.
Journal of the American Chemical Society
1,916
public-domain
3,558
[CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262.1 THE DISSOCIATION PRESSURES OF MERCUROUS CHLORIDE. B Y ALBXANDBR SMITH AND ROBBRT PSYTON CALVBRT. Received February 14, 1916. The vapor pressures of calomel, over limited ranges of temperature, have been studied by Wiedemann, Stelzner, and Niederschultee and by Trans. Faraday Sac., IO, 228 (1915). 2 Bey. deut. physik. Ges.. 3, 159 (1905). DISSOCIATION PRESSWS OF MERCUROUS CHLORIDB. DISSOCIATION PRESSWS OF MERCUROUS CHLORIDB. 801 sharpness of definition in this photomicrograph is due to the fact that the surface was no longer plane, the solution having affected a greater trans- formation at some points of the surface than at others. These photo- micrographs are of interest in connection with the photographs taken by Cohen' of the same phenomenon. While Cohen's photographs are natural size and hence do not reveal the rapidity with which the trans- formation into the gray modification of lead occurs, yet they bring out more satisfactorily than photomicrographs the extent of the disintegration. HILLSIDE LABORATORY, STAMFORD. CONN. Trans. Faraday Sac., IO, 228 (1915). 2 Bey. deut. physik. Ges.. 3, 159 (1905). Trans. Faraday Sac., IO, 228 (1915). Summary of Results. (I) Five different types of lead electrode have been studied in a cell made up in the following manner: + + Pb - sat. sol. Pb(NOa)z 11 0.10 N KCl, HgCl - Hg + Pb - sat. sol. Pb(NOa)z 11 0.10 N KCl, HgCl - Hg Of these five electrodes the only one which proved to be constant and reproducible to within 0.3 millivolt was that in which the lead was de- posited electrolytically on platinum. ( 2 ) Freshly cast sticks of lead, after immersion for varying periods of time in an acidified solution of lead nitrate, lost their ductility and other properties commonly associated with lead. The gray m a s thus obtained undoubtedly is an allotropic modification of the metal. ( 2 ) Freshly cast sticks of lead, after immersion for varying periods of time in an acidified solution of lead nitrate, lost their ductility and other properties commonly associated with lead. The gray m a s thus obtained undoubtedly is an allotropic modification of the metal. (3) Measurements of the electromotive force developed at oo and 25' by c d s in which freshly cast lead and the gray modification formed the electrodes and 0.2 N Pb(NO& served as the electrolyte, failed to give any positive indication of a transition temperature. (4) A calculation of the normal electrode potential of lead was made from the mean value of the electromotive force of cells involving a constant and reproducible lead electrode. The value of this potential was found to be 0.1318 volt at 25'. (5) The electrodes were examined under the microscope and photo- micrographs were made to show the rapidity with which freshly cast lead undergoes transformation when immersed in a solution of acidified lead nitrate. HILLSIDE LABORATORY, STAMFORD. CONN. HILLSIDE LABORATORY, STAMFORD. CONN. Zemczuzng and Rambach, 2 mzorg Chew, 65, 40j (1910) ' Harkins and Clark, Ibzd., 37, 1819 (1915) ' THIS JOURNAL, 32, 1.541 (1910). I! Smith and Calvert, Ibid., 36, 1363 (1914) [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 ALEXANDER SMITH AND ROBBRT PEWON CALVERT. 802 Smith and Menzies-l The pressures of vapor in equilibrium with the solid calomel were determined by the former for temperatures below 180' and by the latter for temperatures above 361'. In the one series, the highest pressure obtained was o 5 mm.; in the other, the lowest pressure was more than 450 mm. For some work now nearing completion in this laboratory it is necessa'ry to have data for certain points in the wide tem- perature interval not previously investigated. In this paper, that in- formation is supplied. Since the entropy of vaporization is a more signifi- cant relation than is 'Srouton's rule, or any of the modifications of that rule which have been so extensively applied to vapor and dissociation pressures, the values of this constant for several dissociating substances have been calculated. We have included, also, a discussion of the various confining fluids which may be used in the static isoteniscope. Smith and Menzies-l The pressures of vapor in equilibrium with the solid calomel were determined by the former for temperatures below 180' and by the latter for temperatures above 361'. In the one series, the highest pressure obtained was o 5 mm.; in the other, the lowest pressure was more than 450 mm. For some work now nearing completion in this laboratory it is necessa'ry to have data for certain points in the wide tem- perature interval not previously investigated. In this paper, that in- formation is supplied. Since the entropy of vaporization is a more signifi- cant relation than is 'Srouton's rule, or any of the modifications of that rule which have been so extensively applied to vapor and dissociation pressures, the values of this constant for several dissociating substances have been calculated. We have included, also, a discussion of the various confining fluids which may be used in the static isoteniscope. Smith and Menzies-l The pressures of vapor in equilibrium with the solid calomel were determined by the former for temperatures below 180' and by the latter for temperatures above 361'. In the one series, the highest pressure obtained was o 5 mm.; in the other, the lowest pressure was more than 450 mm. For some work now nearing completion in this laboratory it is necessa'ry to have data for certain points in the wide tem- perature interval not previously investigated. [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 In this paper, that in- formation is supplied. Since the entropy of vaporization is a more signifi- cant relation than is 'Srouton's rule, or any of the modifications of that rule which have been so extensively applied to vapor and dissociation pressures, the values of this constant for several dissociating substances have been calculated. We have included, also, a discussion of the various confining fluids which may be used in the static isoteniscope. The Apparatus.-The measurements were made by use of the static isoteniscope and the apparatus previously described by the authors.z The isoteniscope was heated in a vigorously stirred bath of the molten nitrates of sodium and potassium. The temperature scale is that based on the ice point, steam point, and sulfur boiling point (taken as 444.7'); it could be reproduced, in successive calibrations of the platinum ther- mometer, to within *0.03'. The pressure readings are corrected to 0' and sea-level at latitude 45 '. Confining Fluids.-In any measurement with the static isoteniscope, the choice of a suitable material to confine the vapors is extremely im- portant. In many cases alloys, or a pure metal, like bismuth, may be used, but they are of such high density that errors due to leveling may correspond to several tenths of a millimeter of mercury. The equimolar mixture of sodium and potassium nitrates is available at temperatures above its melting point, 2 2 2 . 4,3 and below 450' at which the liberation of oxygen becomes appreciable. The mixture gives a light, mobile, perfectly transparent liquid which can be leveled with precision. But, unfortunately, the hot nitrates act chemically on many vapors and thereby disturb the vapor pressure equilibrium. The eutectic of lithium and potassium chlorides* gives, at high temperatures, a liquid having no appreciable vapor pressure and one which would seem admirably suited for confining the vapor of any chloride. But our experience has convinced us of ty of using this mixture because, first, of its st e and, secondly, of its attacking the glass, thus rendering the isoteniscope opaque. The ideal material for confining a vapor is the liquid form of the sub- ' THIS JOURNAL, 32, 1.541 (1910). I! Smith and Calvert, Ibid., 36, 1363 (1914) ' Harkins and Clark, Ibzd., 37, 1819 (1915) DISSOCIATION PRESSURES OF MERCUROUS CHLORIDE. 803 stance whose vapor pressure is being determined. [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 When the subdance sublimes without melting, the temperature of melting can often be low- ered below the boiling point, even for low pressures, by the addition of another salt. Thus, for confining the vapors of the chloride, the bromide and the iodide of ammonium, we have used the eutectic of the ammonium salt and the corresponding silver halide.' Also the eutectic of potassium chloride and the vaporizing substance has proved satisfactory in at least two vapor pressure measurements, the results of which are yet to be pub- lished. In the work of Smith and Menzies on calomel,2 the eutectic mixture of potassium and sodium nitrates was employed as confining liquid. Some interaction of the vapor with the nitrates occurred, causing a slow rise in pressure due to the accumulation of an excess of mercury vapor. The vapor was expelled and replaced by a fresh supply immediately before each reading. In the present work two different confining liquids were used in two series of measurements, namely, in the first, molten nitrates of sodium and potassium and, in the second, the eutectic of potassium chloride and silver chloride containing three mols of the former to seven of the latter and melting at 306 0.3 With the mixture of chlorides, the rise in pressure, caused by the dissolving of the mercuric chloride in the con- fining liquid and the accumulation of an excess of mercury vapor, was very much slower than when nitrates were employed. By distilling the vapor through the confining liquid and practically saturating the latter with mercuric chloride before making an observation, the increase for a five-minute interval was reduced to less than one-tenth of that observed with the nitrates as confining liquid. During this boiling-out process a very large deposit of mercury formed as a mirror in the upper cool part of the isoteniscope, thus affording an- other proof that calomel vapor dissociates giving free mercury. TABLE I.-VAPOR PRESSURES OF CALOMEL- -OBSERVATIONS TABLE I.-VAPOR Temp. OC. 309.52 * 328.28 340.32 * 341 I 56 354.57 356.34* 366.58 367,72* 383.95* 375. IO PRESSURES OF pobs. 1 0 5 . I 180.2 259.1 262 .o 372.3 399.1 507.7 535.5 621.3 781.4 CALOMEL- + 2 . 1 + 5 . 4 AP. 0.0 + 0 . 6 + 8 . 9 + 0 . 1 + 0 . 3 +I3 .O f 2 . 9 $16.7 -OBSERVATIONS. Zemczuzng, 2. anorg. Chem., 57, ~5 (1908). [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 P O 103 .o 180.2 253 7 2 6 1 . 4 372 .a 390.2 507.6 522.5 618.4 764.7 1 Smith and Calvert, LOC. cit. 2 L O C . cit. Zemczuzng, 2. anorg. Chem., 57, ~5 (1908). ALEXANDER SMITH AND ROBE$RT PBYTON C A L W T . 804 The Obsertrrttions.--The pressures were read five minutes after the bding out had been stopped (Pobs) and again five minutes later. The difhmce (AP), when subtracted from Pobs., gives the pressure (Po) corrected to the time zero at which the boiling out ceased. The results are also shown graphically in Fig. I . corrected to the time zero at which the boiling out ceased. The results are also shown graphically in Fig. I . 310 320 330 340 350 360 370 380 Temperature Celsius. Fig. I. An asterisk indicates that the observation was made with the molten nitrates as confining fluid in the bend of the isoteniscope. Much more weight is to be given to the results obtained by the use of the chlorides 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 Log. P. Fig. 2. 310 320 330 340 350 360 370 380 Temperature Celsius. Fig. I. An asterisk indicates that the observation was made with the molten nitrates as confining fluid in the bend of the isoteniscope. Much more weight is to be given to the results obtained by the use of the chlorides 310 320 330 340 350 360 370 380 Temperature Celsius. Fig. I. Fig. I. An asterisk indicates that the observation was made with the molten nitrates as confining fluid in the bend of the isoteniscope. Much more weight is to be given to the results obtained by the use of the chlorides An asterisk indicates that the observation was made with the molten nitrates as confining fluid in the bend of the isoteniscope. Much more weight is to be given to the results obtained by the use of the chlorides 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 Log. P. Fig. 2. 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 Log. P. Fig. 2. DISSOCIATION PFtESSURZS OF MERCUROUS CHLORIDE. 805 for the confining liquid; yet it will be observed that Fig. 2, in which log ,P is plotted against I/T, that the final data for both series gives points lying closely along the same straight line. 3 Chem. Weekblad, 6, 1035 (1909). [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 ALEXANDER SMITH AND ROBERT PEYTON CALVERT. 806 and the temperature calculated by means of the formula and the differ- ence ( A ) between the two values. The arithmetical mean of these devia- tions is 0.15' which, divided by the square root of the number of obser- vations, i. e., by 3, gives *o O j o as indicative of the precision of the measurements. The Entropy of Vaparization.-Hildebrand' has pointed out that Trouton's rule, namely, that the ratio of the heat of vaporization t a the boiling point of the absolute scale is a constant, or, as he states it, that the entropy of vaporization to one atmosphere pressure, is constant, does not hold even for normal substances, especially at extreme tem- peratures. He points out that €or normal liquids entropy of vaporization to a fixed molecular concentration of each vapor is more nearly constant. The constants increase as the boiling points are higher. For any vapor, at pressures so low that the laws of gases are applica- ble, p = RTc, where c is the molecular concentration. Hence, log p = log T + log RG. When c is o.oo5oi mol per liter, log R.c = Q.S. Now, d log p / d log T = I, i R'l'. For the purpose of comparison with Hilde- brand's results, we have calculated the value of the entropy of vaporiza- tion I,/RT, when log p = log 'I' Follo\%ing him, we have also calculated the value of L, RT, = 100 where the vapor pressure is 100 mm. , since at this lower pressure Trouton's rule should give more constant values than at 760 mm. The values at 760 mm. are given as well. 0.5. ABLE TV.-ENTROPY OF VAPORKZATION TO F I X ~ D MOLAR CONCN. (c = 0.00507). TABLE TV.-ENTROPY OF VAPORKZATION TO F I X ~ D MOLAR CONCN. (c = 0.00507). Substance. P H J . . ........ PClr ............. N (CHsfrCl ....... N( CH,)J. ........ NH&!I. .......... Hg&It. . . . . . . . . . . "41. . . . . . . . . . . . NHIBr. . . . . . . . . . . Normal liqs. ...... Assoc. liqs.. ...... 20.7 19.8 17.5 18.7 17.5 17.7 17.2 18.2 16.0-16.7 13.1-13.9 20.5 17.6 20.7 16.8 16.4 25.4 18.7 18.7 17.9 16.9 18.7 14.9 18.0 16.3 20.1 15.9 11-15. [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 This has especial interest in view of the fact that the correction applied for the increase in pressure with time was very large in the one series and almost negligible in the other. Kirchoff-Rankm-Dupre Equation.-For the purpose of interpolation and for testing the accuracy of our readings, we have derived an equation of the Kirchoff-Rankin-Dupr6 type. log 9 = - 7792.10 - 12.2309 log T + 49.2048. T log 9 = - 7792.10 - 12.2309 log T + 49.2048. T From this we have calculated the following dissociation pressures of cal mel in mm of mercury for even temperatures: From this we have calculated the following dissociation pressures of calw mel, in mm. of mercury, for even temperatures: mel, in mm. of mercury, for even temperatures: LE II.-DISSOCIATION PRESSURES OF CALOMEL AT EVEN QMPERATURES. TABLE II.-DISSOCIATION PRESSURES OF CALOMEL AT EVEN QMPERATURES. Degree. Mm. Degree. Mm. Degree. Mm. 300 74.4 340 251.4 380 696.8 3 10 103 .o 350 329.9 383.7 760.0 320 140.5 360 428.0 ... ... 330 189.2 3 70 548.9 ... ... The "boiling point," at 760 mm., is 383.7 O. Harris and Meyer found 357';' Jonker, 373°;2 and Smith and Menzies, 382.5 0.3 Although the solution of the above equation for the vapor pressure at any given temperature is quite easy, the calculation of the tempera- ture corresponding to a given pressure is more'troublesome. No*, in these measurements the chief errors lay in the temperature control and measurement and not in the reading of the pressure. Consequently, in testing the consistency of the result, it is preferable to express the devia- tions of the individual observations from the mathematical curves, repre- sented by the Kirchoff -Rankin-Dupd equation, in degrees rather thaq in mm. Table I11 gives, for each pressure, the temperature observed Pressure. Temp. (obs.). Temp. (cdc.). A. TABLE III.-DISSOCIATION PRESSURES OF CALOMEL. 103 .o 309.52* 309.98 fo.46' 180.2 328.28 328.34 +0.06' 253.7 340.32 * 340.33 S O . O I 0 2 6 1 . 4 341.56 341.38 -0.18 372 .O 354.57 354.56 +.OI 390.2 356.34* 356.40 +0.06 507.6 366.58 366.84 +os 26 522.5 367.72* 367.98 $0.26 618.4 375.10 374.94 -0.16 764.7 383.95 383.98 +o .os 1 Bey., 27, 1484 (1894). 3 Chem. Weekblad, 6, 1035 (1909). 8 L O C . C i t . P T ( b ) T ( d ) A TABLE III.-DISSOCIATION PRESSURES OF CALOMEL. : THIS JOURNAL, 37, 970 (1915). 1 Presented in abstract at the Seattle Meeting of the American Chemical Society, Sept. I , 1915. [CONTRIBUTION FROM THE CKEMIcAt LABORATORY OF COLUMBIA UNIVRRSm, NO. 262 I . . . . . . 301 . s o 395 469 534 562 604 627 616 . . 302.2' 335.6' 390 435.8 450 506.3 519 378.5 582 656.7 544 610.8 5 93 667.6 605 677 9 .. .. .. .. For the purpose of comparison, the corresponding values, as given by Hildebrand for normal and for associated liquids, are indicated. In the case of dissociating substances, such as our table contains, it was to be expected that the heats of vaporization (including heats of dissociation) would be higher. It should be noted that our values refer to a constant THE: OXIDES OF IRON. I. 807 total molecular concentration both of the undissociated molecules and of the molecules of the products of the dissociation. total molecular concentration both of the undissociated molecules and of the molecules of the products of the dissociation. [CONTRIBUTION FROM THE GEOPHYSICAL LABORATORY, CARNEGIE INSTITUTION, WASHINGTON, D. C.] THE OXIDES OF IRON. I. SOLID SOLUTION IN THE SYSTEM F&Os-FEs04l. BY R. B. SOSMAN AND J. C. Hos~'B~~'ER. Received February 4, 1916. CONTENTS.-Introduction. State of Knowledge of the Oxides of Iron. Apparatus Employed. Materials: Ferric Oxide; Merck's "Reagent Iron Oxide;" Oxide from Elec- trolytic Iron; Sibley Ore; Magnetite; Oxygen. Special Problems: Optical Proper- ties of the Oxides of Iron; Reduction of Iron Oxides by Platinum; Disappearance of Oxygen in Furnace; Adsorption of Gases. Questions of Equilibrium: Repro- ducibility of Dissociation Pressures; Accuracy of Temperature and Pressure; Pressures from Ferric Oxide from Different Sources; Effect of Temperature Gradient in Furnace; Comparison of Rising and Falling Temperatures; Pressures Obtained by Oxidation of Magnetite. The System FezOs-FeaOd: Experimental Results: General Plan; Methods of Analysis; Total Iron; Pressure-composition Isotherm at 1200 '; Pressure-composition Isotherm at I 100' ; Form of Dissociation-pressure Curve near Fez08 ; Optical Properties. Comparison with Previous Investigations. Summary. Summary. The dissociation pressures of calomel for temperatures between 309 ' and 384 ' are given. The pressure, 760 mm., is reached at 383.7 '. The KirchoE-Rankin-Dupr6 equation represents the results satisfac- torily. The entropies of vaporization to constant molecular concentration for eight dissociating substances are recorded. Confining liquids are discussed. NICHOLS LABORATORIES OF 1NOP.GANIC CHEMISTRY, COLUMBIA UNIVERSITY, NEW YORK CITY. NICHOLS LABORATORIES OF 1NOP.GANIC CHEMISTRY, COLUMBIA UNIVERSITY, NEW YORK CITY. [CONTRIBUTION FROM THE GEOPHYSICAL LABORATORY, CARNEGIE INSTITUTION, WASHINGTON, D. C.] THE OXIDES OF IRON. I. SOLID SOLUTION IN THE SYSTEM F&Os-FEs04l. BY R. B. SOSMAN AND J. C. Hos~'B~~'ER. Received February 4, 1916. [CONTRIBUTION FROM THE GEOPHYSICAL LABORATORY, CARNEGIE INSTITUTION, WASHINGTON, D. C.] THE OXIDES OF IRON. I. SOLID SOLUTION IN THE SYSTEM F&Os-FEs04l. BY R. B. SOSMAN AND J. C. Hos~'B~~'ER. Received February 4, 1916. Introduction. The silicate systems which have hitherto been the subjects of study in this laboratory have all been made up of oxides which are apparently unaffected by atmospheric oxygen at temperatures below 1600'. They could therefore be melted in platinum-wound furnaces in the open air. The oxides which can be so treated include silica, alumina, magnesia, lime, and the alkalies. But iron, which is one of the most important con- stituents of natural silicates, changes its form of combination and the properties of its compounds according to the amount of oxygen which is combined with it, and. furthermore, the amount of oxygen so combined is constantly changing, especially at high temperatures. It becomes necessary, then, to take account of oxygen as one of the components in any silicate system containing iron.
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FURTHER OBSERVATIONS ON THE PNEUMOTHORAX TREATMENT OF PHTHISIS.
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Rationale. Is phthisis benefited by an artificial pneumothorax, and if so, why? All who have observed a certain number of patients thus treated give an affirmative answer to the first question. But it is more difficult to explain why the results are beneficial. There are many more or less plausible ex- planations forthcoming. y There are two points in my paper which I expect will have struck the reader ; one is the small reliance placed on the rotary and caloric reactions, and the other is the total omission of that so-called group in which increased labyrin- thine pressure is said to exist. It is not, perhaps, quite within the scope of the paper to give at all at length my reasons for not employing the tests, nor for placing more reliance upon them. I am convinced in my own mind that one is able to make one’s diagnoses and one’s prognoses in the class of case under consideration equally well without their employment. I must, however, admit that, especially in cases of low arterial tension, the results were most extra- ordinarily interesting. With regard to increased intra- labyrinthine pressure, although I have operated on at least 20 cases of labyrinthine vertigo where there had been no perforation into the labyrinth-14 of them where there had been no previous suppuration, and the rest where there had-I have only twice seen any fluid on opening the labyrinth. In these two cases there was a considerable amount of fluid ; one of them also had an extremely large external semicircular canal, apparently four or five times greater than normal. But even then I am not prepared to say that these cases contained fluid under pressure, and I have consequently thought it better, as the matter is treated entirely clinically, to omit this possible factor. But it is rather difficult, indeed, to understand, from a purely mechanical point of view, how fluid can be retained under pressure in such a non-vascular bony cavity as the labyrinth. planations forthcoming. Collapse and compression of the diseased lung must rest it as effectively as a well-applied splint rests a tuberculous knee ; ;. in either case auto-inoculation is reduced to a minimum. Another beneficial factor is the expression and drainage of fluid from the lung which, when completely collapsed, scarcely exceeds a normal kidney in size. Rationale. Even when adhesions prevent complete collapse, a partial pneumothorax may greatly aid the drainage of cavities and the expression of fluid from the whole lung. I have seen several patients the apices of whose lungs were firmly adherent to the chest wall, and whose disease was most evident in the upper half of the lung. Yet compression of the lower half was strikingly beneficial, the daily expectoration diminishing to about one-tenth of the former amount in a few weeks. It is therefore probable that, even if a cavity in the apex is not compressed, the compression of the neighbouring lung aids the drainage of pus which, before compression, flowed over from the cavity into the more dependent portions of the lung. That fluid is squeezed out of the lung is shown by the increase of the expectoration which occurs during the first few days of the treatment. days Another favourable factor is the extensive formation of fibrous tissue around the diseased areas, which thus become securely cut off from healthy tissues. It is important to note that healthy pulmonary tissue does not necessarily become disorganised or permeated by scar tissue even after several years of consistent compression ; and when a lung containing both healthy and tuberculous tissue is allowed to re-expand, the expansion is practically limited to the tissues which were originally healthy or but slightly oedematous. This has been repeatedly demonstrated post mortem. It has been recently urged against this treatment that healthy portions of the lung are wasted by compression which fails to act on diseased portions because pleural adhesions prevent their collapse. But this speculative objection is negatived by the post mortem observations already referred to. The assump- tion lately advanced by a consultant in London that tuber- culous foci in the lung are invariably accompanied by adjacent pleural adhesions is indeed extraordinary, for the induction of an artificial pneumothorax has frequently shown that comparatively healthy lung is adherent to the chest wall, while another portion of the lung, occupied by cavities and pneumonic infiltration, is covered by free pleura. y The use of ernutine was suggested by my senior clinical assistant, Mr. A. F. Penny, of London. I found it impossible, even in so lengthy an analysis, to contrast and apply Dr. BY CLAUDE LILLINGSTON, M.B., B.C. CANTAB. BY CLAUDE LILLINGSTON, M.B., B.C. CANTAB. SINCE the publication of three papers on the subject in THE LANCET of July 15th, 1911, Forlanini’s treatment has steadily gained favour in England, where its popularity suffered from a negative phase long after its establishment abroad. Now it is suffering here from a positive phase which is equally unfortunate, as a careful selection of cases is not always made, and the operation is not always -performed with that detailed knowledge and observance of the technique which are imperatively necessary. The induction of an artificial pneumothorax by one whose knowledge of the technique is culled from the brief perusal of a few treatises on the subject shows de- plorably vicarious courage. It may, therefore, be advisable at this stage to take stock of this method, to deal with the broad outlines of the rationale, indications for and against, and complications, and to describe in detail some of the steps in the technique which, if misunderstood, may cause sudden death. strenuously, by dietary. 2. A female, aged 62. A great sufferer from rheumatoid arthritis, and almost complete deafness in the right ear, The patient, despite her severe handicap, was a most energetic and hard-working person, busy with good works. Sept. 3rd, 1912. For about three years she had suffered with left Eustachian obstruction, gradually becoming more and more deaf. On Jan. 1st, 1912, the patient had a severe attack of vertigo in bed in the morning, with objects rotating,clockwise in a vertical plane. She has had four or five severe attacks since, and many mild ones. The attacks are heralded by flushing and by a feeling of surface warmth, and at times this is accompanied by perspiration. The patient has, however, had three attacks of vertigo without these symptoms. She could hear a whisper on the left side 4 inches, and C.32 to C.4096 double vibrations. Her blood pressure was 100. 1 Zeitschrift fur Tuberculose, 1910, vol. xvi. R&Eacute;SUM&Eacute;. Chronic progressive middle-ear deafness and arterio- sclerosis are thus, according to my investigations, the most frequent causes of aural vertigo, and fortunately one finds that a large amount of benefit can be derived from the use of drugs in these cases, though by no means all of them are capable of this relief. We have also seen that operative interference is justified, and where it is used it should be uniformly successful. 1642 DR. C. LILLINGSTON: THE PNEUMOTHORAX TREATMENT OF PHTHISIS. DR. C. LILLINGSTON: THE PNEUMOTHORAX TREATMENT OF PHTHISIS. 1642 DR. C. LILLINGSTON: THE PNEUMOTHORAX TREATMENT OF PHTHISIS. Easter, 1910, he went for a golling holiday, forgetful of his age and of the age of his arteries, and played three rounds a day. A few mornings later he awoke with violent vertigo, retching. and vomiting. His body felt very warm. The vomit was bilious, and the direction of objects left to right. He recovered completely. His blood pressure was 150. Again, in July of the same year the same series of events led to a second attack. They were always in the morning. Yet the patient did not take warning, and late one night he fell, seized with sudden vertigo. He sweated feeely and vomited (note sea-sickness). He could not be moved for three hours. When I saw the gentleman in question he was still going ahead as if he were in the prime of life. He had occasionally a severe attack. He got quite over his attacks by taking his life less strenuously, and by the addition of a little alcohol to his dietary. Rationale. , , a : the absence of which its virulence is reduced. This accounts for the preference shown by the tubercle bacillus for organs, largely composed of fibrous tissue, in which the process of oxidation is low, and in which a considerable quantity of oxygen is therefore available for the tubercle bacillus. Hence, also, the rarity of phthisis in cases of mitral stenosis in which the lungs are congested with venous blood. The nitrogen, therefore, which is injected into the pleural cavity under a positive pressure may act beneficially through its gradual absorption by the diseased lung, to which the supply of arterial blood is largely reduced by the partial compression of the pulmonary arteries. Other observations which support this view are the benefit to tuberculous peritonitis derived from exploratory laparotomy, which favours venous stasis and absorption of atmospheric nitrogen occur ; and the benefit obtained by Kuhn’s suction mask, and by Bier’s treatment by passive congestion. ;Several other arguments are advanced by Goebel in support of his view. , , a : minutes. Patients are frequently either warned against the treatment, or they are recommended it when moribund, and when the disease is extensive in both lungs. To induce a pneumothorax when moist sounds are audible everywhere over both lungs is to hasten the patient’s death and to bring the treatment to disrepute. Yet half the patients in whom I have been requested to induce an artificial pneumothorax belong to this category. p g g y The condition most suitable for the treatment is extensive disease of one lung, little or no disease of the other. But there are also many other suitable conditions. For instance, the disease may be limited to the apices of both lungs, but compression of one lung becomes an urgent necessity on account of recurrent and severe haemoptysis. Again, one lung may be practically healthy, and the disease in the other may extend only from the apex to the third rib in front, and to the spine of the scapula behind; yet if this condition has persisted for over a year in spite of sanatorium and tuberculin treatment, and the patient is incapacitated, an artificial pneumothorax may be the best treatment. Sometimes half of each lung may be involved, and the patient may be going downhill in spite of ordinary treat- ment. Rationale. It should also be remembered that slight movements by a patient breathing with one lung only are equivalent to violent exercise by the possessor of two healthy lungs. It is, however, extraordinary how much exercise can be taken by patients accustomed to harbouring a pneumothorax, and I have sprinted to catch a train when I boasted a large pneumothorax. But till the healthier lung has proved its capacity to do the work of two, it must be watched with the vigilance of a cat at a mouse-hole, and absolute rest must be prescribed as soon as danger signals, as a rise of temperature or other physical signs, appear. If the healthier of the two lungs shows recent signs of disease, such as a pleuritic rub combined with fever, the induction of a pneumothorax on the other side hould be deferred till this condition has subsided, even if the healthier lung appears in other respects to be normal. good , ordinary This theory is ingenious, almost fantastic. But Molle’s arguments are too numerous and carefully weighed to be straightway dismissed. I have seen two patients whose disease was practically unilateral, and on whom several attempts to induce an artificial pneumothorax failed on account of pleural adhesions. The apparent failure of the operation greatly depressed the patients, who thought their last chance of recovery was lost. But both improved rapidly, and one is now walking ten miles a day two years after the attempt to induce an artificial pneumothorax. Both these patients were desperately ill. lung appears respects It is the exception for the pleural surfaces to be adherent throughout, even in old-standing cases complicated by repeated attacks of pleurisy; and I have only in 15 per cent. of my cases failed to induce a pneumothorax on account of adhesions. But it is often difficult to find a point where the pleural surfaces are free, and from which the intrapleural pressure may be raised till soft and recent adhesions else- where give way. When once a small pocket of nitrogen has been formed between the pleural surfaces, it is often astonishing how, with frequent small injections of nitrogen, this pocket may be enlarged till almost all the adhesions have given way. Rationale. The experienced physician who has watched such a case for several months can often give a shrewd estimate of the activity of the disease in each lung, and if he finds it practically stationary in one, the compres- sion of the other may give strikingly good results. Dr. Jane Walker has kindly shown me a patient who developed a spontaneous pneumothorax which she is maintaining by injections of nitrogen. The patient is rapidly improving, although more than half his uncompressed lung is affected ; here, therefore, the disease is evidently stationary. s e . The other explanation, for an account of which I am indebted to Dr. Ernest P. A. Mariette, is advanced by Molle,2 who attributes the success of treatment by an artificial pneumo- thorax to a trophic excitation of the pneumogastric nerves. He holds that there is a danger of ’’ I transferring " the disease to the opposite side in patients whose reflex irritability is abnormally great. He has observed in certain ,phthisical patients a neuromuscular hemihyper&aelig;sthesia at .a spot where other physical signs of disease shortly appeared, and which was symmetrically opposite to the ,original lesion on the other side. He also points out that counter-irritation by the cautery, blisters, or tincture of iodine may cause migration of nervous com- plications from one side of the body to the other. Patients who react thus to counter-irritants are not, in his ’opinion, suitable for an artificial pneumothorax, the stimulus of which is likely to "transfer" the disease to the uncompressed lung. Patients, on the other hand, whose reflex irritability is found by experiments with counter-irritants to be low, are suitable for the pneumo- thorax treatment, provided the disease is more or less ’unilateral. But, he adds, counter-irritation alone will prove as effective as a pneumothorax in this class of patient. He concludes that the new surgical methods of treating phthisis, ;and especially an artificial pneumothorax, constitute a special form of counter-irritation whose effects are akin, for good or evil, to those of ordinary counter-irritation. , , y y Often only after a pneumothorax has been induced is it possible to estimate the degree of activity of the disease in the healthier lung. Whenever the disease in this lung is slight but active, the pneumothorax on the other side must be gradually induced, so as not to throw a sudden excess of work on the uncompressed lung. Presse M&eacute;dicale, Jan. 27th, 1912. Rationale. Kerrison’s classification to that I adopted, so preferred to leave it, hoping at some future time to give it the place it deserves and derive from it the benefits it possesses. possesses. Harley-street, W. Harley-street, W. Harley-street, W. MANCHESTER UNIVERSITY.-Dr. T. Wingate Todd, lecturer in anatomy in this University, has been appointed Henry Wilson Payne Professoi of Anatomy in the Medical Department of the Western Reserve University, Cleveland, Ohio. MANCHESTER UNIVERSITY.-Dr. T. Wingate Todd, lecturer in anatomy in this University, has been appointed Henry Wilson Payne Professoi of Anatomy in the Medical Department of the Western Reserve University, Cleveland, Ohio. TUBERCULOSIS DISPENSARY FOR BIRKENHEAD.- Dr. J. R. Hutchinson, Local Government Board inspector, held an inquiry recently at the Birkenhead town hall into an application by the town council for powers to borrow f:.620 for a tuberculosis dispensary under the Insurance Act. It is proposed to purchase premises in Duncan-street, and convert them into a dispensary. At the conclusion of the inquiry the inspector visited the premises, pneumonic infiltration, by pleura. Two other explanations of the beneficial action of an artificial pneumothorax may be briefly mentioned. Carl Goebel1 has pointed out that as an aerobic organism, the tubercle bacillus grows best in a free supply of oxygen, in 1643 PNEUMOTHORAX TREATMENT OF PHTHISIS. DR. C. LILLINGSTON: THE PNEUMOTHORAX TREATMENT OF PHTHISIS. 1643 R. C. LILLINGSTON: THE PNEUMOTHORAX TREATMENT OF PHTHISIS. the absence of which its virulence is reduced. This accounts for the preference shown by the tubercle bacillus for organs, largely composed of fibrous tissue, in which the process of oxidation is low, and in which a considerable quantity of oxygen is therefore available for the tubercle bacillus. Hence, also, the rarity of phthisis in cases of mitral stenosis in which the lungs are congested with venous blood. The nitrogen, therefore, which is injected into the pleural cavity under a positive pressure may act beneficially through its gradual absorption by the diseased lung, to which the supply of arterial blood is largely reduced by the partial compression of the pulmonary arteries. Other observations which support this view are the benefit to tuberculous peritonitis derived from exploratory laparotomy, which favours venous stasis and absorption of atmospheric nitrogen occur ; and the benefit obtained by Kuhn’s suction mask, and by Bier’s treatment by passive congestion. ;Several other arguments are advanced by Goebel in support of his view. Rationale. patients desperately Though not prepared to dismiss the two latter explana- tions as mere speculative conjectures, I hold that immobilisa- ’tion and drainage of the diseased lung are the chief causes - of arrest of the disease. The objection has been raised ’that if collapse and rest are beneficial to the diseased lung, the extra work thrown on the other lung must injure it. But, as Saugman has pointed out, the two lungs are simultaneously treated by different methods. Under ordinary sanatorium treatment patients with active disease are kept strictly at rest, while patients with slight and afebrile disease are exercised. Similarly, with the pneumo- ’thorax treatment, the severely diseased lung is given absolute rest, while the slightly diseased lung is exercised. given way. So far from being a contraindication, tuberculous laryngitis is, oeteris paribus, an indication for the treatment which favours its arrest by reducing cough and expectoration and by improving the general condition. Dr. S. V. Pearson’s success at Mundesley Sanatorium with a case of severe laryngeal disease combined with almost unilateral pulmonary tuberculosis has been most striking with this treatment. It is unlikely that the life of a patient suffering from intestinal tuberculosis and persistent diarrhoea can be appreciably prolonged, even if the pulmonary disease be arrested by a pneumothorax. It is deplorable how many patients ultimately die from tuberculosis of the intestine, kidneys, meninges, and other organs whose pulmonary disease was found at the necropsy to be completely arrested by an artificial pneumothorax. In most of these cases the disease had lasted for several years before the pneumothorax was induced, and it is certain that when this A CASE OF COMPLETE ERADICATION OF EXTENSIVE RECURRENT CANCER AFFECTING BOTH BREASTS AND AXILL&AElig;; g;; DEATH 15 YEARS LATER FROM HEART DISEASE AND DROPSY AT THE AGE OF 82 YEARS. BY R. CLEMENT LUCAS, B.S. LOND., F.R.C.S. ENG., CONSULTING SURGEON TO GUY’S HOSPITAL AND TO THE EVELINA HOS- PITAL FOR CHILDREN; MEMBER OF THE COUNCIL OF THE ROYAL COLLEGE OF SURGEONS AND OF THE EXECUTIVE COMMITTEE OF THE IMPERIAL CANCER RESEARCH FUND. g ew IT is not only by the consent, but also by the desire, of the relatives of this patient that I publish the details of her case, and its publication may perhaps assist other surgeons to persuade their patients to avail themselves of the only known means of cure for cancer, rather than gamble away their only available chance of safety by resorting to useless methods of treatment so temptingly put forward by designing persons, who play upon the fears and encourage the natural dread that everyone feels at the idea of having an operation performed. ad a tageous pat e t. Gaseous embolism may happen in yet another way. Durin a first injection the operator thrusts the needle through the chest wall till characteristic manometric oscillations show that its point is in the pleural cavity. He now holds the needle securely at this depth lest it should pass deeper and penetrate the visceral pleura. But though neither the patient nor the needle has apparently been moved, the oscillations may suddenly cease, owing, as a rule, to the parietal pleura having slipped over the point of the needle, which consequently is again superficial to both layers of the pleura. The operator who, because he has once obtained typical oscillations, and because he thinks the needle has not moved since, injects nitrogen in the face of a motionless manometer, will sooner or later have a sudden death to explain. operation performed. E. J. was a patient of Mr. J. A. Hosker, of Boscombe, Bournemouth, and she had been operated upon some years before I saw her by a Bournemouth surgeon, who had amputated her left breast for cancer, but had not carried his incision into the axilla to clear away the lymphatics in that situation. In the interval between the operation and the recurrence of the disease the operating surgeon had died, and this fact may have made the patient hesitate some time before consulting her own medical man as to her tumours when recurrence took place. p Results. In such a chronic and capricious disease as phthisis an estimation of the value of any form of treatment is worthless unless the number of patients treated is considerable, and the improvement effected has stood the test of time. My experience, therefore, with 18 patients treated for periods ranging from 6 months to 3 years, is insufficient per se to build pretentious claims on, and I will but briefly touch on the following points. The disease was in the third stage and active, and the prognosis was very bad in every case. Yet the disease is either arrested or is undergoing arrest in 13. Five patients are either dead or dying ; but I sincerely believe that the treatment has prolonged their lives. I have seen no fatal accident from the treatment. More valuable are Dr. L. Spengler’s results, for they have stood the test of a longer probation period.4 He has excluded all recent cases, confining himself to those in which a pneumothorax had been induced nine months to four years earlier. As evidence of arrest of disease he adopted the following conditions : absence of pyrexia, cough, and ex- pectoration ; if the latter be present, it must not contain tubercle bacilli. Fifteen patients fulfilled these conditions, and were also capable of a full day’s work. In 12 of these the prognosis before the operation was almost hopeless, and in the remaining 3 it was very bad. Dr. Spengler con- siders that these results are a satisfactory answer to the objection that though a pneumothorax may cause temporary improvement, it does not often enough produce permanent results to warrant the risk of operation. A useful review of recent literature on the treatment is given by Dr. W. T. Ritchie,5 who has collected the results of several workers in this field. Interesting original articles on this subject have also lately been published by Dr. S. Vere Pearson 6 and Mr. NnhPrt. r!hitt-t7 7 Sudden death from gaseous embolism continues to occur, as recent publications show. But, as indicated by the following case recently published by Balvay and Arcelin,3 these deaths may be the operator’s-and not the opera- tion’s-fault. The patient had acquired a fairly large artificial pneumothorax, but her lung was still adherent to the chest wall in places. g y g Indications for and against the Treatment. g The present attitude of many general practitioners and ’consultants towards the treatment is one of blank ignorance -or of horror and contempt. To them an artificial pneumo- thorax is identical with a spontaneous, accidental pneumo- ’thorax with its concomitant distress and risks of sepsis. Yet the former can be induced so gradually that the patient is put to little discomfort, dyspn&oelig;a, or pain ; and, if necessary, the gas can be withdrawn from the pleural cavity, and stat2c.s cluo ante restored in a few 1644 MR. R. C. LUCAS: ERADICATION OF CANCER OF BOTH BREASTS. 1644 MR. R. C. LUCAS: ERADICATION OF CANCER OF BOTH BREASTS. typical oscillations appeared. By this procedure hopelessly ill patients have regained their health ; but the risk entailed is considerable, even when the needle is aspirated to ascer- tain the presence of blood in its lumen before any gas is injected. No novice should attempt this method. treatment is adopted earlier in the disease, the tale of fatalities due to tuberculosis in organs other than the lung will be appreciably diminished. eciably Coniplications and their Avoidance. p Much has been written about pleural reflex and gaseous embolism, the two causes of sudden death during the operation. With care, both risks are negligible. The risk of pleural reflex is reduced by injecting eucaine or stovaine into the pleura. Two points in this connexion are im- portant. The solution of anaesthetic must be concentrated so that only a little fluid escapes into the tissues ; otherwise fluid in the track of the pneumothorax needle will fill its lumen, and thus prevent the registration by the mano- meter of changes in the intrathoracic pressure. If the operator holds the hypodermic needle lightly he can also probe with it to ascertain the pleura’s exact depth from the surface and its state, whether it be thickened or not. I usually inject eucaine at three or four different points in the chest, and select for my first puncture with the pneumothorax needle that point at which the hypodermic needle has encountered least resistance. Unfortunately, however, firm pleural adhesions sometimes exist even where the exploring hypodermic needle has encountered but slight resistance. 3 Archives G&eacute;n&eacute;rales de M&eacute;decine de Lyon, May 29th, 1911. Edinburgh Medical Journal, July, 6 Brit. Med. Jour., Oct. 12th, 1912. uuml;nchener Medizinische Wochenschrift, Feb. 28 5 Edinburgh Medical Journal, July, 1912. 4 M&uuml;nchener Medizinische Wochenschrift, Feb. 28th, 1911. g 6 Brit. Med. Jour., Oct. 12th, 1912. 7 Medical Press and Circular, Nov. 13th, 1912. 4 M&uuml;nchener Medizinische Wochenschrift, Feb. 28th, 1911. 5 Edinburgh Medical Journal, July, 1912. 6 Brit. Med. Jour., Oct. 12th, 1912. 7 Medical Press and Circular, Nov. 13th, 1912. Brit. Med. Jour., Oct. 12th, 1912. 7 Medical Press and Circular, Nov. 13th, 1912. p Results. On introducing the pneumothorax needle at a different point from that at which the first injec- tions had been given, the operator failed to obtain charac- teristic manometric oscillations. In spite of this ominous warning the gas was introduced under considerable pressure. The patient immediately became unconscious. She coughed up blood, and in 48 hours she was dead. The needle had been introduced over the site of an adhesion which held a portion of the lung to the chest wall, and thus gas was forced into a pulmonary vein. Gorleston-on-Sea. Gorleston-on-Sea. pulmonary A similar accident may occur even if the pneumothorax is complete and no pleural adhesions exist, if the operator is careless enough to ignore a motionless mano- meter. For the needle may catch in a blood-vessel super- ficial to the parietal pleura, and with this vessel impaled on ts point, it may invaginate the parietal pleura without puncturing it, and may reach to the depth at which the operator on previous occasions has penetrated to the pleural cavity, and has obtained characteristic oscillations. He may now argue that he is working at the right depth, and that no manometric oscillations appear because the needle has become obstructed by a small particle of tissue in its transit through the chest wall. He therefore proceeds to blow out this hypothetical particle by forcing gas through the needle. The result is more instructive to himself than advantageous to the patient. 5 Edinburgh Medical Journal, July, 1912. 6 Brit. Med. Jour., Oct. 12th, 1912. A CASE OF COMPLETE ERADICATION OF EXTENSIVE RECURRENT CANCER AFFECTING BOTH BREASTS AND AXILL&AElig;; She, however, had a nephew in our profession, Mr. W. Watkin Leigh, of Treharris, who’ at one time had been my dresser at Guy’s Hospital, and p It follows that however secure the operator may feel. his only safe course is to watch every movement d f the manometer, and to cut off the gas as soon as satisfactory oscillations cease. When, hour after hour, he has tried in vain to obtain satisfactory oscillations by puncturing in several places, the temptation to 11 shove in gas and see what happens " is great. Cases have, in fact, been recorded in which no oscillations were obtained till a little gas had been forced into the pleural cavity, after which
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Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe
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Article Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe Matteo Miceli 1, Silvana Casati 1 , Roberta Ottria 1 , Simone Di Leo 2, Ivano Eberini 3, Luca Palazzolo 3 , Chiara Parravicini 3 and Pierangela Ciuffreda 1,* Matteo Miceli 1, Silvana Casati 1 , Roberta Ottria 1 , Simone Di Leo 2, Ivano Eberini 3, Luca Palazzolo 3 , Chiara Parravicini 3 and Pierangela Ciuffreda 1,* 1 Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy; matteo.miceli@unimi.it (M.M.); silvana.casati@unimi.it (S.C.); roberta.ottria@unimi.it (R.O.) 1 Dipartimento di Scienze Biomediche e Cliniche “Luigi Sacco”, Università degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy; matteo.miceli@unimi.it (M.M.); silvana.casati@unimi.it (S.C.); roberta.ottria@unimi.it (R.O.) 2 Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate (MI), Italy; simone.dileo@unimi.it 2 Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Via Fratelli Cervi 93, 20090 Segrate (MI), Italy; simone.dileo@unimi.it g y 3 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy; ivano.eberini@unimi.it (I.E.); luca.palazzolo@unimi.it (L.P.); chiara.parravicini@unimi.it (C.P.) 3 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy; ivano.eberini@unimi.it (I.E.); luca.palazzolo@unimi.it (L.P.); chiara.parravicini@unimi.it (C.P.) * Correspondence: pierangela.ciuffreda@unimi.it; Tel.: +39-02-50319695 Academic Editor: Anton Simeonov Received: 7 May 2019; Accepted: 14 June 2019; Published: 15 June 2019 Abstract: Monoacylglycerol lipase (MAGL) is a serine hydrolase that has a key regulatory role in controlling the levels of 2-arachidonoylglycerol (2-AG), the main signaling molecule in the endocannabinoid system. Identification of selective modulators of MAGL enables both to provide new tools for investigating pathophysiological roles of 2-AG, and to discover new lead compounds for drug design. The development of sensitive and reliable methods is crucial to evaluate this modulatory activity. In the current study, we report readily synthesized long-wavelength putative fluorogenic substrates with different acylic side chains to find a new probe for MAGL activity. 7-Hydroxyresorufinyl octanoate proved to be the best substrate thanks to the highest rate of hydrolysis and the best Km and Vmax values. In addition, in silico evaluation of substrates interaction with the active site of MAGL confirms octanoate resorufine derivative as the molecule of choice. The well-known MAGL inhibitors URB602 and methyl arachidonylfluorophosphonate (MAFP) were used for the assay validation. The assay was highly reproducible with an overall average Z′ value of 0.86. Article Set-Up and Validation of a High Throughput Screening Method for Human Monoacylglycerol Lipase (MAGL) Based on a New Red Fluorescent Probe The fast, sensitive and accurate method described in this study is suitable for low-cost high-throughput screening (HTS) of MAGL modulators and is a powerful new tool for studying MAGL activity. Keywords: high-throughput screening; endocannabinoid; monoacylglycerol lipase; assay development molecules molecules molecules molecules 1. Introduction The endocannabinoid system (ES) is a modulatory system that plays a critical role in a variety of neuronal functions including motor activity, nociception, and appetite control as well as peripheral functions such as energy metabolism and inflammation [1]. This wide range of effects makes it an interesting target for drug development although a difficult one to act upon due to life-threatening psychological adverse effects [2] linked to the direct antagonist action against the endocannabinoid receptors. Molecules 2019, 24, 2241; doi:10.3390/molecules24122241 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules 2 of 14 Molecules 2019, 24, 2241 Inhibition or enhancement of the ES hydrolytic regulatory enzymes seems a very promising strategy to act on the ES in a more fine-tuned way [3]. Monoacylglycerol lipase (MAGL), a soluble serine hydrolase that associates with cell membranes, is a very promising target for pharmacological inhibition, since it is the main hydrolase implicated in the degradation of the main signaling molecule in the ES, 2-AG. The hydrolysis of 2-AG is catalyzed also by two additional serine hydrolases, α-β hydrolase domain 6 and 12 (ABHD6 and ABHD12). MAGL, however, is responsible for approximately 85% of 2-AG hydrolysis [4]. y y Identification of new promising lead compounds for MAGL inhibition from large libraries of molecules needs simple, highly sensitive, specific, inexpensive and reliable assays suitable for a high-throughput format. The most commonly assay procedures used to screen and characterize MAGL inhibitors quantify the hydrolysis products of radiolabeled MAGL substrates [5,6] or the arachidonic acid released upon 2-AG hydrolysis by using high-performance liquid chromatography (HPLC) with UV [7] or with mass spectrometric detection [8]. Another method assesses the rate of MAGL-catalyzed hydrolysis of p-nitrophenyl alkyl esters by monitoring the liberation of p-nitrophenol [9]. Furthermore, a fluorescence-based assay has been developed applying 7-hydroxycoumarinyl arachidonate (7-HCA) [10], as well as an HPLC method with fluorescence detection, using as fluorogenic probe 1,3-dihydroxypropan-2-yl-4-pyren-1-ylbutanoate [11]. We have previously reported an efficient protocol for a MAGL continuous assay, based on a new long-wavelength fluorogenic substrate, 7-hydroxyresorufinyl arachidonate 1g [12]. Here we report the synthesis and the evaluation, as MAGL assay substrates, of 10 alkyl esters of fluorescent resorufin with the aim to propose a high-throughput screening (HTS) method for MAGL activity based on a new red fluorogenic probe. 2.1. Synthesis of 1a–k For the synthesis of fluorogenic compounds, two different methods have been followed. While the synthesis of the esters 1a–g involves passing through the formation of the acyl chloride, the compounds 1h–k required Steglich esterification [16]. In the case of fatty acid with branched acyl chain such as 2-methylhexanoic acid, 2-ethylhexanoic acid and 2-butyloctanoic acid the symmetrical anhydrides were isolated instead of the esters. Therefore, the use of oxalyl chloride has to be avoided with sterically hindered carboxylic acids. In the Steglich esterification indeed, the addition of 3 mol % DMAP accelerates the DCC-activated esterification of carboxylic acids with alcohols to such an extent that formation of side products is suppressed. Synthesized molecules were completely characterized by 1D NMR, as well as 2D NMR homocorrelation (COSY) and heterocorrelation (HMQC and HMBC). The purity of all compounds measured with NMR was greater than 98%. 1. Introduction Essential to the success of any HTS program that seeks to discover modulators of the function of specific proteins, is the development of a high-quality screening, which requires an accurate, and homogeneous biochemical readout of protein activity, robust assay reproducibility and appropriate sensitivity [13,14]. Furthermore, possible interferences due to the concentrations of library compounds used as protein activity modulators in the assay could occur, so shifting the emission wavelength to the red reduces these interferences [15]. It must also be underlined that a suitable substrate has to be readily accessible, stable in solution and with low rate of spontaneous hydrolysis. We have previously reported an efficient protocol for a MAGL continuous assay, based on a new long-wavelength fluorogenic substrate, 7-hydroxyresorufinyl arachidonate 1g [12]. Here we report new esters of fluorescent resorufin with different classes of acyl chains such as linear, i.e., acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), branched, i.e., 2-methylhexanoate (1h), 2-ethylhexanoate (1i) and 2-butyloctanoate (1j), and aromatic, i.e., benzoate (1k) (Figure 1) and compared them with 1g. Moreover, we investigated the MAGL substrate specificity depending on the acyl chain, and determined the kinetic constants. The differences of substrate interaction with MAGL active site were also analyzed using structural in-silico techniques. 3 of 14 bstrate nces of Molecules 2019, 24, 2241 (1k) (Figure 1) and specificity dependin bstrate interaction with MAGL active site were also analyzed using structural in silico technique O N O O O R a R= CH3; b R= CH2CH2CH3; c R= CH2(CH2)5CH3; d R= CH2(CH2)9CH3; e R= CH2(CH2)17CH3; 1 f R= g R= h R= i R= j R= k R= Figure 1. Structures of 7-hydroxyresorufynil esters: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), 2- ethylhexanoate (1i) 2-butyloctanoate (1j), benzoate (1k). Figure 1. Structures of 7-hydroxyresorufynil esters: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), 2-ethylhexanoate (1i) 2-butyloctanoate (1j), benzoate (1k). a R= CH3; b R= CH2CH2CH3; c R= CH2(CH2)5CH3; d R= CH2(CH2)9CH3; e R= CH2(CH2)17CH3; Figure 1. Structures of 7-hydroxyresorufynil esters: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), 2- ethylhexanoate (1i) 2-butyloctanoate (1j), benzoate (1k). Figure 1. Structures of 7-hydroxyresorufynil esters: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), icosanoate (1e), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), 2-ethylhexanoate (1i) 2-butyloctanoate (1j), benzoate (1k). 2.2. Substrates Screening Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96- Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96-well black plate, at room temperature, with 5 µM of each compound in total volume of 100 µL/well, as described in the experimental section. Fluorescence was measured at indicated time points with a Jasco FP-8300 fluorometer (Jasco Europe, Cremella (LC) Italy) using the kinetic mode (λex = 571 nm, λem = 588 nm, slit = 5.0). Data are mean ± standard error of independent experiments. Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96- Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96-well black plate, at room temperature, with 5 µM of each compound in total volume of 100 µL/well, as described in the experimental section. Fluorescence was measured at indicated time points with a Jasco FP-8300 fluorometer (Jasco Europe, Cremella (LC) Italy) using the kinetic mode (λex = 571 nm, λem = 588 nm, slit = 5.0). Data are mean ± standard error of independent experiments. 2.2. Substrates Screening Screening experiment were performed at 5 µM in Tris-HCl containing EDTA (10% DMSO) in the presence of 25 ng of human recombinant MAGL (hMAGL). Typical MAGL assays use bovine serum albumin (BSA) since it assists in the solubility of the substrate and might prevent its non-specific binding to the plastic labware [10]. We decided not to use BSA because it is not compatible with lipase assay due to its well-known esterase-like activity [17]. As reported in Figure 2, at 5 µM compound 1a shows a considerable spontaneous hydrolysis, compound 1b a smaller one while all the other esters seem to be stable. During the set-up of the assay conditions also spontaneous hydrolysis of 1a–d, and 1h–k at all concentration used for kinetic experiments (data not shown) have been evaluated. At 25 µM, the highest concentration assessed (Supplementary Figure S1), compounds 1a–c show a hydrolysis rate increment proportional to the concentration as expected, even if almost negligible for 1c. Compounds 1i–k seems to be still stable with no significant increment in hydrolysis rate, while compounds 1d–h show an atypical behavior, indeed, to a five-fold increase of the ester concentration correspond a less increment in the rate of hydrolysis. For this reason, the solubility in aqueous buffer of substrates 1d–h at 25 µM was verified by dynamic light scattering (DLS) analyses (Supplementary Figure S3). DLS analyses revealed the presence of micelles and/or aggregates that are formed at this concentration, due to the hydrophobic nature of the substrates. 4 of 14 nalyses es that Molecules 2019, 24, 2241 aqueous buffer of su (Supplementary Fig e formed at this concentration, due to the hydrophobic nature of the substrates. Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96- Figure 2. Time course of 1a–d, 1f, and 1h–k hydrolysis by human recombinant monoacylglycerol lipase (hMAGL). 25ng/well of hMAGL (circles) or buffer alone (empty squares) were incubated in 96-well black plate, at room temperature, with 5 µM of each compound in total volume of 100 µL/well, as described in the experimental section. Fluorescence was measured at indicated time points with a Jasco FP-8300 fluorometer (Jasco Europe, Cremella (LC) Italy) using the kinetic mode (λex = 571 nm, λem = 588 nm, slit = 5.0). Data are mean ± standard error of independent experiments. Figure 2. 2.3. Kinetic Studies The amount of enzyme used in the assay was optimized to 25 ng/well to maintain linearity over time and to maintain substrate consumption below 10% to adhere to the assumptions of the Michaelis–Menten equation [12]. Enzyme kinetics were analyzed by measuring product formation rates in the presence of different amounts of the substrates. Standard curves, showing fluorescence response vs. fluorophore concentration, were constructed from a resorufin dilution series, transforming relative fluorescence units (RFU) into picomoles of resorufin. Figure 3 reports the action of hMAGL on different concentrations of all synthesized substrates. To evaluate the real enzymatic activity, the amount of free resorufin released from the spontaneous hydrolysis of the substrate was subtracted. The initial linear part of the resulting curve and its slope were considered to measure the enzyme activity [18]. Michaelis–Menten kinetic parameters, Km and Vmax values, were derived with Prism GraphPad software, Version 6.0c applying a nonlinear regression analysis and Michaelis-Menten fit (Table 1). As shown in Figure 3, fluorometric determination of the kinetic constants for compounds 1a and 1b was impossible. For all other compounds, Michaelis-Menten curves were constructed considering hMAGL hydrolysis at five concentrations (0.5, 1.0, 2.5, 5.0, 10.0 µM) and the obtained results for Km and Vmax are reported in Table 1. Among new compounds, 1j has the best fit with Michaelis-Menten regression but has a very low rate of hydrolysis. The amount of enzyme used in the assay was optimized to 25 ng/well to maintain linearity over time and to maintain substrate consumption below 10% to adhere to the assumptions of the Michaelis–Menten equation [12]. Enzyme kinetics were analyzed by measuring product formation rates in the presence of different amounts of the substrates. Standard curves, showing fluorescence response vs. fluorophore concentration, were constructed from a resorufin dilution series, transforming relative fluorescence units (RFU) into picomoles of resorufin. Figure 3 reports the action of hMAGL on different concentrations of all synthesized substrates. To evaluate the real enzymatic activity, the amount of free resorufin released from the spontaneous hydrolysis of the substrate was subtracted. Th l l f h l d l d d h The initial linear part of the resulting curve and its slope were considered to measure the enzyme activity [18]. Michaelis–Menten kinetic parameters, Km and Vmax values, were derived with Prism GraphPad software, Version 6.0c applying a nonlinear regression analysis and Michaelis-Menten fit (Table 1). 2.3. Kinetic Studies The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and Vmax were determined via computer assisted nonlinear regression analysis using GraphPad Prism Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and Vmax were determined via computer-assisted nonlinear regression analysis using GraphPad Prism 6.0c (GraphPad Software, San Diego, CA 92108). p g y g ad Software, San Diego, CA 92108). Table 1. List of tested substrates with kinetic and modeling parameters. 6.0c (GraphPad Software, San Diego, CA 92108). The initial linear part of the resulting curve and its slope were considered to measure the enzyme activity [18]. Michaelis–Menten kinetic parameters, Km and Vmax values, were derived with Prism GraphPad software, Version 6.0c applying a nonlinear regression analysis and Michaelis-Menten fit (Table 1). As shown in Figure 3, fluorometric determination of the kinetic constants for compounds 1a and 1b was impossible. For all other compounds, Michaelis-Menten curves were constructed considering hMAGL hydrolysis at five concentrations (0.5, 1.0, 2.5, 5.0, 10.0 μM) and the obtained results for Km and Vmax are reported in Table 1. Among new compounds, 1j has the best fit with Michaelis-Menten regression but has a very low rate of hydrolysis. Kinetic parameters reported in Table 1 highlight the substrate preference of hMAGL. Of all the compounds newly synthesized, the fastest hydrolyzed was 1c. Compound 1e has a very low solubility in DMSO (< 0.5 nM) and could not be assessed as hMAGL substrate. Actually, even LogD value (Table 1) highlights higher affinity of this compound to non-polar organic solvents. Substrate 1k was excluded from further investigations due to its inappreciable hydrolysis by hMAGL. All Table 1. List of tested substrates with kinetic and modeling parameters. 2.3. Kinetic Studies Compound LogD at pH 7.4 a Km (µM) Vmax (nmol/min/mg protein) Docking Score MM-GBSA Fitting Model r2 1a 1.69 n/a n/a −6.7 −52.4 n/a n/a 1b 2.83 n/a n/a −7.7 −60.5 n/a n/a 1c 4.61 0.66 ± 0.14 106 ± 5.4 −8.2 −72.0 M-M 0.8488 1d 6.39 0.31 ± 0.09 24 ± 1.2 −10.5 −81.4 M-M 0.6731 1e 9.94 n/a n/a −9.8 −67.8 n/a n/a 1f 8.69 0.42 ± 0.1 13 ± 0.64 −10.8 −73.2 M-M 0.7391 1g 8.50 0.87 ± 0.13 25.8 ± 0.88 −10.7 −76.0 M-M 0.9908 1h 4.26 1.09 ± 0.02 33 ± 2.509 −8.5 −60.3 M-M 0.8859 1i 4.71 2.8 ± 0.7 1.3 ± 0.1 −8.8 −62.8 M-M 0.9236 1j 6.49 1.1 ± 0.1 0.67 ± 0.03 −8.4 −65.4 M-M 0.9450 1k 3.74 n/a n/a −8.9 −63.2 n/a n/a a Data generated using the Chemicalize https://chemicalize.com/ (developed by ChemAxon, http://www.chemaxon. com). Kinetic parameters reported in Table 1 highlight the substrate preference of hMAGL. Of all the compounds newly synthesized, the fastest hydrolyzed was 1c. Compound 1e has a very low solubility in DMSO (<0.5 nM) and could not be assessed as hMAGL substrate. Actually, even LogD value (Table 1) highlights higher affinity of this compound to non-polar organic solvents. Substrate 1k was excluded from further investigations due to its inappreciable hydrolysis by hMAGL. All substrates have Km values ranging from 0.31 to 2.8 µM, in accordance to that of 0.87 µM already calculated for 1g [12]. Maximum velocities are similar as well, ranging from 0.67 to 33 nmol/min/mg protein, with the exception of 1j that displays a very low hydrolysis rate with a Vmax of 0.67 nmol/min/mg protein. Interestingly, 1c has a Vmax of 106 nmol/min/mg protein. For this reason, compound 1c was selected as fluorogenic substrate for the set-up of a new method. 2.3. Kinetic Studies As shown in Figure 3, fluorometric determination of the kinetic constants for compounds 1a and 1b was impossible. For all other compounds, Michaelis-Menten curves were constructed considering hMAGL hydrolysis at five concentrations (0.5, 1.0, 2.5, 5.0, 10.0 µM) and the obtained results for Km and Vmax are reported in Table 1. Among new compounds, 1j has the best fit with Michaelis-Menten regression but has a very low rate of hydrolysis. 5 of 14 5 of 14 Molecules 2019, 24, 2241 Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and Vmax were determined via computer-assisted nonlinear regression analysis using GraphPad Prism Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and Vmax were determined via computer-assisted nonlinear regression analysis using GraphPad Prism 6.0c (GraphPad Software, San Diego, CA 92108). Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and V were determined via computer assisted nonlinear regression analysis using GraphPad Prism Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). The reactions were conducted in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA) with 25 ng/well of hMAGL. Values are the means of triplicates ± standard error. The kinetic parameters Km and Vmax were determined via computer-assisted nonlinear regression analysis using GraphPad Prism 6.0c (GraphPad Software, San Diego, CA 92108). Figure 3. Action of hMAGL on different concentrations of 1a–d, 1f, 1h–j, in the presence of DMSO (10%, v/v). in assay conditio 2.5. Docking Studies 2.5. Docking Studies The identified binding site corresponds to the catalytic active site identified by Lauria et al. [21] Table 1 reports the Glide XP docking score and MM-GBSA binding energies for the top scoring pose of each tested compound, showing that all of them are able to bind MAGL in its catalytic site. Docking The identified binding site corresponds to the catalytic active site identified by Lauria et al. [21] Table 1 reports the Glide XP docking score and MM-GBSA binding energies for the top scoring pose of each tested compound, showing that all of them are able to bind MAGL in its catalytic site. Docking simulations provided overlapping poses for all the tested compounds and a good superposition of their common resorufin group, confirming the accuracy of this approach. Moreover, the carbonyl group for all the tested compounds, except for 1j, overlaps the carbonyl group of the natural substrate 2-AG in proximity to Ser122, according to Lauria et al. [21]. In particular, Ala51, Ser122 and Met123 establish hydrogen bond interactions with the carbonyl and ester oxygen atoms (Figure 5), while His121 and His269 are involved in π–π interaction with the resorufin group, as reported in Table S2 of Supplementary. As shown in Figure 5, the alkyl chains of the resorufine ester accommodate in a well-shaped pocket surrounded by hydrophobic/aromatic residues, such as Leu148, Leu213, Leu214, Ile179, Ala151, Ala156, Phe159, Phe209. Accordingly, in silico results also underline the importance of the contribution of the hydrophobic interactions in the stability of the complexes. Molecules 2019, 24, x FOR PEER REVIEW 6 of 13 simulations provided overlapping poses for all the tested compounds and a good superposition of their common resorufin group, confirming the accuracy of this approach. Moreover, the carbonyl group for all the tested compounds, except for 1j, overlaps the carbonyl group of the natural substrate 2-AG in proximity to Ser122, according to Lauria et al. [21]. In particular, Ala51, Ser122 and Met123 establish hydrogen bond interactions with the carbonyl and ester oxygen atoms (Figure 5), while His121 and His269 are involved in π–π interaction with the resorufin group, as reported in Table S2 of Supplementary. As shown in Figure 5, the alkyl chains of the resorufine ester accommodate in a well-shaped pocket surrounded by hydrophobic/aromatic residues, such as Leu148, Leu213, Leu214, Ile179, Ala151, Ala156, Phe159, Phe209. 2.4. Validation of 1c for Screening Assay Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 in assay conditions. Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 in assay conditions. 2.4. Validation of 1c for Screening Assay In order to validate compound 1c for HTS two known MAGL inhibitors, URB602 and Methyl arachidonylfluorophosphonate (MAFP) [7,19] were used. The dose-response curves are shown in 6 of 14 Molecules 2019, 24, 2241 2.4. Validation of 1c Figure 4. After incubation of hMAGL with the inhibitor, 1c was rapidly added and fluorescence read for 30 min. The activity of hMAGL was calculated as described for the kinetic assay. IC50 for URB602 was found to be 8.1 µM, which is in line with literature data [20]. As already known, MAFP had the lowest IC50 value, our experiments gave an IC50 of 15.3 nM, which is in the range of values indicated in literature [12,20]. arachidonylfluorophosphonate (MAFP) [7,19] were used. The dose-response curves are shown in Figure 4. After incubation of hMAGL with the inhibitor, 1c was rapidly added and fluorescence read for 30 min. The activity of hMAGL was calculated as described for the kinetic assay. IC50 for URB602 was found to be 8.1 μM, which is in line with literature data [20]. As already known, MAFP had the lowest IC50 value, our experiments gave an IC50 of 15.3 nM, which is in the range of values indicated in literature [12 20] 20]. [ , ] Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 in assay conditions Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. Data derived from two independent experiments performed in triplicate and calculated as non-linear regressions using sigmoid dose-response setting with variable Hill slope by GraphPad Prism 6.0c. In panel B 100% inhibition is not achieved due to the low solubility of URB 602 in assay conditions. Figure 4. Inhibition of human recombinant MAGL with known inhibitors: methyl arachidonyl fluorophosphonate (A), URB602 (B). The assay was carried out as described in Experimental procedures section. 3. Discussion Here we present 7-hydroxyresorufinyl octanoate (1c) as a new fluorogenic substrate to evaluate MAGL activity by HTS. Although numerous assay methodologies have been published in the past decades for measurement of MAGL activity, few of them allow the screening of a large number of compounds showing high sensibility, high throughput, and low assay cost. In order to overcome these issues, we investigated the use of an alternative substrate for measuring MAGL activity. The native substrates for MAGL include 2-AG (C20:4) and 2-oleoyl glycerol (2-OG, C18:1) both containing long hydrocarbon fatty acid chains with relatively unstable cis olefins. Nevertheless, MAGL is able to hydrolyze a vast array of esters structurally very different, such as p-nitrophenyl esters [9] or 7-hydroxycoumarinyl esters [22]. We chose resorufine as fluorescent probe because its derivatives have proven to be an excellent fluorescence marker and have the advantages to be hardly susceptible to background signals. Often pharmacological and biochemical research need to measure enzymatic activity in crude cell lysate or turbid solution [23], the shift of emission wavelengths at higher values give less interference [24]. In the current approach, 10 fluorogenic alkyl esters of resorufin with different acyl chains are synthesized and evaluated as MAGL assay substrates (Figure 1). Selected compound 1c ensure lower background signals, thanks to resorufine in comparison with 7-methylumbelliferone, higher stability and lower costs if compared to arachidonoyl or oleyl derivatives. g y p y y MAGL fluorescence assays were carried out in the presence of 5 µM substrate, 10 % DMSO and at pH 7.4, a value that attempts to mimic the native intracellular conditions. Diverging too far from the physiological pH could affect the interactions between the enzyme and the substrates, thus giving an altered view of the potential biochemical and cellular activity of these compounds. Moreover, this pH value can be maintained by various buffers, such as Tris-HCl, PBS, sodium phosphate, Mops and Hepes, and allowed us a prompt comparison with previous data [12]. Among the synthesized compounds, linear acylic chains of up to 12 carbon atoms are substrates for MAGL, as well as the derivative of oleic acid, as expected, since 2-OG, an oleic acid derivative, is one of the native substrates of the protein. The addition of just two more carbons to the side chain makes the hydrolysis of this compound almost non-existent. The same result is obtained when the branching becomes as long as four carbon atoms. in assay conditio 2.5. Docking Studies Accordingly, in silico results also underline the importance of the contribution of the hydrophobic interactions in the stability of the complexes. Figure 5. A) Docking poses of compound 1c into the MAGL binding site. MAGL is shown in ribbon representation; tested compound and Ser122 are shown in stick representation and hydrogens are hidden B) Ligand interaction diagram of compound 1c into the MAGL binding site Figure 5. (A) Docking poses of compound 1c into the MAGL binding site. MAGL is shown in ribbon representation; tested compound and Ser122 are shown in stick representation and hydrogens are hidden. (B) Ligand interaction diagram of compound 1c into the MAGL binding site. Figure 5. A) Docking poses of compound 1c into the MAGL binding site. MAGL is shown in ribbon representation; tested compound and Ser122 are shown in stick representation and hydrogens are Figure 5. (A) Docking poses of compound 1c into the MAGL binding site. MAGL is shown in ribbon representation; tested compound and Ser122 are shown in stick representation and hydrogens are hidden. (B) Ligand interaction diagram of compound 1c into the MAGL binding site. Molecules 2019, 24, 2241 7 of 14 7 of 14 3. Discussion Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 μM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), d 2 b t l t t (1j) Th ifi ti it f th hMAGL ti 241 9 U/ Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 µM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), and 2-butyloctanoate (1j). The specific activity of the hMAGL preparation was 241.9 U/mg. y ( j) p y p p g The presence of aggregates in the substrates’ solutions at 25 μM concentration prompted us to consider only solutions until 10 μM concentration for calculations of kinetic parameters. Observing obtained results (Figure 3), 1f gives a good fit with Michaelis-Menten type kinetics indeed, its side chain, the same of natural substrate 2-OG, seems to allow a better accommodation in the enzyme active site. It is possible that the enhanced rigidity of the alkyl chain, conferred by the presence of the double bond, guides the substrate to a conformation favorable to enzymatic hydrolysis, hindering The presence of aggregates in the substrates’ solutions at 25 µM concentration prompted us to consider only solutions until 10 µM concentration for calculations of kinetic parameters. Observing obtained results (Figure 3), 1f gives a good fit with Michaelis-Menten type kinetics indeed, its side chain, the same of natural substrate 2-OG, seems to allow a better accommodation in the enzyme active site. It is possible that the enhanced rigidity of the alkyl chain, conferred by the presence of the double bond, guides the substrate to a conformation favorable to enzymatic hydrolysis, hindering disadvantageous folding in the enzyme active site. 3. Discussion When in aqueous buffer, 1a and 1b show the highest rate of spontaneous hydrolysis among our compounds (Figure 2), to the point that fluorometric determination of their kinetic constants proved impossible (Figure 3). For resorufin acetate 1a, this behavior was already reported by Maeda and colleagues [25], leading to the impossibility of using that compound as a probe. Even if 1b showed an inferior hydrolysis rate than 1a (Figure 1), this compound is unsuitable for the achievement of Michaelis-Menten conditions in an aqueous environment (Figure 3) [26]. On the other hand, compounds with a long linear chain or with a branched side chain, showed low to negligible hydrolysis. These differences may be explained with the susceptibility of acyl resorufins towards nucleophilic attack [27]. Short linear side chains (1a, 1b) leave the α carbon exposed to the attack of water, while side chains with branches next to the carboxyl group (1h, 1i, 1j) exert a steric hindrance on water molecules, protecting the ester group from hydrolysis. A similar phenomenon reasonably happens with medium (1c, 1d) and long (1f, 1g) alkyl chains. When in an aqueous environment, the alkyl chain folds on itself via van der Waals interactions next to the carboxyl group, thus mimicking the side branch protecting effect. Moreover, the formation of aggregates, as highlighted by DLS analyses (Supplementary Figure S2), could be a reasonable explanation for the unusual behavior observed in spontaneous hydrolysis experiments of compounds 1d–h where to a five-fold increase of ester concentration corresponds a less increase in the rate of the hydrolysis. In Figure 6, hydrolysis of different substrates by MAGL, normalized against compound 1c, is reported to better underline the high difference between the hydrolysis rate displayed by 1c and the other proposed MAGL substrates. ecules 2019, 24, 2241 8 o ure 6, hydrolysis of different substrates by MAGL, normalized against compound 1c, is report better underline the high difference between the hydrolysis rate displayed by 1c and the oth oposed MAGL substrates. Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 μM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. 3. Discussion The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), and 2-butyloctanoate (1j). The specific activity of the hMAGL preparation was 241.9 U/mg. Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 µM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), and 2-butyloctanoate (1j). The specific activity of the hMAGL preparation was 241.9 U/mg. 8 of 14 e other Molecules 2019, 24, 2241 to better underline proposed MAGL su Figure 6 Normali ed hydrolysis of different substrates by hMAGL Release of 7 hydroxyresorufin Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 μM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), d 2 b t l t t (1j) Th ifi ti it f th hMAGL ti 241 9 U/ Figure 6. Normalized hydrolysis of different substrates by hMAGL. Release of 7-hydroxyresorufin upon hydrolysis of different resorufynil esters (each at a concentration of 5 µM) by 25 ng hMAGL was measured in triplicates, the slopes of the regression lines were averaged and then normalized against the slope of the most efficiently hydrolyzed compound. The error bars represent the standard deviation of the results. The following compounds were used as drawn below the bars: acetate (1a), butyrate (1b), octanoate (1c), dodecanoate (1d), oleate (1f), arachidonate (1g), 2-methylhexanoate (1h), and 2-butyloctanoate (1j). The specific activity of the hMAGL preparation was 241.9 U/mg. Figure 6. Normalized hydrolysis of different substrates by hMAGL. 4.2.1. Synthesis of 7-Hydroxyresorufinyl-Derivatives 1a–1g To a solution of the opportune carboxylic acid (0.46 mmol) in dry CH2Cl2 (2 mL), oxalyl chloride (58 µL, 0.69 mmol) in dry CH2Cl2 (0.5 mL) was added dropwise at 0 ◦C under stirring. N,N-dimethylformamide (DMF, 1 drop) was added next. The reaction mixture was stirred at room temperature for 3 h and then concentrated under vacuum, giving crude acyl chloride. This residue was dissolved in dry CH2Cl2 (1 mL) and added dropwise to an ice-cold suspension of resorufin (50 mg, 0.23 mmol) and triethylamine (48 µL, 0.35 mmol) in dry CH2Cl2 (3 mL) and then stirred overnight at room temperature. After dilution with CH2Cl2 the salts residues were removed by filtration obtaining a brick-red solution; that was washed with 0.5 M HCl (2 mL) and saturated NaHCO3 (2.5 mL), dried on anhydrous Na2SO4 and concentrated to give the crude product that was purified with column chromatography on silica gel (Supplementary Table S1). Yields 62–92%. 3. Discussion In order to evaluate and validate the assay for its potential in high-throughput screening applications, we further determined the Z-factor [28], a dimensionless statistical parameter that reflects both the assay signal dynamic range and the data variation associated 9 of 14 Molecules 2019, 24, 2241 with the signal measurements. The assay was highly reproducible with an overall average Z′ value of 0.86. The objective of this study was to identify a substrate readily accessible, stable in solution and with low rate of spontaneous hydrolysis for realizing an HTS method for MAGL activity using human recombinant MAGL. It should be considered that MAGL belongs to the group of serine hydrolases (SHs), a superfamily of enzymes able to cleavage ester, amide, or thioester bonds of protein, peptide, and small molecule substrates [29] including esterases, lipases, peptidases, and amidases [30]. Within this family, there are the SHs that regulate the biosynthesis and degradation of two major endocannabinoids, AEA and 2-AG, as well as other lipid-metabolizing serine hydrolases that are ubiquitously distributed [31]. Therefore, in the potentiality to apply 1c as a probe for experiments performed in complex biological systems, as living cells or cell lysates, the hydrolysis of the probe 1c by other SHs enzymes should be considered. These results demonstrated that our fluorometric method could be successfully applied to identify compounds that modulate MAGL activity. 3. Discussion disadvantageous folding in the enzyme active site. The scale of Y axis (Figure 3), and the Vmax and Km values (Table 1), highlight how steric hindrance, due to the different length of acyl side chain on C2, reduce the rate of substrate hydrolysis. Interestingly, 1j shows a Michaelis-Menten kinetic behavior even if with very low hydrolysis rate. In this case, the bulkier side chain likely exerts an even stronger effect of steric hindrance, which does not allow reaching the concentration needed to manifest the inhibitory effect. Compound 1c proved to be a very interesting substrate for MAGL. Indeed, with respect to the previously described arachidonate 1g [12] and oleate 1f, the two resorufine derivatives of the MAGL natural substrates, 1c shows slightly higher XP Glide docking score, MM-GBSA binding energy and Km values. In the molecular docking poses, 1c resorufin perfectly overlaps the 1g one, while the linear side chain of 1c mimics the 2-AG side chain. Focussing our attention specifically on the difference in activity pointed out for compound 1c versus 1b, as already reported in the Results section and in Figure S4, a significant relative difference in the extension of the hydrophobic interactions can be one of the factors contributing to the different stability of the complexes, as shown by the different of the solvation/desolvation energy reported in the Prime MM-GBSA energy column in Table 1. The Vmax for 1c, however, is much higher than that of 1g, as indicated also by the direct comparison of the catalytic speed of the two substrates. In addition, the smaller substrate 1c has superior aqueous solubility properties and contains no potential labile cis olefins. Therefore, the signal to background ratio of 1c (seven to one) is suitable for the screening for MAGL inhibitors in high-throughput screening. For all these reasons, 1c was selected and validated as new red fluorogenic probe for the HTS method. MAFP and URB602, two well-known serine hydrolase and MAGL inhibitors, were chosen for validation [7,19]. After a 60 min pre-incubation of the enzyme with the inhibitor, the IC50 values for both MAGL inhibitors using hMAGL and 1c (5 µM) were obtained and compared with those reported in literature [12,20]. 4.1. Chemicals Arachidonic acid, resorufin, and all other reagents and solvents were purchased from Sigma-Aldrich. Reactions progress was monitored by analytical thin-layer chromatography (TLC) on pre-coated aluminum foils. Monoacylglycerol lipase (human recombinant, 50 µg) was purchased from Cayman Chemical. All steps, which included resorufin, were carried out protecting the compound from light. Fluorescence signals were recorded by a Jasco FP-8300 fluorometer using the kinetic mode (λex = 571 nm, λem = 588 nm, slit = 2.5 nm in both cases) in black, flat-bottomed, 96-well polystyrene microtiter plates. 1H-NMR spectra were recorded in CDCl3 (isotopic enrichment 99.95%) solutions at 300 K using a Bruker AVANCE 500 instrument (Bruker Italia Srl, Milan, Italy) (500.13 MHz for 1H, 125.76 MHz for 13C) using 5 mm inverse detection broadband probes and deuterium lock. Chemical shifts (δ) are given as parts per million relative to the residual solvent peak (7.26 ppm for 1H) and coupling constants (J) are in Hertz (Supplementary Table S4). 4.2. Synthesis 4.6. Data Analysis All experiments were performed in triplicate and independently replicated at least once. The curve generated from the hydrolysis of the substrates was used to convert raw fluorescence data into nmol/mL/min of resorufin produced. The values of negative controls were subtracted from the enzymatic curve. The initial velocities were determined from the linear portion of the resulting curve. Kinetic data were elaborated using GraphPad Prism 6.0c and Microsoft Excel graphing software; kinetic parameters Km and Vmax were calculated using GraphPad, applying a nonlinear regression analysis (Michaelis-Menten). The quantitative data were calculated as means ± standard errors. The Z′-factor was calculated using the equation Z′ = 1 −(3σh + 3σl)/|µh −µl|, where σh and σl are the standard deviations of the high and low signal controls, respectively, and µh and µL are the mean signal intensities of the high and low signal controls, respectively [28]. 4.4. Substrates Screening hMAGL activity was monitored following the increase of resorufin fluorescence (λex = 571 nm, λem = 588 nm), at intervals of 1 min. hMAGL (specific activity 241.9 U/mg) was diluted to 250 ng/mL in 50 mM Tris-HCl buffer (pH 7.4), with 1mM EDTA (reaction buffer). Each reaction well contained 80 µL Tris-HCl 50 mM with EDTA 1 mM, 10 µL of reaction buffer with 25 ng hMAGL and the putative substrate dissolved in 10 µL of DMSO (final concentrations 5 µM or 25 µM for all other substrates), total volume 100 µL [12]. In addition, resorufin calibration curve and negative control, containing 90 µL of reaction buffer and no hMAGL, were measured. All resorufin ester solutions were freshly prepared in DMSO. 4.5. Kinetic Assays of hMAGL Reagent solutions for each substrate were prepared diluting first 5 mM stock solution with DMSO (5 µM, 1 µM, 25 µM, 50 µM, 100 µM and 250 µM), and then 1:9 in the reaction buffer. For kinetic experiments 10 µL of the reaction buffer, containing 25 ng of hMAGL, and 90 µL of the appropriate reagent solution were added to each well. The final concentrations were for hMAGL 25 ng/100 µL (7.6 nM), for all substrates 0.5 µM, 1 µM, 2.5 µM, 5 µM, 10 µM, and 25 µM, and DMSO 10%. For negative controls 10 µL of reaction buffer were added instead of enzyme. In addition, blank samples (90 µL of reaction buffer, 10 µL DMSO) were analyzed. Fluorescence was recorded at room temperature for 50 cycles with a cycle time of 3 min. A standard curve was generated by plotting fluorescence of five concentrations of resorufin (0.02 µM, 0.1 µM, 0.5 µM, 1 µM, 5 µM) prepared by diluting DMSO stocks in reaction buffer (10% DMSO). 4.2.2. Synthesis of Resorufin Esters 1h–1k To a stirred solution of the opportune carboxylic acid (0.46 mmol) in dry CH2Cl2 (3 mL), were added DMAP (2.5 mg. 0.02 mmoli) and resorufin (50 mg, 0.23 mmol). DCC (72 mg, 0.35 mmoli) was added to the reaction mixture at 0 ◦C. Stirring was continued for 5 min at 0 ◦C and for 3 h at 20 ◦C. Precipitated urea was then filtered offand the filtrate evaporated down in vacuo. The residue was taken up in CH2Cl2 and, if necessary, filtered free of any further precipitated urea. The CH2Cl2 solution 10 of 14 Molecules 2019, 24, 2241 was washed twice with 0.5 M HCl (2 mL) and saturated NaHCO3 (2.5 mL), dried on anhydrous Na2SO4 and concentrated to give the crude product that was purified by column chromatography on silica gel, yields 82–92% (Supplementary Table S1). 4.3. Stability of the Substrates The stability of all substrates was tested in 50 mM Tris-HCl buffer (pH 7.4, 1 mM EDTA), as previously reported [12]. Briefly, 10 µL of the substrates dissolved in DMSO (final concentration 5 µM) were added to 90 µL of the buffer. The fluorescence increase at 588 nm was monitored at intervals of 1 min over 90 min at rt., using a Jasco FP-8300 fluorometer (λex = 571 nm, λem = 588 nm). 4.8. In Silico Molecular Docking Simulations All the computational procedures were carried out by the Schrödinger Small-Molecule Drug Discovery Suite 2018-01 (Schrödinger, Cambridge, USA). The crystallographic structure of the catalytic domain of hMAGL was downloaded from the RCSB PDB (PDB ID: 3PE6, resolution of 1.35 Å) [32]. Since the selected MAGL crystallographic structure presents three engineered mutations for increasing the quality of the diffracting crystal, the Schrödinger Protein Refinement tool was used to mutate Ala36, Ser169 and Ser176 in Lys36, Leu169 and Leu176, respectively. The wild-type MAGL structure was then energy minimized using the Schrödinger Protein Preparation Wizard in order to fix structural issues in the three-dimensional (3D) structure. Tested ligands were built through the Schrödinger Maestro Build Toolbar and prepared for docking by the Schrödinger Ligand Preparation, generating the stereoisomers of 1h, 1i, and 1j. A receptor grid, which defines the MAGL active site, was generated via the Schrödinger Receptor Grid Generation, centring a cubic box, with an edge of 20 Å, on the co-crystallized inhibitor. The molecular docking procedure was carried out by the Schrödinger Glide Docking in the “extra precision (XP)” mode in order to evaluate the ability of the tested ligand to bind the MAGL catalytic domain, keeping only the 20 top-scoring poses. The top-scoring solution for each ligand was submitted to the Schrödinger Prime MM-GBSA, which integrates molecular mechanics energies combined with the generalized Born and surface area continuum solvation [33] in order to compute ligand binding and ligand strain energies for a set of ligands and a single receptor. 4.9. Dynamic Light Scattering Analyses Dynamic light scattering experiments were performed in a custom modified setup (Scitech 100). The wavelength of the excitation light is λ = 532 nm and the scattered light was collected at a constant angle θ = 90◦. Both the excitation light and the scattered light were introduced and fetched by means of polarization-maintaining fibers. The sample was put in a cylindrical glass capillary (O.D. = 3 mm I.D. 2.4 mm) and sealed with a silicon cap. The intensity time autocorrelation was obtained using g2 (τ) a digital correlator (flex-03d Correlator.com). Each g2 (τ) was obtained after averaging 600 s and for each experiment three g2 (τ) were acquired. 4.7. Validation of 1c for Screening Assay MAFP and URB602 were chosen for the method validation due to their well-known MAGL inhibitory activity [7,19]. To prepare inhibitors stock solutions, commercial MAFP solution (10 mg/mL in ethanol) was diluted to 200 µM in DMSO and 15 µM URB602 solution in DMSO was obtained from powder. Eight different working solution were then prepared by dilution with DMSO. 10 µL of diluted 11 of 14 Molecules 2019, 24, 2241 hMAGL solution containing 25 ng of the enzyme and 10 µL of the appropriate MAFP or URB602 solution were added to wells of a 96-well plate and the volume was adjusted to 95 µL with reaction buffer (Tris-HCl 50 mM with EDTA 1 mM). In control wells, 10 µL of DMSO were added instead of inhibitor solution and the black samples containing only reaction buffer and DMSO (10%) also were prepared. Final concentrations of MAFP were 1.0 µM, 500.0 nM, 100.0 nM, 50.0 nM, 10.0 nM, 5.0 nM, and 0.1 nM; final concentrations of URB602 were 75.0 µM, 50.0 µM, 25.0 µM, 10.0 µM, 5.0 µM, 1.0 µM, and 100.0 nM. A 100.0 µM 1c working solution was prepared by diluting a 5.0 mM DMSO stock 1:50 in DMSO. After 60 min of incubation at 25 ◦C, 5.0 µL of 1c working solution was added to each well to give a final substrate concentration of 5.0 µM (10% DMSO). Fluorescence was recorded at room temperature for 30 cycles, with a cycle time of 1 min. All experiments were performed in triplicate and independently replicated at least once and the mean of the three obtained values was used for calculation. The mean fluorescence value of a blank was subtracted from the value of each sample and control well to normalize data at each time point, the mean value of control wells was subtracted to the mean value of each sample. From the slop of kinetic curves, residual enzymatic activity was calculated and IC50 values were obtained by non-linear regression analysis of log[concentration]/inhibition curves. IC50 was determined as the concentration of inhibitor that results in an initial velocity 50% that of the sample containing no inhibitor. IC50 was used along with previously calculated Km to determine Ki. 5. Conclusions We present here the set-up and validation of a new HTS method for MAGL activity based on a red fluorogenic probe. Starting from the synthesis of a few long-wavelength fluorogenic compounds, characterized by different acylic side chains, and their application to the fluorometric determination 12 of 14 Molecules 2019, 24, 2241 of MAGL activity, 7-hydroxyresorufinyl octanoate (1c) was selected as the best substrate for the HTS method. 1c is the eligible substrate among the compound tested, being the substrate with the higher rate of hydrolysis and the best Km and Vmax values. in-silico docking studies show the favorable interactions between 1c and MAGL active site. Moreover, compound 1c can be easily prepared in milligram and gram scale and is very stable in solution as demonstrated by the low rate of spontaneous hydrolysis. The probe 1c was validated using the well-known MAGL inhibitors URB602 and MAFP, in assay condition. y In conclusion, the fast, sensitive, and accurate method described in this study is a powerful new tool for the high-throughput assay of MAGL activity and screening of MAGL modulators. Supplementary Materials: The following are available online. Table S1. Mobile phases used for the separation of synthesis products. Table S2. Ligand: MAGL interactions. Table S3. Decay times and corresponding radius. Table S4. Assignment of 1H and 13C-NMR Chemical Shift in CDCl3. Figure S1. Comparison between spontaneous hydrolysis of 1a–d, 1f, and 1h–k. Figure S2: Time correlation functions for the scattered intensity (at θ = 90◦). Figure S3. Binding poses of tested compounds into the MAGL binding site. 1H- and 13C-NMR spectra of compounds 1a–k. Figure S4. MAGL complexes with compounds 1b and 1c. Author Contributions: Conceptualization P.C.; Investigation, M.M., S.C., R.O., C.P., L.P., S.D.L.; Validation, M.M.; Formal Analysis, M.M., R.O., L.P., C.P; Resources P.C. and I.E.; Writing—Original Draft Preparation, M.M., R.O., S.C., I.E. and P.C.; Writing—Review & Editing, R.O., S.C., and P.C.; Funding Acquisition, P.C. and I.E. Funding: This work was supported by “Università degli Studi di Milano, Linea 2 – Azione A 2017-2018” to P.C. and MIUR Progetto Eccellenza to I.E. Acknowledgments: The authors thank the University of Milan for financial support. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Nat. Med. 2018, 16, 113–124. [CrossRef] [PubMed] 14. Yu, Y.; Li, Z.; Guo, R.; Qian, J.; Zhang, H.; Zhang, J.; Zhao, X.; Wang, S.; Wang, Y. 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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/). © 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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Nitroalkanes as Central Reagents in the Synthesis of Spiroketals
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Roberto Ballini 1,*, Marino Petrini 1 and Goffredo Rosini 2 Roberto Ballini 1,*, Marino Petrini 1 and Goffredo Rosini 2 1 Dipartimento di Scienze Chimiche, Università di Camerino, via S.Agostino, 1, I-62032 Camerino, Italy; E-mail: marino.petrini@unicam.it. 2 Dipartimento di Chimica Organica ‘A. Mangini’, Alma Mater Studiorum–Università di Bologna, Viale del Risorgimento n. 4, 40136 Bologna, Italy; E-mail: goffredo.rosini@unibo.it. * Author to whom correspondence should be addressed; E-mail: roberto.ballini@unicam.it; Fax: +39 0737 402297 * Author to whom correspondence should be addressed; E-mail: roberto.ballini@unicam.it; Fax: +39 0737 402297 * Author to whom correspondence should be addressed; E-mail: roberto.ballini@unicam.it; Fax: +39 0737 402297 Received: 8 January 2008; in revised form: 2 February 2008 / Accepted: 4 February 2008 / Published: 7 February 2008 Received: 8 January 2008; in revised form: 2 February 2008 / Accepted: 4 February 2008 / Published: 7 February 2008 Received: 8 January 2008; in revised form: 2 February 2008 / Accepted: 4 February 2008 / Published: 7 February 2008 Abstract: Nitroalkanes can be profitably employed as carbanionic precursors for the assembly of dihydroxy ketone frameworks, suitable for the preparation of spiroketals. The carbon-carbon bond formation is carried out exploiting nitroaldol and Michael reactions, while the nitro to carbonyl conversion (Nef reaction) ensures the correct introduction of the keto group. Several spiroketal systems endowed with considerable biological activity can be prepared using this synthetic strategy. Keywords: Conjugate addition, Nef reaction, nitroaldol reaction, nitroalkanes, spiroketa molecules ISSN 1420-3049 © 2008 by MDPI www.mdpi.org/molecules Review Molecules 2008, 13, 319-330 Molecules 2008, 13, 319-330 molecules ISSN 1420-3049 © 2008 by MDPI www.mdpi.org/molecules 1. Introduction The spiroketal moiety is a key motif embodied in a large number of natural products present in plants, fungi, insect secretions, shellfish toxins and other living organisms [1-5]. Many of these compounds also display a considerable biological activity as antibiotics and pheromones. From a synthetic standpoint, among various practical strategies currently available for the assembling of the spiroketal unit, the one based on the acid promoted intramolecular acetalization of dihydroxy ketone derivatives 1 firmly occupies a prominent position (Scheme 1). 320 Molecules 2008, 13 Scheme 1 O O R OH R1 OH m n R O R1 m n 1 2 H+ Scheme 1 Scheme 1 O R O R1 m n 2 H+ 2 The ring size of the resulting spiro compounds 2 can be suitably tuned by a careful choice of the carbon tethers linking the hydroxy and the carbonyl functions. The enhanced thermodynamic stability of the ketal system in compounds 2 usually makes unnecessary the continuous removal of water from the reaction mixture as usually needed for the intermolecular version of this process. Syntheses of dihydroxy ketone precursors of spiroketals enjoy the rich chemistry pertaining to the carbonyl group. Nucleophilic substitution onto carboxylic acid derivatives, alkylation of ketone enolates, aldol condensations and conjugate addition of ketone enolate equivalents to Michael acceptors are the most exploited synthetic routes to access dihydroxy ketone derivatives [6-7]. Dithianes are well known acyl anion equivalents that can be profitably employed in alkylation reactions after metalation using strongly basic reagents. Their utilization in the synthesis of natural products including spiroketal systems has been recently reviewed [8]. Nitroalkanes represent a formidable source of stabilized carbanions since the high electron-withdrawing power of the nitro group provides an outstanding enhancement of the hydrogen acidity at the α-position [9]. Nitronate anions 4 that can be generated in mild basic condition from nitroalkanes 3 act as carbon nucleophiles with common electrophilic reagents including haloalkanes, aldehydes and Michael acceptors, leading to carbon–carbon bond formation (Scheme 2) [10-12]. 1. Introduction Scheme 2 R NO2 B 3 4 R1X R NO2 R1 EWG R NO2 EWG R1CHO R NO2 R1 OH 5 6 7 R N O O R N O O Scheme 2 R NO2 B 3 4 R1X R NO2 R1 EWG R NO2 EWG R1CHO R NO2 R1 OH 5 6 7 R N O O R N O O The ability of the carbon-nitrogen bond in adducts 8 to be converted into a carbonyl group 9 by means of a Nef reaction [13], ultimately leads to a reversal of polarity of the neighboring carbon atom from nucleophilic to electrophilic, making nitroalkanes effective acyl anion equivalents (Scheme 3). Scheme 2 R NO2 B 3 4 R1X R NO2 R1 EWG R NO2 EWG R1CHO R NO2 R1 OH 5 6 7 R N O O R N O O Scheme 2 3 The ability of the carbon-nitrogen bond in adducts 8 to be converted into a carbonyl group 9 by means of a Nef reaction [13], ultimately leads to a reversal of polarity of the neighboring carbon atom from nucleophilic to electrophilic, making nitroalkanes effective acyl anion equivalents (Scheme 3). 321 Molecules 2008, 13 This account reports on the utilization of nitroalkanes as versatile reagents capable of promoting e carbon-carbon bond formation and then, upon their conversion into a carbonyl group, allowing the ti f th i d l t hili f ti t bt i th i k t l f k Scheme 3 R NO2 3 E+ R NO2 8 E Nef R O 9 E R O Scheme 3 R NO2 3 E+ R NO2 8 E Nef R O 9 E R O Scheme 3 Scheme 3 R NO2 3 E+ R NO2 8 E Nef R O 9 E R O 9 8 This account reports on the utilization of nitroalkanes as versatile reagents capable of promoting the carbon-carbon bond formation and then, upon their conversion into a carbonyl group, allowing the formation of the required electrophilic function to obtain the spiroketal framework. This account reports on the utilization of nitroalkanes as versatile reagents capable of promoting the carbon-carbon bond formation and then, upon their conversion into a carbonyl group, allowing the formation of the required electrophilic function to obtain the spiroketal framework. 2. 1,6-Dioxaspiro[4.4]nonanes Conjugate addition of nitroalkanes to enones provides a rapid entry to 1,4-difunctionalized derivatives. Particularly, nitromethane (10) can be regarded as a d1,d1 multiple coupling reagent corresponding to a carbonyl dianion synthon (Scheme 4) [14]. Scheme 4 CH3 NO2 O + O Al2O3 basic 62% O NO2 Al2O3 basic 53% O O NO2 O NaBH4 EtOH, 85% OH NO2 OH TiCl3 H2O, 65% O O O O O O + Chalcogran 10 11 12 13 14 15 (E)-16 (Z)-16 39/61 d1,d1 Scheme 4 Scheme 4 A double Michael addition of nitromethane using two different α,β-unsaturated carbonyl derivatives can be used as key strategy for the preparation of racemic 2-ethyl-1,6-dioxaspiro[4.4]nonane (16), known as chalcogran, the main component in the aggregation pheromone of the bark beetle Pityogenes chalcografus (L) [15]. Addition of nitromethane (10) to 1-penten-3-one (11) in the presence of basic Molecules 2008, 13 322 alumina under solventless conditions, affords the corresponding 4-nitro ketone 12 that is suitable for a subsequent 1,4-addition to acrolein (13) under the same conditions leading to nitro diketone 14. Reduction of the carbonyl groups with NaBH4 generates the diol system 15, while the nitro to carbonyl conversion is easily accomplished by a reductive procedure using an aqueous solution of TiCl3. This transformation is followed by a spontaneous spiroketalization that produces chalcogran 16 as a mixture of stereoisomers in 20% overall yield. The two conjugate additions can be combined in a single operation when symmetrical 2,7-dialkyl- 1,6-dioxaspiro[4.4]nonane systems are needed as demonstrated in the examples portrayed in Scheme 5 [16]. Basic alumina can be profitably substituted by a macroreticular resin with basic character (Amberlyst A21) also working under solventless conditions. Washing the resin with acetonitrile affords a solution of the nitro diketone that upon addition of water and NaBH4 leads to the nitro diol 18. The Nef conversion is then carried out under classical hydrolytic conditions converting the nitro group into the corresponding nitronate anion that suffers a cleavage in strongly acidic conditions. The obtained spiroketals 19 are components of the pheromone mixture in insects of the Andrena species. CH3 NO2 + R O R OH NO2 OH 1. NaOH 2. H2SO4 O O O O 10 17 18 19 2 1. Amberlyst A21 2. 2. 1,6-Dioxaspiro[4.4]nonanes NaBH4, CH3CN/H2O R R R Me Et n-Pr 18 19 70 60 80 71 71 80 yield (%) (E,E/E,Z/Z,Z) (48:35:17) (50:50:0) (48:33:19) Scheme 5 CH3 NO2 + R O R OH NO2 OH 1. NaOH 2. H2SO4 O O O O 10 17 18 19 2 1. Amberlyst A21 2. NaBH4, CH3CN/H2O R R R Me Et n-Pr 18 19 70 60 80 71 71 80 yield (%) (E,E/E,Z/Z,Z) (48:35:17) (50:50:0) (48:33:19) Scheme 5 10 10 Enantioselective reduction of nitro diketones 20 using baker’s yeast or diisopinocampheyl- chloroborane (DIP-ClTM) provides a straightforward entry to optically active nitro diols 21 (Scheme 6) [17-18]. The level of enantioselection is usually high (ee > 95%) and, quite interestingly, reduction of each carbonyl group seems to proceed independently, since no meso forms of nitro diols 21 are found in the reaction mixture. Since DIP-ClTM is commercially available in both enantiometic forms, it is possible with this reagent to prepare (R,R)- and (S,S)-21. The nitro to carbonyl conversion of some compounds (S,S)-21 generates the corresponding spiroketals 22 in moderate to good yields, but with modest stereoselectivity. 323 Molecules 2008, 13 R OH NO2 OH 1. NaOH 2. H2SO4 21 (Z,Z)-22a R Scheme 6 R O NO2 O 20 R (E,E)-22a 41%(1:3) a: R = Me; baker's yeast, 58%, (S,S), ee= 95% reduction b: R = Ph;(+)-DIP-ClTM, 74%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% c: R =2-OMe-Ph;(+)-DIP-ClTM, 48%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% (S,S)-21a (Z,Z)-22b (E,E)-22b 56%(1:1.25) (Z,Z)-22c (E,E)-22c 70%(1:2.5) (S,S)-21b,c OO O Ar O O O O Ar Ar O O Ar R OH NO2 OH 21 R Scheme 6 R O NO2 O 20 R a: R = Me; baker's yeast, 58%, (S,S), ee= 95% reduction b: R = Ph;(+)-DIP-ClTM, 74%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% c: R =2-OMe-Ph;(+)-DIP-ClTM 48% (R R) ee= 95% R OH NO2 OH 21 R Scheme 6 R O NO2 O 20 R reduction Scheme 6 R OH OH 21 R R O 2 O 20 R a: R = Me; baker's yeast, 58%, (S,S), ee= 95% reduction b: R = Ph;(+)-DIP-ClTM, 74%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% TM 21 a: R = Me; baker's yeast, 58%, (S,S), ee= 95% b: R = Ph;(+)-DIP-ClTM, 74%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% c: R =2-OMe-Ph;(+)-DIP-ClTM, 48%, (R,R), ee= 95% (-)-DIP-ClTM, 59%, (S,S), ee= 95% 3. 1,6-Dioxaspiro[4.5]decanes and 1,6-dioxaspiro[4.6]undecanes H2SO4, n-C5H12 O (E)-28 O 74% O 13 Amberlyst A21 78% Scheme 7 23 24 Br O O PdCl2/CuCl2, DMF 87% Br O NaNO2 DMF, 75% Scheme 7 23 24 Br O O PdCl2/CuCl2, DMF 87% Br O NaNO2 DMF, 75% NO2 O NO2 O O NaBH4 MeOH, 85% O 13 Amberlyst A21 78% Scheme 7 Scheme 7 Scheme 7 23 Br 23 24 25 26 PdCl2/CuCl2, DMF 87% DMF, 75% NO2 O NO2 O O NaBH4 MeOH, 85% O 13 Amberlyst A21 78% 23 24 25 26 NO2 O NO2 O O NaBH4 MeOH, 85% O 13 Amberlyst A21 78% 25 26 27 NO2 OH OH 1.NaOH, EtOH 2. H2SO4, n-C5H12 74% O (E)-28 O (E)-28 27 2-Nitrocycloalkanones can be considered as direct precursors of ω-nitro esters by way of an alcoholic retro-Claisen cleavage promoted by mild bases [21]. Therefore, conjugate addition of 2- nitro-cyclopentanone (29) to 1-buten-3-one (30) produces the expected adduct 31, which upon cleavage with KF in methanol affords ketoester 32 (Scheme 8). Complete reduction of the ester and keto groups in 32 using NaBH4 followed by usual nitro to carbonyl transformation gives a diastereomeric mixture of 2-methyl-1,6-dioxaspiro[4.5]decane (33), another component of the pheromone mixture of Paravespula vulgaris. 2-Nitrocycloalkanones can be considered as direct precursors of ω-nitro esters by way of an alcoholic retro-Claisen cleavage promoted by mild bases [21]. Therefore, conjugate addition of 2- nitro-cyclopentanone (29) to 1-buten-3-one (30) produces the expected adduct 31, which upon cleavage with KF in methanol affords ketoester 32 (Scheme 8). Complete reduction of the ester and keto groups in 32 using NaBH4 followed by usual nitro to carbonyl transformation gives a diastereomeric mixture of 2-methyl-1,6-dioxaspiro[4.5]decane (33), another component of the pheromone mixture of Paravespula vulgaris. The previous synthetic approach can be extended to 2-nitrocyclohexanone (34, n = 2) that like compound 29, can be coupled with different enones 35 (Scheme 9) [22]. The following three synthetic operations, namely retro-Claisen cleavage, reduction and nitronate formation can be carried out on adducts 36 in a single step using NaBH4 in a mixture of acetonitrile/water (3:2). The intermediate Scheme 8 O NO2 O basic Al2O3 ether, 78% O NO2 O MeOH, Δ KF 90% 32 MeO NO2 O O 1. NaBH4 MeOH/THF 2.NaOH 3. 3. 1,6-Dioxaspiro[4.5]decanes and 1,6-dioxaspiro[4.6]undecanes The assembly of the 1,6-dioxaspiro[4.5]decane skeleton requires a different synthetic approach since the oxane ring can only be formed through a ring closure involving a 1,5 relationship between the carbon atoms bearing the hydroxy and the nitro groups. A component of odors of the common wasp Paravespula vulgaris, namely (E)-7-methyl-1,6-dioxaspiro[4.5]decane (28) can be efficiently prepared starting from 1-bromo-5-hexene (23) that upon a Wacker-type oxidation with benzoquinone in the presence of Pd(II)/Cu(II) salts gives ketone 24 (Scheme 7) [19]. The required 1-nitrohexan-5- one (25) obtained by nucleophilic displacement of the bromine atom using sodium nitrite, is then made to react with acrolein in the presence of Amberlyst A21, leading to nitro diketone 26. Reduction of the carbonyl groups and Nef reaction complete the synthesis of spiroketal 28 in good overall yield and high stereoselectivity. The exclusive formation of the E stereoisomer for compound 28 can be rationalized accounting for the strong conformational stabilization caused by the anomeric effect that forces the oxygen atom of the furanose ring to occupy an axial position, while the methyl on the piranose ring is equatorial [20]. 324 Molecules 2008, 13 2-Nitrocycloalkanones can be considered as direct precursors of ω-nitro esters by way of an alcoholic retro-Claisen cleavage promoted by mild bases [21]. Therefore, conjugate addition of 2- nitro-cyclopentanone (29) to 1-buten-3-one (30) produces the expected adduct 31, which upon cleavage with KF in methanol affords ketoester 32 (Scheme 8). Complete reduction of the ester and keto groups in 32 using NaBH4 followed by usual nitro to carbonyl transformation gives a diastereomeric mixture of 2-methyl-1,6-dioxaspiro[4.5]decane (33), another component of the pheromone mixture of Paravespula vulgaris. 23 24 25 26 27 Br O O PdCl2/CuCl2, DMF 87% Br O NaNO2 DMF, 75% NO2 O NO2 O O NaBH4 MeOH, 85% NO2 OH OH 1.NaOH, EtOH 2. 3. 1,6-Dioxaspiro[4.5]decanes and 1,6-dioxaspiro[4.6]undecanes H2SO4, 61% OO 33 (E:Z = 2:1) O 29 30 31 Scheme 8 O NO2 O basic Al2O3 ether, 78% O NO2 O MeOH, Δ KF 90% 32 MeO NO2 O O 1. NaBH4 MeOH/THF 2.NaOH 3. H2SO4, 61% OO 33 (E:Z = 2:1) O 29 30 31 Scheme 8 31 32 The previous synthetic approach can be extended to 2-nitrocyclohexanone (34, n = 2) that like compound 29, can be coupled with different enones 35 (Scheme 9) [22]. The following three synthetic operations, namely retro-Claisen cleavage, reduction and nitronate formation can be carried out on adducts 36 in a single step using NaBH4 in a mixture of acetonitrile/water (3:2). The intermediate 325 Molecules 2008, 13 nitronate 37 upon rapid hydrolysis directly affords spiroketals 38. Thus in conclusion, this versatile strategy also allows for a rapid entry to 1,6-dioxaspiro[4.6]undecane systems. Scheme 9 O NO2 R O basic Al2O3 O NO2 O R MeCN-H2O (3:2) NaBH4 37 HO N OH R O 38 O R 34 35 36 n n O O 2N HCl n n n R yield (%) E:Z 1 Me 65 3:2 a b 1 Et 70 3:2 c 2 Me 75 3:2 d 2 H 60 _ rt Scheme 9 O NO2 R O basic Al2O3 O NO2 O R MeCN-H2O (3:2) NaBH4 34 35 36 n n rt Scheme 9 Scheme 9 rt 34 38 n R yield (%) E:Z 1 Me 65 3:2 a b 1 Et 70 3:2 c 2 Me 75 3:2 d 2 H 60 _ γ-Rubromycin is a pigment isolated from Streptomyces collinus particularly active against the reverse transcriptase of HIV-1. Like many other members of this class of compounds, γ-rubromycin is characterized by a bisbenzanellated 1,6-dioxaspiro[4.5]decane system that presents some challenging aspects from a synthetic standpoint. O O O O O OMe OH OH OH O MeO2C γ-Rubromycin 39 O O O O O OMe OH OH OH O MeO2C γ-Rubromycin 39 γ-Rubromycin 39 Preliminary attempts at using a dithiane based strategy for the assembly of the dihydroxycarbonyl moiety were unsuccessful. Therefore, a different procedure involving the utilization of the nitrocompound chemistry was pursued. Starting from aldehyde 40, a nitroaldol reaction with Molecules 2008, 13 326 nitromethane in aqueous media promoted by cetyltrimethylammonium bromide (CTABr), gives quantitatively the corresponding nitroalcohol 41 that upon dehydration and double bond reduction leads to 4-arylnitroalkane 42 (Scheme 10) [23-24]. 3. 1,6-Dioxaspiro[4.5]decanes and 1,6-dioxaspiro[4.6]undecanes A further nitroaldol reaction between nitro derivative 42 and arylaldehyde 43 represents the key step for the convergent synthesis of the carbon unit of the target molecule. Nitroalkene 44, which is directly obtained by this process, is reduced using Pearlman’s catalyst [Pd(OH)2/C] and after the Nef reaction, transformed into the spiroketal system 45 together with lesser amounts of uncyclized derivative 46. The latter compound slowly converts into 45 on standing, thus demonstrating that the rate of formation of the spiroketal framework is strongly affected by the nature of substituents present nearby the hydroxy groups. 4. 1,7-Dioxaspiro[5.5]undecanes and 1,7-Dioxaspiro[5.6]dodecanes Scheme 10 OMe OBn O MeNO2, CTABr 0.025M NaOH 100% OMe OBn OH 1. MsCl, i-Pr2NEt 2. NaBH4,MeOH NO2 OMe OBn NO2 + OMe OMe BnO O NH4OAc 67% OMe OBn O2N OMe OMe BnO AcOH 57% O O OMe OMe OMe OMe OH O OMe OMe HO + Pd(OH)2/C EtOH, conc.HCl on standing 40 41 42 43 44 45 (64%) 46 (18%) Scheme 10 OMe OBn O MeNO2, CTABr 0.025M NaOH 100% OMe OBn OH 1. MsCl, i-Pr2NEt 2. NaBH4,MeOH NO2 67% 40 41 Scheme 10 Scheme 10 MeNO2, CTABr 0.025M NaOH 100% OMe OBn OH 1. MsCl, i-Pr2NEt 2. NaBH4,MeOH NO2 67% 41 40 41 OMe OBn NO2 + OMe OMe BnO O NH4OAc OMe OBn O2N OMe OMe BnO AcOH 57% 42 43 44 42 EtOH, conc.HCl 4. 1,7-Dioxaspiro[5.5]undecanes and 1,7-Dioxaspiro[5.6]dodecanes As previously observed, a major advantage pertaining to the chemistry of nitro compounds compared to that of strongly anionic systems (i.e. organometallic reagents and related unstabilized carbanions), concerns the relatively mild conditions needed to generate the corresponding nitronate anion. This important feature makes the protection of many common functional groups including keto and hydroxyl groups unnecessary. The Henry reaction between nitro alcohol 47 and O-protected Molecules 2008, 13 Molecules 2008, 13 327 hydroxyaldehydes 48 occurs efficiently under heterogeneous conditions leading directly to nitroalkenes 49 (Scheme 11) [25]. As usual, reduction of the nitroalkene double bond is also coupled with formation of the nitronate anion 50, that upon acidification undergoes succesive THP deprotection, Nef reaction and ketalization affording spiroketals 51. These compounds are the main component of the sex pheromones of the olive fruit fly (Dacus oleae) (51a) and Andrena haemorrhoa (51b). Following a related strategy in which a 5-nitroketone and a 4-hydroxyaldehyde are used for the nitroaldol reaction, other dialkyl-1,7-dioxaspiro[5.5]undecanes can be readily obtained [26]. 5. Conclusions Nitroalkanes bearing oxygenated functional groups are suitable reagents for the assembling of the carbon skeleton of spiroketal systems. New carbon-carbon bonds can be generated exploiting the nucleophilic character of nitronate anions, easily generated from nitroalkanes using mild bases. Nitroaldol as well as conjugate addition reactions can be carried out on nitroalkanes using aldehydes and electron-poor olefins as reagents. Once the appropriate hydroxy groups are inserted in the right place of the structural framework, the nitro to carbonyl conversion ensures a rapid spiroketalization of the resulting dihydroxy ketone. The obtained spiroketals are compounds of relevant practical interest as pheromones, antibiotics and enzyme inhibitors. 3. 1,6-Dioxaspiro[4.5]decanes and 1,6-dioxaspiro[4.6]undecanes Scheme 11 R OH NO2 + OTHP O basic Al2O3 CH2Cl2, 40°C R OH NO2 OTHP R OH N OTHP n n n O O NaBH4 MeOH 2N HCl O O R n 47 48 49 50 51 n R yield (%) 1 53 a b 2 Me 54 51 H 49 64 66 Scheme 11 R OH NO2 + OTHP O basic Al2O3 CH2Cl2, 40°C R OH NO2 OTHP R OH N OTHP n n n O O NaBH4 MeOH 2N HCl O O R n 47 48 49 50 51 Scheme 11 49 The previous synthetic methodologies as a whole, are based on the nitro to carbonyl conversion to generate the necessary keto group for the spiroketalization step. However, beside nitroaldol and conjugate addition reactions, other processes such as C-acylation of nitroalkanes are possible. This strategy entails a reductive removal of the nitro group once it has served for the insertion of the carbonyl moiety in the molecular framework. This strategy has been successfully applied to the preparation of optically active 2-methyl-1,7-doxaspiro[5.6]dodecane (58), a pheromone of Andrena haemorrhoa F starting from (S)-4-nitro-2-(t-butyldimethylsilyloxy)pentane (52, Scheme 12) [27]. Reaction of compound 52 with N-acetylimidazole (53) under basic conditions leads to nitroketone 54 that can be efficiently denitrated under radical conditions using tributyltin hydride. The introduction of a further carbon framework containing the second hydroxy group is realized by a regioselective alkylation of ketone 55 with chloroalkane 56. O-Protected dihydroxy ketone 57 albeit obtained in modest yield, can be readily hydrolyzed to give spiroketal 58 with excellent enantiomeric excess. 328 Molecules 2008, 13 Scheme 12 NO2 OTBDMS 52 53 54 O N N DBU, THF, rt OTBDMS NO2 O Bu3SnH, AIBN 62% C6H6, Δ 92% OTBDMS O Cl OTHP 1. LDA, THF, -78°C 2. 55 OTBDMS O OTHP O O HCl MeOH 43% 58 ee = 97% 46% 57 56 Scheme 12 NO2 OTBDMS 52 53 54 O N N DBU, THF, rt OTBDMS NO2 O Bu3SnH, AIBN 62% C6H6, Δ 92% OTBDMS O Cl OTHP 1. LDA, THF, -78°C 2. 55 OTBDMS O OTHP O O HCl MeOH 43% 58 ee = 97% 46% 57 56 Scheme 12 Scheme 12 OTBDMS O Cl OTHP 1. LDA, THF, -78°C 2. 55 OTBDMS O OTHP 57 56 57 Acknowledgements Financial assistance by MIUR, Univeristy of Camerino and University of Bologna is gratefully acknowledged. Franke, W.; Kitching, W. Spiroacetals in Insects. Curr. Org. Chem. 2001, 5, 233–251. 1. Perron, F.; Albizati, K. F. Chemistry of Spiroketals. Chem. Rev. 1989, 89, 1617–1661. 2. Brimble, M. A.; Farès, A. F. 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Nitro Ketones in Organic Synthesis: A New, Short Synthesis of Racemic trans-2-Methyl-1,7-dioxaspiro[5.5]undecane, trans,trans- and trans,cis-2,8- Dimethyl-1,7-dioxaspiro[5.5]undecane by Henry Reaction. Liebigs Ann. Chem. 1994, 1235–1237. 27. Nakamura, K.; Kitayama, T.; Inoue, Y.; Ohno, A. Asymmetric Synthesis of a Pheromone for Andrena Haemorrhoa F from a Chiral Nitro Alcohol Obtained by the Yeast Reduction of a Nitro Ketone. Tetrahedron 1990, 46, 7471–7481. Samples availability: Contact the author.
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Extrasolar planets and brown dwarfs around AF-type stars
Astronomy & astrophysics
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To cite this version: S. Borgniet, A.-M. Lagrange, N. Meunier, F. Galland, L. Arnold, et al.. Extrasolar planets and brown dwarfs around AF-type stars. Astronomy and Astrophysics - A&A, 2019, 621, pp.A87. ￿10.1051/0004- 6361/201833431￿. ￿hal-01994794￿ Extrasolar planets and brown dwarfs around AF-type stars X. The SOPHIE sample: combining the SOPHIE and HARPS surveys to compute the close giant planet mass-period distribution around AF-type stars⋆,⋆⋆ S. Borgniet1, A.-M. Lagrange1, N. Meunier1, F. Galland1, L. Arnold2, N. Astudillo-Defru3, J.-L. Beuzit1, I. Boisse4, X. Bonfils1, F. Bouchy4,5, K. Debondt1, M. Deleuil4, X. Delfosse1, M. Desort1, R. F. Díaz5, A. Eggenberger5, D. Ehrenreich5, T. Forveille1, G. Hébrard2,6, B. Loeillet6, C. Lovis5, G. Montagnier2,6, C. Moutou4,7, F. Pepe5, C. Perrier1, F. Pont5, D. Queloz5,8, A. Santerne4, N. C. Santos9,10, D. Ségransan5, R. da Silva11,12, J. P. Sivan2, S. Udry5, and A. Vidal-Madjar6 Vidal-Madjar6 1 CNRS, IPAG, Université Grenoble Alpes, 38000 Grenoble, France e-mail: simon.borgniet@obspm.fr 2 2 Observatoire de Haute-Provence, Institut Pythéas UMS 3470, CNRS, Aix-Marseille Université, 04870 St-Michel-l’Observatoire, France 3 Departamento de Astronomía, Universidad de Concepción, Casilla 160-C, Concepción, Chile p p p 4 LAM (Laboratoire d’Astrophysique de Marseille) UMR 7326, CNRS, Aix Marseille Université, 13388 Mars 5 5 Observatoire Astronomique de l’Université de Genève, 51 Chemin des Maillettes, 1290 Versoix, Switzerland 6 5 Observatoire Astronomique de l’Université de Genève, 51 Chemin des Maillettes, 1290 Versoix, Switzerland 6 Institut d’Astrophysique de Paris UMR 7095 CNRS Université Pierre & Marie Curie 98 bis Boulevard Arago 75014 Par 5 Observatoire Astronomique de l’Université de Genève, 51 Chemin des Maillettes, 1290 Versoix, Switzerland 6 Institut d’Astrophysique de Paris, UMR 7095 CNRS, Université Pierre & Marie Curie, 98 bis Boulevard Ar France 7 Canada-France-Hawaii Telescope Corporation, 65-1238 Mamalahoa Hwy, Kamuela, HI 96743, USA endish Laboratory, J J Thomson Avenue, Cambridge CB3 0 y g 9 Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, 4150-762 Porto, P 10 D d Fí i A i F ld d d Ciê i U i id d d P R d C Al 9 Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, 4150-762 Porto, Portugal 10 Departamento de Física e Astronomia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, 10 Departamento de Física e Astronomia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre Portugal g 11 ASI – Space Science Data Center (SSDC), Via del Politecnico snc, 00133 Rome, Italy 11 ASI – Space Science Data Center (SSDC), Via del Politecnico snc, 00133 Rome, Italy 12 11 ASI – Space Science Data Center (SSDC), Via del Polite 12 12 INAF – Osservatorio Astronomico di Roma, Via Frascati 33, 00078 Monte Porzio Catone, Rome, Italy 12 INAF – Osservatorio Astronomico di Roma, Via Frascati 33, 00078 Monte Porzio Cat Received 15 May 2018 / Accepted 30 August 2018 Received 15 May 2018 / Accepted 30 August 2018 ABSTRACT Context. The impact of stellar mass on the properties of giant planets is still not fully understood. Main-sequence (MS) stars more massive than the Sun remain relatively unexplored in radial velocity (RV) surveys, due to their characteristics which hinder classical RV measurements. Aims. Our aim is to characterize the close (up to ∼2 au) giant planet (GP) and brown dwarf (BD) population around AF MS stars and compare this population to stars with different masses. Aims. Our aim is to characterize the close (up to ∼2 au) giant planet (GP) and brown dwarf (BD) population around AF MS stars and compare this population to stars with different masses. Aims. Our aim is to characterize the close (up to ∼2 au) giant planet (GP) and brown dwarf (BD) population around AF MS stars and compare this population to stars with different masses. compare this population to stars with different masses. Methods. We used the SOPHIE spectrograph located on the 1.93 m telescope at Observatoire de Haute-Provence to observe 125 north- ern, MS AF dwarfs. We used our dedicated SAFIR software to compute the RV and other spectroscopic observables. We characterized the detected substellar companions and computed the GP and BD occurrence rates combining the present SOPHIE survey and a similar HARPS survey. Methods. We used the SOPHIE spectrograph located on the 1.93 m telescope at Observatoire de Haute-Provence to observe 125 north- ern, MS AF dwarfs. We used our dedicated SAFIR software to compute the RV and other spectroscopic observables. We characterized the detected substellar companions and computed the GP and BD occurrence rates combining the present SOPHIE survey and a similar HARPS survey. Results. We present new data on two known planetary systems around the F5-6V dwarfs HD 16232 and HD 113337. For the latter, we report an additional RV variation that might be induced by a second GP on a wider orbit. We also report the detection of 15 binaries or massive substellar companions with high-amplitude RV variations or long-term RV trends. Based on 225 targets observed with SOPHIE and/or HARPS, we constrain the BD frequency within 2–3 au around AF stars to be below 4% (1σ). For Jupiter-mass GPs within 2–3 au (periods ≤103 days), we find the occurrence rate to be 3.7+3 −1% around AF stars with masses <1.5 M⊙, and to be ≤6% (1σ) around AF stars with masses >1.5 M⊙. ⋆Based in part on observations made at Observatoire de Haute Provence (CNRS), France. ⋆⋆RV time series of the full sample are only available at the CDS via anonymous ftp to cds http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/621/A87 HAL Id: hal-01994794 https://hal.sorbonne-universite.fr/hal-01994794v1 Submitted on 25 Jan 2019 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. Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics A&A 621, A87 (2019) https://doi.org/10.1051/0004-6361/201833431 © ESO 2019 ⋆Based in part on observations made at Observatoire de Haute Provence (CNRS), France. ⋆⋆RV time series of the full sample are only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/621/A87 Key words. techniques: radial velocities – stars: early-type – planetary systems – stars: variables: general Extrasolar planets and brown dwarfs around AF-type stars X. The SOPHIE sample: combining the SOPHIE and HARPS surveys to compute the close giant planet mass-period distribution around AF-type stars⋆,⋆⋆ S. Borgniet1, A.-M. Lagrange1, N. Meunier1, F. Galland1, L. Arnold2, N. Astudillo-Defru3, J.-L. Beuzit1, I. Boisse4, X. Bonfils1, F. Bouchy4,5, K. Debondt1, M. Deleuil4, X. Delfosse1, M. Desort1, R. F. Díaz5, A. Eggenberger5, D. Ehrenreich5, T. Forveille1, G. Hébrard2,6, B. Loeillet6, C. Lovis5, G. Montagnier2,6, C. Moutou4,7, F. Pepe5, C. Perrier1, F. Pont5, D. Queloz5,8, A. Santerne4, N. C. Santos9,10, D. Ségransan5, R. da Silva11,12, J. P. Sivan2, S. Udry5, and A. Vidal-Madjar6 Extrasolar planets and brown dwarfs around AF-type stars X. The SOPHIE sample: combining the SOPHIE and HARPS surveys to compute the close giant planet mass-period distribution around AF-type stars⋆,⋆⋆ S. Borgniet1, A.-M. Lagrange1, N. Meunier1, F. Galland1, L. Arnold2, N. Astudillo-Defru3, J.-L. Beuzit1, I. Boisse4, X. Bonfils1, F. Bouchy4,5, K. Debondt1, M. Deleuil4, X. Delfosse1, M. Desort1, R. F. Díaz5, A. Eggenberger5, D. Ehrenreich5, T. Forveille1, G. Hébrard2,6, B. Loeillet6, C. Lovis5, G. Montagnier2,6, C. Moutou4,7, F. Pepe5, C. Perrier1, F. Pont5, D. Queloz5,8, A. Santerne4, N. C. Santos9,10, D. Ségransan5, R. da Silva11,12, J. P. Sivan2, S. Udry5, and A. ⋆Based in part on observations made at Observatoire de Haute Provence (CNRS), France. ⋆⋆RV time series of the full sample are only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/621/A87 A87, page 1 of Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4. which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction More than three thousand exoplanets and brown dwarfs (BDs) have now been confirmed1, while thousands of other Kepler can- didates await confirmation. Most of these substellar companions are close (≤5–10 au) to their host star and have been detected via radial velocity (RV) and transit surveys (see e.g. Borucki et al. 2010; Howard et al. 2011; Fressin et al. 2013; Mayor et al. 2014). Giant gaseous planets are at the core of the planetary sys- tems as they carry most of their mass. These close giant planets (GPs) have revealed an unexpected diversity of orbital (period, eccentricity, inclination) and physical (mass, composition) prop- erties. This variety emphasizes the complexity of the different processes and interactions that shape the GP population in terms of formation, migration, dynamical interactions, or influence of the primary host star. While our understanding of the GP formation and evolu- tion mechanisms has dramatically improved over the past twenty years, the influence of stellar properties on GP distribution remains a key topic. For example, it has been clearly estab- lished that the GP frequency increases with stellar metallicity for solar-like, main-sequence (MS) FGK dwarfs (Santos et al. 2004; Fischer & Valenti 2005). p y This paper first presents our SOPHIE survey and its results, and then presents a statistical analysis combining our SOPHIE and HARPS targets from Paper IX to make a single, global study. The SOPHIE-HARPS combination is possible here because (i) the HARPS and SOPHIE instruments work on the same principles, (ii) they have a similar instrumental RV pre- cision (less than 1 m s−1 for HARPS versus a few m s−1 for SOPHIE), (iii) our surveys have the same overall characteristics, and (iv) we use exactly the same reduction and analysis that we used for our HARPS sample in Paper IX. In Sect. 2, we describe our SOPHIE sample and observations and give a brief outline of our computational tool, our observables, and our RV variability diagnosis. Section 3 is dedicated to the detection and character- ization of giant planets and other long-term companions within our SOPHIE survey. In Sect. ABSTRACT For periods shorter than 10 days, we find the GP occurrence rate to be below 3 and 4.5% (1σ), respectively. Our results are compatible with the GP frequency reported around FGK dwarfs and are compatible with a possible increase in GP orbital periods with stellar mass as predicted by formation models. Results. We present new data on two known planetary systems around the F5-6V dwarfs HD 16232 and HD 113337. For the latter, we report an additional RV variation that might be induced by a second GP on a wider orbit. We also report the detection of 15 binaries or massive substellar companions with high-amplitude RV variations or long-term RV trends. Based on 225 targets observed with SOPHIE and/or HARPS, we constrain the BD frequency within 2–3 au around AF stars to be below 4% (1σ). For Jupiter-mass GPs within 2–3 au (periods ≤103 days), we find the occurrence rate to be 3.7+3 −1% around AF stars with masses <1.5 M⊙, and to be ≤6% (1σ) around AF stars with masses >1.5 M⊙. For periods shorter than 10 days, we find the GP occurrence rate to be below 3 and 4.5% (1σ), respectively. Our results are compatible with the GP frequency reported around FGK dwarfs and are compatible with a possible increase in GP orbital periods with stellar mass as predicted by formation models. Key words. techniques: radial velocities – stars: early-type – planetary systems – stars: variables: general ⋆Based in part on observations made at Observatoire de Haute Provence (CNRS), France. ⋆⋆RV time series of the full sample are only available at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/621/A87 A87, page 1 of 30 Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A87, page 1 of 30 A&A 621, A87 (2019) From 2005 to 2016, we carried out a survey targeting GPs and BDs orbiting within ∼3 au around southern AF MS dwarfs with the High-Accuracy Radial velocity Planet Searcher (HARPS, see Pepe et al. 2002) spectrograph on the 3.6 m ESO telescope at La Silla Observatory in Chile. During this survey, we detected three new GPs around two F5-6V stars (Desort et al. 2008), showed that GPs more massive than Jupiter and BDs are often detectable at periods of up to a thousand days around MS AF dwarfs more massive than the Sun (Lagrange et al. 2009), and derived the first constraints on the occurrence rates of such companions (Borgniet et al. 2017, hereafter Paper IX). Meanwhile, we carried out a survey of northern AF MS stars with the SOPHIE echelle fibre-fed spectrograph (Bouchy & Sophie Team 2006), which is the successor of the ELODIE spectrograph (Baranne et al. 1996) on the 1.93 m telescope at Observatoire de Haute-Provence in France. Most of the observations were made in the course of the SP4 programme within the SOPHIE consortium (Bouchy et al. 2009). The first results of our SOPHIE survey, regarding the complex RV variations of HD 185395 and the detection of a ∼3 MJup GP orbiting with a ∼320-day period around HD 113337, were presented in Desort et al. (2009) and Borgniet et al. (2014), respectively. 1. Introduction 4 we make a global analysis of the combined SOPHIE + HARPS sample: (i) we characterize the RV intrinsic variability of our targets, (ii) we derive the search com- pleteness of our combined survey, and (iii) we combine the com- panion detections and the detection limits to compute the close companion (GP and BD) occurrence rates and estimate their (mass, period) distribution around AF MS stars. We conclude in Sect. 5. However, the impact of the stellar host mass on its compan- ion’s properties is still not fully understood. The core-accretion formation process, which is believed to be at the origin of most of the RV and transit planets, predicts an increase in GP fre- quency with stellar mass (up to 3–3.5 M⊙, Kennedy & Kenyon 2008). This was confirmed by comparing the GP occurrence rates derived from RV surveys of low-mass M dwarfs (see e.g. Bonfils et al. 2013) and of solar-mass FGK dwarfs (see e.g. Cumming et al. 2008). Subgiant and giant stars seem to confirm this trend for higher stellar masses, as higher GP occurrence rates than those found around solar-mass dwarfs have been reported for these evolved stars (see e.g. Johnson et al. 2010; Reffert et al. 2015). However, the actual mass and origin of these evolved stars is subject to an ongoing controversy, as several studies argue that these giants do not significantly differ from solar-type stars in terms of mass, and are consequently their descendants (instead of being the descendants of AF MS stars; see the list of references in Borgniet et al. 2017). AF stars with well-established masses above 1 M⊙on the main sequence are generally rejected from RV surveys because their specific characteristics (fewer spectral lines and faster rota- tion) hinder classical RV measurements. A recent survey has targeted chemically peculiar Ap stars, which exhibit lower rota- tional velocities and more narrow spectral lines than typical AF MS dwarfs (Hartmann & Hatzes 2015). 2. Description of the SOPHIE survey Twelve years ago our group developed a tool, Software for the Analysis of the Fourier Interspectrum Radial velocities (SAFIR, Galland et al. 2005b), specifically dedicated to deriv- ing RV measurements of AF MS stars as precisely as possible. For one target, SAFIR cross-correlates each observed spectrum with a reference spectrum built from all the acquired spectra instead of a binary mask, allowing one to reach a better RV pre- cision (closer to the photon noise limit defined by Bouchy et al. 2001). Even if this RV precision remains intrinsically degraded compared to cooler and/or slower rotating stars, we developed SAFIR (and other specific tools) to demonstrate that it is indeed possible to search for close (≲3 au) massive (≥1 MJup) GP and BD companions around AF MS stars (Galland et al. 2005a, 2006; Lagrange et al. 2009; Meunier et al. 2012). 1 http://exoplanet.eu, Schneider et al. (2011). 2.2. Observations As was done for our HARPS sample, we computed the RV and, whenever possible, the line profiles and the chromospheric emission in the calcium lines of our targets with our software SAFIR (Chelli 2000; Galland et al. 2005b). The main principles of SAFIR are fully detailed in Paper IX. Our main observables are the RV, the spectrum cross-correlation function (CCF) and its full width at half maximum (FWHM), the bissector velocity span (hereafter BIS; Queloz et al. 2001) and the logR′ HK. Our main (but not the only) diagnosis for classifying the RV variability of our targets and distinguishing between its possible sources is the (RV, BIS) diagram; see Paper IX for a fully detailed descrip- tion of our various observables and of our variability criteria and diagnosis. Very briefly, we classify our targets as RV vari- able if the total RV amplitude and the RV standard deviation are respectively at least six and two times larger than the mean RV uncertainty. A RV–BIS anti-correlation is most likely the indication of stellar magnetic activity, a RV–BIS vertical dia- gram indicates pulsations and a flat RV–BIS diagram points toward companionship; CCF distortions and/or RV–FWHM cor- relation might indicate the presence of a SB2 or SB1 binary. More detailed results on our target observables can be found in Appendix A. 1 We observed our 125 SOPHIE targets mainly between Novem- ber 2006 and April 2014. We made some additional observa- tions in 2015–2016 to complete our survey for the targets with the less data and to further monitor the most interesting tar- gets. We acquired the SOPHIE spectra in high-resolution mode (R ∼75000), in the 3872–6943 Å wavelength range. As was done with our HARPS targets, we adapted the exposure times depend- ing on the observing conditions and on the target magnitude in order to get a high signal-to-noise ratio (S/N), of at least 100 at λ = 550 nm. We made most of the exposures in the simultaneous- Thorium mode, for which the SOPHIE A fibre is centred on the target while the B fibre is fed by a Thorium lamp. This allowed us to follow and correct for a potential instrumental RV drift induced by local temperature or pressure variations, if needed. 2.1. Sample Our SOPHIE sample is made up of 125 AF MS dwarf stars with spectral types in the range A0V to F9V. We detail the physical properties of our sample in Appendix A. We selected our tar- gets using the same selection process as for our HARPS survey (see details in Lagrange et al. 2009, Paper IX and Appendix B). Briefly, our selection relies on the following criteria: (i) the spectral type; (ii) the distance to the Sun, and (iii) the exclu- sion of known SB2 binary, chemically peculiar stars, or con- firmed δ Scuti or γ Doradus pulsators. We ended up with 125 targets with B −V in the range −0.02 to 0.56, v sin i in the range 3 to 250 km s−1, and masses in the range 1 to 2.5 M⊙(Fig. 1). We note that the exclusion of known pulsators results in a visible dichotomy between our ∼A- and ∼F-type targets. 1 http://exoplanet.eu, Schneider et al. (2011). A87, page 2 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. g p yp Fig. 1. Main physical properties of our sample. Left panel: HR diagram of our sample, in absolute V-magnitude vs B−V. Each black dot corresponds to one target. The Sun (red star) is displayed for comparison. Middle panel: v sin i vs B −V distribution. Right panel: mass histogram of our sample (in M⊙). Fig. 1. Main physical properties of our sample. Left panel: HR diagram of our sample, in absolute V-magnitude vs B−V. Each black dot corresponds to one target. The Sun (red star) is displayed for comparison. Middle panel: v sin i vs B −V distribution. Right panel: mass histogram of our sample (in M⊙). Fig. 2. Observation summary. Left panel: histogram of the spectrum number per target. Middle panel: histogram of the separate observation epoch number per target. Right panel: histogram of the time baselines. Fig. 2. Observation summary. Left panel: histogram of the spectrum number per target. Middle panel: histogram of the separate observation epoch number per target. Right panel: histogram of the time baselines. 2.4. SOPHIE+ In June 2011 (BJD 2455730), SOPHIE was updated (and called SOPHIE+) by implementing octagonal fibres at the fibre link (Bouchy et al. 2013). Prior to this update, the SOPHIE RV were known to exhibit a systematic bias induced by the insufficient scrambling of the old fibre link. This bias was called the “see- ing effect” as seeing variations at the fibre entrance were causing RV variations (Boisse et al. 2011). One way to correct for this bias is to measure the RV in the blue and red halves of each SOPHIE spectral order and compute their difference δRV, which should be correlated to the original RV if affected by the seeing effect, as detailed in Díaz et al. (2012). We applied the same cor- rection in SAFIR, computing δRV for all spectra acquired prior to June 2011, and correcting the RV from the δRV correlation if needed (i.e. if the Pearson’s correlation coefficient was above 0.5 in absolute value). This happened for 16 of our targets for which the δRV correlation was >0.5. y p g g The BIS data has a 24.5 m s−1 dispersion and does not show any high-amplitude variations. However, a slight long- term variation may be present with a BIS extremum between BJD 2455600 and BJD 2456500, approximately (Fig. D.1, sec- ond row). Except for two peaks at ∼2 and ∼3 days which are probably induced by the stellar rotation, the BIS Lomb–Scargle periodogram does not exhibit any significant signal for periods up to 1000 days. However, it shows power between 2000 and 4000 days, i.e. in roughly the same period range as the observed RV long-term variations. When looking at the BIS CLEAN periodogram, the long-term peak is centred at a ∼2000-day peri- odicity (i.e. quite distinct from the ∼3200-day RV periodicity) and is no longer the highest peak in the periodogram (the high- est peak being at ∼2.5 days). The (RV, BIS) diagram is nearly flat (with a −0.12 ± 0.02 linear slope and a Pearson correlation coefficient of -0.32), thus ruling out stellar activity as the only source of the RV variations. The FWHM time series exhibits a high-amplitude (∼250 m s−1) long-term variation, with a minimum between BJD 2455700 and BJD 2456200 (Fig. D.1, third row). 2.2. Observations For very bright targets (very small exposure times), we acquired the spectra in the SOPHIE standard spectroscopy mode (which is appropriate for very bright targets): the A fibre is centred on the target while the B fibre is not illuminated. Our observing strategy and analysis are fully detailed in Paper IX. Briefly, we acquired at least two consecutive spectra at each epoch/visit during sev- eral consecutive nights to estimate the short-term stellar jitter of our ∼F-type targets, whereas we acquired multiple consecutive spectra per epoch for our ∼A-type targets, to mitigate the effect of pulsations. It should be noted that we quadratically add 5 m s−1 of sys- tematic error to the SOPHIE uncertainties to take into account the SOPHIE instrumental error (Díaz et al. 2012; Bouchy et al. 2013; Borgniet et al. 2014). Such an error may be slightly excessive for the latest SOPHIE measurements (see below), but we pre- fer to be conservative and keep the same uncertainty for all our data. The median time baseline of our SOPHIE survey is 1448 days or ∼4 yr (the mean time baseline being 1640 days or 4.5 yr), with a median spectrum number per target of 23 (36 on average) acquired during a median number of 11 visits (17 on average, Fig. 2). A87, page 3 of 30 A87, page 3 of 30 A&A 621, A87 (2019) Table 1. Stellar properties of our targets with detected GPs. Table 1. Stellar properties of our targets with detected GPs. We showed that the periodic RV variations are induced by a mp sin i ∼2.8 MJup giant planet orbiting around HD 113337 with a 324-day period on an eccentric orbit (e ≃0.46). Based on weak long-term variations observed in the BIS and FWHM, we finally supposed that the long-term quadratic RV variation was more probably induced by a stellar activity cycle than by an additional companion. Table 1. Stellar properties of our targets with detected GPs. 3.1. Giant planets In this section we present two planetary systems with three GPs, either confirmed or probable candidates. The host stars’ fundamental properties are detailed in Table 1, and the orbital parameters of the detected and/or candidate GPs in Table 2. The RV and line profile data of these systems are shown in detail in Appendix D. 2.2. Observations Parameter Unit HD 16232 HD 113337 Spectral type F6Va F6Vb V 7.09c 6.03c B −V 0.5c 0.37c v sin i (km s−1) 30c 6c π (mas) 24.52 ± 0.68d 27.11 ± 0.29d [Fe/H] −0.03e 0.09e Teff (K) 6396 ± 95e 6669 ± 80e log g (dex) 4.44 ± 0.08 f 4.21 ± 0.08 f M⋆ (M⊙) 1.20 ± 0.09 f 1.41 ± 0.09 f Radius (R⊙) 1.10 ± 0.05 f 1.55 ± 0.07 f Age (Gyr) 0.9 ± 0.8a 0.15+0.1 −0.05 g References. (a)Guenther et al. (2009). (b)Boesgaard & Tripicco (1986). (c)Taken from the CDS. The uncertainties on the v sin i are not provided. (d)van Leeuwen (2007). (e)Casagrande et al. (2011). The catalog does not provide the uncertainties on the metallicity. (f)Allende Prieto & Lambert (1999). (g)Borgniet et al. (2014). New SOPHIE data. As a target of great interest, we contin- ued to observe HD 113337 with SOPHIE in 2014–2016, adding 35 additional spectra to our database and consequently expand- ing the time baseline by ∼3 yr, to 9.2 yr (3369 days). We display the main SOPHIE spectroscopic observables for this target in Fig. D.1. Most interestingly, the new RV data exhibits a clear rebound on the long-term scale around Julian Day (hereafter JD) ∼2457000. It means that the long-term RV variations can no longer be modelled by a quadratic fit and could possibly be periodic. We computed the RV periodograms according to the Lomb–Scargle definition (Scargle 1982; Press & Rybicki 1989), and also with the CLEAN algorithm (which deconvolves the Lomb–Scargle periodogram from the window function; see Roberts et al. 1987). Both periodograms exhibit two clear peaks (Fig. D.1, top row), one at ∼320 days corresponding to the already-known GP (hereafter HD 113337b), and another at ∼3200 days corresponding to the long-term variations. References. ( )Guenther et al. (2009). ( )Boesgaard & Tripicco (1986). (c)Taken from the CDS. The uncertainties on the v sin i are not provided. (d)van Leeuwen (2007). (e)Casagrande et al. (2011). The catalog does not provide the uncertainties on the metallicity. (f)Allende Prieto & Lambert (1999). (g)Borgniet et al. (2014). 2.4. SOPHIE+ In the same way as the BIS, the FWHM Lomb–Scargle periodogram exhibits power between 2000 and 4000 days, and the FWHM CLEAN periodogram exhibits a clear, single peak around 2200 days. There is no other significant signal at shorter periods; the peaks present in the Lomb–Scargle periodogram around 200–400 days and at ∼30 days are aliases induced by the tem- poral sampling (see window function). The Pearson coefficients for the (RV, FWHM) and (BIS, FWHM) couples are 0.25 (no correlation) and −0.49 (anti-correlation), respectively. This suggests a common origin for the long-term (high-amplitude) FWHM and (low-amplitude) BIS variations (possibly an activity cycle) while giving further credence to another origin to the RV variations. 3.1.1. HD 113337 system 2009, and Paper IX). ; g g , p ) In this hypothesis, an activity cycle with a duration approxi- mating that of the candidate planet c period would explain both the FWHM and BIS long-term variations (and their correlation) as well as the RV long-term variations. We consider though that this interpretation has an important weakness: the RV corrected from the Keplerian fit of planet b only and the FWHM are not correlated (Pearson’s coefficient <0.1, see Figs. D.1 and 3). This is due to the RV and FWHM long-term variations being signifi- cantly shifted in phase (considering a period of ∼3265 days from the two-planet Keplerian fit). A phase shift of 15 to 30◦between RV, BIS, and FWHM activity-induced variations can be expected in the case of short-term stellar activity linked to the stellar rota- tional period (see e.g. Santos et al. 2014; Dumusque et al. 2014, who show that an active region crossing the visible stellar disk induces a RV shift and a line profile deformation with a slight phase shift). However, such a phase shift cannot be present in the case of a long-term activity cycle where the RV and line profile long-term variations are induced by the changing active region filling factor of the stellar disk along the cycle (Borgniet et al. 2015), being thus correlated (Dumusque et al. 2011). In the case of HD 113337, we derived the Pearson’s coefficient of the RV residuals of planet b versus FWHM while shifting in phase the FWHM along the ∼3265-day period deduced from the two- planet Keplerian fit (Fig. 3). The highest correlation (∼0.65) is reached for a phase shift of ∼0.45 (or 1470 days), while the high- est anti-correlation (approximately −0.65) is reached for a phase shift of ∼0.7 (or 2290 days). While we still do not understand everything about stellar magnetic activity cycles, we consider that the observed shift between the RV and FWHM long-term variations is a strong argument against the activity cycle origin for the RV long-term variations, given our present knowledge (Dumusque et al. 2011). We note that long-period RV planets have already been distinguished from activity cycles by look- ing for the presence of a phase shift between the RV and the logR′ HK taken as an activity proxy (Wright 2016). 3.1.1. HD 113337 system (2015) and the stellar mass value from Allende Prieto & Lambert (1999). (b)The number in parentheses refers to the model assuming a constant velocity. Notes. (a)The orbital parameters for HD 16232b were computed with yorbit based on the RV measurements given by Kane et al. (2015) and the stellar mass value from Allende Prieto & Lambert (1999). (b)The number in parentheses refers to the model assuming a constant velocity. To perform the Keplerian fits, we used the yorbit software (Ségransan et al. 2011), as we did in our previous studies (see e.g. BO14 and Paper IX). Our two-planet best solu- tion gives orbital parameters for planet b that are in agree- ment with the values that we derived in BO14 in terms of period (323 ± 1 days against 324 ± 2 days, respectively) and minimal mass (3 ± 0.3 MJup against 2.8 ± 0.3 MJup, respectively). Interestingly, the eccentricity of planet b is sig- nificantly reduced by going from a one-planet to a two-planet solution (from 0.46±0.04 to 0.36±0.03, respectively). If consid- ering that the RV long-term variations are induced by a second GP (hereafter planet c), our best model would correspond to a period of ∼3265 ± 134 days and a 6.9 ± 0.6 MJup minimal mass on a slightly eccentric (e ≃0.2) orbit. The RV residuals of the two-planet Keplerian fit show a remaining dispersion of ∼23 m s−1 (Fig. D.1, fifth row), with a significant anti-correlation with the BIS (Pearson coefficient of −0.44). The periodograms of the RV residuals mainly show small peaks at ∼2 and ∼4 days, and at ∼90 days. None of these periodicities can be properly fit- ted with yorbit. We attribute the short-period peaks to the stellar rotation as they are also present in the BIS periodogram. The origin of the remaining ∼90-day peak is less clear, but a similar peak is present in the BIS periodograms (Fig. D.1, second row). Thus, we do not find any significant sign of a third companion- induced periodicity. The orbital parameters we deduced from the two-planet Keplerian fit are fully detailed in Table 2. solar cycle 23 (Meunier et al. 2010). Given that HD 113337 v sin i of 6 km s−1 is higher than SOPHIE instrumental resolution (∼4 km s−1), magnetic stellar activity should induce both RV and line profile (BIS and/or FWHM) variations (see e.g. Desort et al. 2007; Lagrange et al. 3.1.1. HD 113337 system Summary of first planet detection. We reported the detec- tion of a giant planet orbiting around HD 113337 and charac- terized it in Borgniet et al. (2014, hereafter BO14). HD 113337 (HIP 63584) is a F6V star with a 1.41 M⊙stellar mass (Allende Prieto & Lambert 1999), a slight infrared excess attributed to a cold debris disk (Rhee et al. 2007), and may be relatively young (possible age of 150 ± 100 Myr; see BO14). Based on 266 SOPHIE RV measurements spanning six years, we reported clear ∼320-day periodic RV variations along with a longer term, quadratic-shaped RV drift and a flat (RV, BIS) diagram. HD 113337 does not show any chromospheric emission in the calcium H and K lines (Borgniet et al. 2014). The logR′ HK has a mean value of −4.76 and does not exhibit any significant signal. A second GP around HD 113337. Given the new SOPHIE RV data, we tried to fit HD 113337 RV varia- tions with a two-planet Keplerian orbital model (Fig. D.1). A87, page 4 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. Table 2. Best orbital solutions. Parameter Unit HD 16232ba HD 113337b HD 113337c Status Probable Confirmed Possible P day 345.4 ± 3.7 323.4 ± 0.8 3264.7 ± 134.3 T0 BJD-2453000 912.8 ± 42.5 2757.5 ± 4 5236.8 ± 217.1 e 0.18 ± 0.13 0.36 ± 0.03 0.18 ± 0.04 ω ◦ −22.9 ± 44.1 −130.9 ± 4.9 −46 ± 13 K m s−1 176.6 ± 31.5 76.1 ± 2.9 76.7 ± 2.4 Lin. m s−1 yr−1 −44.1 ± 10.2 0 – Quad. m s−1 yr−2 0 0 – Nm 110 (from Kane et al. 2015) 301 – σO−C m s−1 128 (228.4)b 22.3 (67.4)b – Reduced χ2 4.4 (25.4)b 2.9 (8.6)b – mp sin i MJup 6.8 ± 1.4 3 ± 0.3 6.9 ± 0.6 aP au 1.03 ± 0.01 1.03 ± 0.02 4.8 ± 0.23 Notes. (a)The orbital parameters for HD 16232b were computed with yorbit based on the RV measurements given by Kane et al. (2015) and the stellar mass value from Allende Prieto & Lambert (1999). (b)The number in parentheses refers to the model assuming a constant velocity. Table 2. Best orbital solutions. Notes. (a)The orbital parameters for HD 16232b were computed with yorbit based on the RV measurements given by Kane et al. 3.1.1. HD 113337 system While the long-term RV variation can be modelled well by a Keplerian fit corresponding to a second GP, other possible origins can also be considered, due to the presence of FWHM (and, on a much smaller scale, BIS) long-term variations. We review below the different hypotheses and their pros and cons (second GP included), and explain why we consider the second GP hypothesis to be the most convincing one. Case of an activity cycle. Magnetic activity cycles are char- acterized by the rise and fall in the number of magnetically active structures on the stellar photosphere (such as dark spots and bright faculae) over durations much longer than the stellar rotation period. They can induce RV and/or line profile and/or logR′ HK variations. Activity cycles with associated timescales of a few years are relatively common among FGK MS dwarfs (see e.g. Schröder et al. 2013). For example, reconstructed solar RV exhibit a peak-to-peak RV amplitude of 8–10 m s−1 over Case of a faint binary. A faint, low-mass stellar compan- ion to HD 113337 in a (nearly) pole-on configuration could A87, page 5 of 30 A87, page 5 of 30 A&A 621, A87 (2019) Fig. 3. Top panel: HD 113337 RV residuals from the Keplerian fit of planet b only vs time (black dots) and HD 113337 FWHM vs time (green triangles, same scale as RV). The Keplerian fit of candidate planet c is overplotted to the RV residuals of planet b (red). The average FWHM be seen inclined (assuming that the star rotates in the same plane as the planetary system). Furthermore, HD 113337 is host to a cold debris disk that is most likely also inclined (K. Su, priv. comm.). However, in such a configuration, the RV and FWHM companion-induced variations would also have to be correlated (see Sect. 3.3.11), which is not the case here. Moreover, given the ∼7 MJup minimal mass of the sec- ond candidate companion, the system inclination would have to be lower than 3◦to allow for an actual companion mass of ∼150 MJup, which makes it quite improbable statistically. Stellar mass-luminosity relations (Baraffe et al. 1998) taken for an age between 120 and 500 Myr show that a com- panion mass of 150 MJup would translate into a contrast of 6 magnitudes with the primary in the H band, and so even greater in the V band. 3.1.1. HD 113337 system The Keplerian fit of candidate planet c is overplotted to the RV residuals of planet b (red). The average FWHM (∼13.7 km s−1) has been set to 0 for clarity. Middle panel: FWHM vs RV residuals of planet b. Bottom panel: Pearson’s correlation coefficient of RV residuals of planet b vs FWHM for different FWHM phase shifts, considering a ∼3265-day period (black solid line). The actual Pearson’s coefficient (for no FWHM phase shift) is displayed as a black dot. Fig. 3. Top panel: HD 113337 RV residuals from the Keplerian fit of planet b only vs time (black dots) and HD 113337 FWHM vs time (green triangles, same scale as RV). The Keplerian fit of candidate planet c is overplotted to the RV residuals of planet b (red). The average FWHM (∼13.7 km s−1) has been set to 0 for clarity. Middle panel: FWHM vs RV residuals of planet b. Bottom panel: Pearson’s correlation coefficient of RV residuals of planet b vs FWHM for different FWHM phase shifts, considering a ∼3265-day period (black solid line). The actual Pearson’s coefficient (for no FWHM phase shift) is displayed as a black dot. 3.1.1. HD 113337 system This would make the effect of the companion light on the primary spectrum completely negligi- ble (especially when using a reference built from the primary spectra for the cross-correlation). Thus, we find the hypothesis of a low-mass stellar companion difficult to support given the observations. Case of planet c. The RV and FWHM long-term variations are phase-shifted, which is not coherent with a magnetic activity cycle (see above). Furthermore, if plotting the BIS versus the RV residuals of planet b (Fig. D.1, fourth row), we find a still nearly flat diagram with a linear slope of −0.14 ± 0.02 and a correla- tion coefficient of −0.29. These values are very close to those we find for the (RV, BIS) diagram (see above), meaning that the horizontal spread in the (RV, BIS) diagram (characteristic of the presence of a companion) may well be induced by more than one planet. On the contrary, the RV residuals of the two-planet fit plotted versus the BIS show a linear slope of −0.49 ± 0.05 and an enhanced anti-correlation (−0.44, see above), meaning that the remaining RV dispersion is likely induced by stellar activity. We consider that the presence of a second GP in the system is thus a very convincing explanation for the RV long-term vari- ations and the shapes of the RV and RV residuals versus BIS diagrams. The additional presence of a long-term activity cycle would then explain the long-term FWHM and BIS variations, and the anti-correlation present between the RV residuals and the BIS. To conclude here, we decided to classify HD 113337 as a can- didate two-planet system (planet b being confirmed and planet c still a candidate). Additional remarks. In addition to the SOPHIE observations, we are carrying out a multi-technique study (optical interferome- try, disk imaging, and deep imaging of the outer environment) of the HD 113337 system. The aim of this study is to bring as many constraints as possible on the system characteristics (stellar fun- damental parameters, age, inclination, etc.) and to fully cover the companion (mass, separation) space. This will be the topic of a forthcoming, dedicated paper. Fig. 3. Top panel: HD 113337 RV residuals from the Keplerian fit of planet b only vs time (black dots) and HD 113337 FWHM vs time (green triangles, same scale as RV). 3.2. θ Cyg: a system with complex RV variations Keplerian+linear fit performed by K15 thus gives mp sin i = 6.6 ± 0.9 MJup, P = 345.4 ± 3.8 days, and e = 0.18 ± 0.11, cor- responding to a 1.01 ± 0.01 au semi-major axis (sma) for HD 16232b. As part of our SOPHIE survey, we acquired 27 spectra on HD 16232, covering a 1893-day baseline. These data are enough to allow us to clearly see the long-term RV trend first detected by K15 in our SOPHIE RV (Fig. D.2, third row). Furthermore, we find that the SOPHIE BIS versus RV diagram looks composite, with a horizontal spread indicative of a companion and a verti- cal spread induced by stellar activity and/or low-level pulsations. The SOPHIE BIS and FWHM do not show any significant sig- nal. We characterize the binary companion at the origin of the long-term RV trend in more detail in Sect. 3.3.2. However, we do not detect any signal other than the binary-induced trend in our SOPHIE RV (Fig. D.2, sixth row). This non-detection may be induced by our smaller number of observations compared to K15 and because our GP detectability is significantly degraded by the presence of the additional RV trend induced by the distant stellar companion. When plotting both RV data sets phased along the period from K15 (Fig. 4), we find that our SOPHIE RV and the best model from K15 are not in perfect agreement; however, it is unclear whether this is induced by a phase shift between our data and the fit or by the relatively high stellar noise in the data. If combining both RV data sets with yorbit, we do not achieve a good one-planet Keplerian fit. We note that we obtain a much better RV accuracy than K15 thanks to the use of SOPHIE and SAFIR. The Lomb–Scargle periodogram of the SOPHIE RV is completely dominated by the window function, due to the pres- ence of the RV linear drift. On the contrary, the Lomb–Scargle and CLEAN periodograms of the more consequent K15 RV data set exhibit a clear peak at the planet period. Finally, θ Cyg has been a target of interest in optical and IR interferometry. Multiple VEGA observations allowed Ligi et al. (2012) to make a first estimation of θ Cyg angular diameter. 3.2. θ Cyg: a system with complex RV variations We detail here our results on HD 185395 or θ Cyg, a target that we observed extensively along our SOPHIE survey and that exhibits intriguingly complex RV and line profile variations. The RV and line profile data of θ Cyg are illustrated in detail in Appendix D. Fig. 4. HD 16232 phased RV. RV from K15 are displayed as blue dots, while our SOPHIE RV are displayed as black diamonds. The data are phased along the orbit of HD 16232b according to the Keplerian fit of K15 RV in Table 2 (the corresponding best model is displayed in red). The HD 185395 system. We presented our first results on θ Cyg (F4-5V, 1.37 M⊙) in Desort et al. (2009, hereafter D09). We made a first analysis of the RV based on 91 ELODIE spectra and our first 162 SOPHIE spectra. We showed in D09 that both ELODIE and SOPHIE data sets exhibit a strong quasi-periodic RV signal, with a ∼220 m s−1 amplitude and a main periodic- ity of ∼150 days. Along with these RV variations was a flat (RV, BIS) diagram, the BIS variations being of much smaller amplitude (∼50 m s−1) than the RV. We argued in D09 that given the 7 km s−1 v sin i of the target, such a flat (RV, BIS) diagram would usually be a clear sign that the RV variations are induced by a planetary companion and are not of stellar origin. However, we also showed that despite this flat (RV, BIS) diagram, the BIS exhibits a strong periodicity of around 150 days, making the ori- gin of this periodicity puzzling. Furthermore, the ∼150-day RV variations could not be easily fitted with Keplerian models, with no stable and/or satisfying solution. We concluded in D09 that the origin of these complex RV and BIS variations could not yet be assessed. In addition, we reported the detection by imaging of a wide stellar companion to θ Cyg, that yet could not be respon- sible for the ∼150-day RV and line profile variations. θ Cyg was also independently followed in RV by another team (Howard & Fulton 2016). Based on 223 RV measurements obtained at the Lick Observatory, these authors also detected the long-term RV drift, as well as the quasi-periodic RV variations at ∼150 days, that they deemed statistically non-significant. 3.1.2. Giant planet around HD 16232 HD 16232 (30 Ari B, HIP 12184) is a F6V dwarf star around which Guenther et al. (2009) reported the detection of a sub- stellar companion of ∼10 MJup minimal mass, based on RV observations. This companion was further characterized thanks to new RV measurements (for a total of 110 spectra; see Kane et al. 2015, hereafter K15). These authors made a new esti- mation of the companion’s orbital parameters, making it of planetary nature, and additionally detected a long-term linear RV trend likely induced by a distant stellar companion. The be just bright enough to slightly blend the primary spectrum, and thus induce the long-term FWHM and (low-amplitude) RV variations. HD 113337 v sin i is quite low for a F6V spectral type (see Borgniet et al. 2014), meaning that the system could A87, page 6 of 30 A87, page 6 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. Fig. 4. HD 16232 phased RV. RV from K15 are displayed as blue dots, while our SOPHIE RV are displayed as black diamonds. The data are phased along the orbit of HD 16232b according to the Keplerian fit of K15 RV in Table 2 (the corresponding best model is displayed in red). extrapolated RV look very much the same as for our SOPHIE RV (i.e. it is dominated by the window function), meaning that the GP detected by K15 is most probably non-detectable here, given our temporal sampling, our small number of observations compared to K15, and the level of short-term RV stellar jitter. Additional SOPHIE observations sampled over the orbital period of HD 16232b could raise the ambiguity. In the context of the following statistical analysis, we nonetheless decided to include HD 16232b as a strong can- didate GP. Based on the Keplerian fit by K15, we computed HD 16232b minimal mass considering a stellar mass of 1.2 M⊙to be fully consistent with our stellar mass values, finding a slightly increased mp sin i = 6.8 ± 1.4 MJup. We include the detailed parameters of the Keplerian fit in Table 2. 3.2. θ Cyg: a system with complex RV variations Furthermore, the unusually high jitter and a possible periodic variability in the squared visibility measurements led the authors to speculate on the presence of a low-mass, unseen close stellar companion as the possible source of the RV and interferomet- ric jitter. However, the more recent study of White et al. (2013), based notably on IR closure phase (CP) measurements made with the Michigan Infrared Combiner (MIRC, Monnier et al. 2004) interferometer on CHARA, found no evidence for a close stellar companion to θ Cyg. Based on the CP, these authors derived upper limits on a potential companion brightness, and found that a companion would have to be fainter than the primary by at least 4.7 magnitudes in the H band between 0.2 and 0.4 au, and fainter by at least 3.4 magnitudes between 0.4 and 0.7 au. To investigate the impact of our temporal sampling on HD 16232b detectability, we computed the expected RV given K15 orbital fit at the epochs of our SOPHIE observations. To do so, we extrapolated the Keplerian+linear fit from K15 to our observation epochs, and then added RV white noise with the same dispersion as the short-term (one night) RV jitter we obtained in our SOPHIE RV data. These extrapolated RV look the same as our SOPHIE RV, the RV linear drift being detectable, but not the planet-induced periodic RV variations (Fig. D.2, second row). Moreover, the Lomb–Scargle periodogram of the A87, page 7 of 30 A87, page 7 of 30 A&A 621, A87 (2019) Using stellar mass-luminosity relations (Baraffe et al. 1998) for an age of ∼1 Gyr, this translates into upper masses of 0.3 and 0.5 M⊙, respectively. (i.e. negligible compared to the primary) and 0.5 M⊙, such a companion on a (nearly circular) 150-day orbital period would have a sma between 0.6 and 0.7 au, respectively. This falls into the separation range covered by the interferometric detection limits (≤0.5 M⊙) from White et al. (2013). The H-band contrasts provided by these authors may well translate into larger con- trasts in the V band (i.e. the SOPHIE range) that would exclude significant effects on the CCF and RV. While not conclusive, this is another argument against the stellar companion hypothesis. New SOPHIE data. 3.3. RV long-term trends and stellar binaries In this section, we describe fourteen massive and/or distant com- panions that we confidently identified in our survey. Given our data, most of them must be spectroscopic binaries (SB), while the others are candidate SB. We proceeded in the same way as done and explained before in Paper IX: Origin of the RV variations. As already shown by D09, a purely stellar origin to the ∼150-day RV and line pro- file variations is unlikely. Given θ Cyg v sin i of ∼7 km s−1, the rotational modulation of stellar magnetic activity should induce RV-correlated BIS variability, which is inconsistent with the flat (RV, BIS) diagram. Stellar granulation is not known to induce such high-amplitude RV variations at such a long period, whereas magnetic activity cycles last much longer given our present knowledge (several years, see Baliunas et al. 1995). Finally, most stellar pulsations (i.e. solar-like or γ Dor-type) happen at much shorter periodicities; Kepler data have revealed solar-like oscillations on θ Cyg, but no other clear pulsation pattern (Guzik et al. 2011). 1. we identified the companion presence based on various diagnosis – RV variations, flat (RV, BIS) diagram, CCF variability, RV-FWHM correlation; y 2. we distinguished between double-lined (SB2) and single- lined (SB1) binaries based on the presence or absence, respectively, of CCF distortions and/or RV-FWHM correla- tion; 3. if possible, we fitted the RV with various models (linear, quadratic, or Keplerian) using both our SOPHIE data and, whenever possible, other RV data sets available in the lit- erature. We then explored the RV residuals looking for lower-mass companions; 4. for our targets with long-term (linear or quadratic) RV trends, we constrained the companion properties (mp sin i, sma) given the available data, as we did in Paper IX. Briefly, we assumed the companion period to be at least equal to the observation time baseline, and the RV amplitude induced by the companion to be at least equal to the span of the observed RV trend, considering a circular orbit. We then deduced the corresponding minimal mass versus sma rela- tion (see Paper IX for details). If appropriate, we looked for additional constraints from the literature. p A second hypothesis could be the presence of a close, unseen low-mass stellar companion, which was not explored by D09. 3.2. θ Cyg: a system with complex RV variations We acquired 164 additional spectra on θ Cyg from 2009 to 2016, raising the total number of SOPHIE spectra to 326, and extending the SOPHIE time baseline to 3482 days (∼9.5 yr). We display the main SOPHIE spectro- scopic observables in Fig. D.3. The new SOPHIE RV data set show both a long-term drift of slightly quadratic shape as well as the quasi-periodic ∼150-day variations. The quadratic trend is probably induced by the wide stellar companion imaged by D09 (see details in Sect. 3.3.10). Once corrected from this quadratic trend, the remaining RV variations have a total ampli- tude of 275 m s−1 and a dispersion of 64.5 m s−1. The RV Lomb–Scargle periodogram exhibits several peaks above the 1% FAP between 100 and 500 days, but only a single peak at 150 days remains when cleaning the periodogram from the window function (CLEAN, Fig. D.3). Finally, the planetary (or multi-planetary) hypothesis was already well explored by D09, but with no satisfying results. We tried to fit various Keplerian models with yorbit to θ Cyg RV (from 1 to 4 planets, with/without trends) and considered the cases detailed by D09, but to no avail. Furthermore, we also considered the ELODIE RV data set from D09, and the RV data set published by Howard & Fulton (2016). Both these data sets exhibit RV variations of ∼300 m s−1 amplitude with a periodic character at about 150 days. We also tried to fit these different RV data sets with yorbit, either separately or by combining them. However, we did not obtain a satisfying solution. Furthermore, the main ∼150-day RV periodicity seems rather variable on our timebase, going from ∼125 to ∼180 days if dividing our timebase in several slices (Appendix D). ( g ) HD 185395 BIS shows only low-amplitude variations (rms 12.5 m s−1), thus leading to a flat (RV, BIS) diagram, indicative of a companion. However, the BIS Lomb–Scargle periodogram exhibits a single peak at a 145-day periodicity (in agreement with D09) which is also present in the CLEAN periodogram. The FWHM shows periodic variations with a 207 m s−1 total amplitude (rms 38 m s−1), corresponding to a single peak at 149 days on the FWHM periodograms. The amount of corre- lation between the RV corrected from the quadratic fit and the FWHM is low (−0.3), but slightly higher than between the uncor- rected RV and the FWHM (−0.2). 3.3. RV long-term trends and stellar binaries Such a companion would be just bright enough to slightly blend the primary spectrum and induce the ∼150-day periodic varia- tion in both the RV and the line profile observables without sig- nificantly altering the CCF shape. Santerne et al. (2015) showed that under some configurations, such an unresolved double-lined spectroscopic binary (hereafter SB2) would produce only a very weak correlation between the RV and the BIS, making it looking like a planetary signal. However, these authors also emphasized that even in such configurations, the FWHM should still be cor- related to the RV, while here the θ Cyg RV-FWHM correlation is weak. In contrast, a more convincing example of a RV–FWHM correlation probably induced by a spectroscopic binary can be found for HD 191195 (Sect. 3.3.11). For masses between ∼0 3.2. θ Cyg: a system with complex RV variations Both the BIS and FWHM data can be fitted fairly well with a nearly sinusoidal model with yorbit, with periods of 144 and 148 days, respectively, and marginally compatible phases. Finally, we recall that θ Cyg does not show any chromospheric emission in the calcium lines (⟨logR′ HK⟩= −4.82). In conclusion, we consider that the origin of θ Cyg mid-term RV variations cannot be definitely determined. The variability of the RV variation period, the similarity of the RV, BIS and FWHM periodograms and the flat (RV, BIS) diagram make it a truly puzzling case. 3.3.2. HD 16232 BIS and FWHM variations (Fig. 5) that are characteristic of a SB2 binary. We tried to fit the RV variations with yorbit using a Keplerian model. When letting free all the orbital parameters, the best model corresponds to a companion with mp sin i = 31.1 MJup, P = 249.2±20.5 days and e ≃0.76. However, given the obvious SB2 nature of this companion considering the line profile variations, and that our temporal sampling far from covers the orbital phase of the binary secondary component, we consider that the actual companion mass has to be much larger than this estimation. We note that if running yorbit with various forced periods in the range of 100–3000 days, the solutions we find are almost as good as the free-parameter solution in terms of χ2, highlighting our incomplete temporal sampling. As presented in Sect. 3.1.2, we detect a long-term linear trend in our SOPHIE RV, along with a composite (RV, BIS) diagram indicative of a companion-induced trend (Fig. D.2). A linear RV trend was already reported by Kane et al. (2015). To derive the best constraints on this companion, we combined our SOPHIE RV data set with the RV from K15 and fitted a linear RV drift with yorbit. Such a combination of different RV data sets is possible as yorbit includes the RV offsets between the data sets as fitting parameters. The best solution is a linear trend with a −77.4 ± 3.2 m s−1 yr−1 slope and a 802 m s−1 amplitude over the combined RV data set (Fig. 6). The companion responsible for this RV trend has then to be more massive than 10 MJup and has to be orbiting further out than 5.2 au (Fig. 7). HD 10453 is a known binary. A stellar companion was resolved by speckle interferometry at a projected separation of ∼0.05” (∼2 au, Hartkopf et al. 2012) while Riddle et al. (2015) imaged a stellar companion at a projected separation of ∼0.18” (∼7 au). Given the uncertainties on our orbital parameters, we cannot conclude whether the RV variations are induced by the companion detected by Hartkopf et al. (2012) or Riddle et al. (2015), or whether the HD 10453 system has more components. A stellar companion to 30 Ari B has been imaged at a projected separation of ∼0.54” (∼22 au, Riddle et al. 3.3.1. HD 10453 HD 10453 (HIP 7916, F5V) shows high-amplitude (∼2.2 km s−1 peak-to-peak) RV variations over our 1590-day time baseline, along with a distorted CCF and high-amplitude A87, page 8 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF type stars. X. . 5. Candidate binary companions (first part). From top to bottom rows: HD 10453, HD 20395, HD 43318, HD 82328, HD 143584. From lef ht columns: RV time series, BIS vs RV, FWHM vs RV, stacked CCF. The best yorbit fit of the RV variations is overplotted in red to the RV. RV–FWHM diagram, the RV–FWHM correlation is overplotted in red if the absolute Pearson correlation coefficient is ≥0.6. d i i i 5 h h i i f Fig. 5. Candidate binary companions (first part). From top to bottom rows: HD 10453, HD 20395, HD 43318, HD 82328, HD 143584. From left to right columns: RV time series, BIS vs RV, FWHM vs RV, stacked CCF. The best yorbit fit of the RV variations is overplotted in red to the RV. On the RV–FWHM diagram, the RV–FWHM correlation is overplotted in red if the absolute Pearson correlation coefficient is ≥0.6. 3.3.2. HD 16232 (2015) for the companion they imaged are compatible with our constraints on the minimal mass and sma of the companion responsible for the long-term RV trend detected both in our SOPHIE data and in K15 data (Fig. 7). dispersion two times smaller than the RV, and the Lomb–Scargle periodogram of the residuals show significantly less power at periods >100 days. Given the small amplitude of the RV trend over our timebase, the candidate distant companion can be either of GP, BD or stellar nature (Fig. 7). HD 43318 has not been reported as a binary before. 3.3.2. HD 16232 2015, K15), with a mass estimated to be roughly 0.5 M⊙(Roberts et al. 2015). In addition, Kane et al. (2015) estimated that the mass of the imaged companion would have to be higher than 0.29 M⊙(∼304 MJup) to explain the RV linear trend they detected. A87, page 9 of 30 A&A 621, A87 (2019) A&A 621, A87 (2019) Fig. 6. Targets with combined RV data sets. In all panels, RV data from our SOPHIE survey are plotted as black dots, and the yorbit fit we derived from the combined RV data sets (either linear, quadratic, or Keplerian) is overplotted in red. Top left panel: HD 16232 – RV data from Kane et al. (2015) are plot- ted as blue triangles. Top right panel: HD 162003 – RV data from Endl et al. (2016) are plotted as blue triangles. Bottom left panel: HD 185395 – our ELODIE RV data (Desort et al. 2009) are plotted as blue triangles and RV data from Howard & Fulton (2016) are plotted as orange dia- monds. Bottom right panel: HD 199254 – our HARPS RV data (Paper IX) are plotted as blue triangles. F Fig. 6. Targets with combined RV data sets. In all panels, RV data from our SOPHIE survey are plotted as black dots, and the yorbit fit we derived from the combined RV data sets (either linear, quadratic, or Keplerian) is overplotted in red. Top left panel: HD 16232 – RV data from Kane et al. (2015) are plot- ted as blue triangles. Top right panel: HD 162003 – RV data from Endl et al. (2016) are plotted as blue triangles. Bottom left panel: HD 185395 – our ELODIE RV data (Desort et al. 2009) are plotted as blue triangles and RV data from Howard & Fulton (2016) are plotted as orange dia- monds. Bottom right panel: HD 199254 – our HARPS RV data (Paper IX) are plotted as blue triangles. The mass and separation estimated by Roberts et al. (2015) for the companion they imaged are compatible with our constraints on the minimal mass and sma of the companion responsible for the long-term RV trend detected both in our SOPHIE data and in K15 data (Fig. 7). The mass and separation estimated by Roberts et al. 3.3.5. HD 82328 HD 20395 (14 Eri, HIP 15244, F5V) shows high-amplitude (∼6.4 km s−1) RV variations along with a flat (RV, BIS) dia- gram, large variations in the CCF, and an apparent RV–FWHM anti-correlation (Fig. 5) that are characteristic of a spectroscopic binary. We used the yorbit software to fit the RV variations. The best solution corresponds to a low-mass stellar companion with P = 675.2 ± 5.9 days, mp sin i ∼163 MJup, and e = 0.33 ± 0.02 (corresponding to a ∼1.7 au sma). However, given that our orbital phase coverage is not complete, we cannot exclude longer periods (and higher masses) for HD 20395B. If con- straining the companion orbital period to wider ranges (up to 2.104–3.104 days) with yorbit, reliable orbital solutions can be found (with sma up to ∼15 au, mp sin i up to ∼350 MJup, and eccentricities up to 0.8). HD 20395 has been reported as an astro- metric binary based on its proper motion (Makarov & Kaplan 2005; Frankowski et al. 2007), but without estimations of the orbital parameters. θ UMA (HIP 46853, F7V) shows a linear RV drift with a 78 m s−1 amplitude over our 3368-day timebase, which is best fitted with a long-term linear trend (Fig. 5). Assuming that the period of the companion responsible for the RV trend is at least equal to the total observation timebase, the companion can then be either of BD or stellar nature and has to be orbit- ing further than 5.3 au from the primary (Fig. 7). HD 82328 is a known physical binary with a ∼5′′ projected separation (Allen et al. 2000). Recently, Tokovinin (2014) has estimated the orbital parameters of the companion based on the absolute V-magnitudes of the components and assuming that the pro- jected separation corresponds to the actual sma of the compan- ion, finding P ∼322 yr and mp sin i = 0.16 M⊙. Such a companion is compatible with the detected RV trend (Fig. 7). 3.3.6. HD 91312 HD 91312 (HIP 51658, A7IV) exhibits a large RV drift (of ∼4.1 km s−1 amplitude) over our 2635-day timebase (Fig. 8). As we cannot compute the line profiles for this target (too few spectral lines are available for SAFIR computation), we cannot check the (RV, BIS) diagram. Even so, we conclude that this RV drift is most probably induced by a companion because its ampli- tude is far larger than the short-term RV dispersion induced by S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. 10 1 100 101 102 10 1 100 101 102 103 Minimal mass (Mjup) HD16232 10 1 100 101 102 10 1 100 101 102 103 HD43318 10 1 100 101 102 10 1 100 101 102 103 HD82328 10 1 100 101 102 10 1 100 101 102 103 Minimal mass (Mjup) HD91312 10 1 100 101 102 10 1 100 101 102 103 HD143584 10 1 100 101 102 10 1 100 101 102 103 HD148048 10 1 100 101 102 10 1 100 101 102 103 Minimal mass (Mjup) HD185395 10 1 100 101 102 10 1 100 101 102 103 HD196524 10 1 100 101 102 Separation (au) 10 1 100 101 102 103 HD199254 10 1 100 101 102 Separation (au) 10 1 100 101 102 103 Minimal mass (Mjup) HD210715 10 1 100 101 102 Separation (au) 10 1 100 101 102 103 HD212754 Fig. 7. Constraints on the mp sin i and sma of the companions to our targets with RV linear or quadratic trends. Dark grey: possible (mass, sma) domain for the companion given the available RV data, assuming a circular orbit and an orbital period at least equal to our total time baseline. Light grey: extended (mass, sma) domain in the case of orbital periods smaller than our total time baseline can still be considered, given the observation temporal sampling. Red diamonds: previously imaged companions from the literature: HD 16232 – DI from Roberts et al. (2015); HD 185395 – DI from Desort et al. (2009); HD 199254 – DI from De Rosa et al. (2014). Blue dots: companions previously detected with astrometry and/or RV from the literature: HD 82328 – astrometry from Tokovinin (2014); HD 212754 – RV from Griffin (2010), and astrometry from Goldin & Makarov (2007) and Tokovinin (2014). 10 1 100 101 102 10 1 100 101 102 103 HD82328 HD148048 10 1 100 101 102 Separation (au) 10 1 100 101 102 103 HD199254 10 1 100 101 102 10 1 100 101 102 103 HD196524 HD212754 101 102 5 5 Separation (au) 10 1 100 101 102 Separation (au) 1 0 1 2 3 HD210715 10 1 100 101 102 Separation (au) 10 1 100 101 102 103 HD212754 Separation (au) Fig. 7. Constraints on the mp sin i and sma of the companions to our targets with RV linear or quadratic trends. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. Dark grey: possible (mass, sma) domain for the companion given the available RV data, assuming a circular orbit and an orbital period at least equal to our total time baseline. Light grey: extended (mass, sma) domain in the case of orbital periods smaller than our total time baseline can still be considered, given the observation temporal sampling. Red diamonds: previously imaged companions from the literature: HD 16232 – DI from Roberts et al. (2015); HD 185395 – DI from Desort et al. (2009); HD 199254 – DI from De Rosa et al. (2014). Blue dots: companions previously detected with astrometry and/or RV from the literature: HD 82328 – astrometry from Tokovinin (2014); HD 212754 – RV from Griffin (2010), and astrometry from Goldin & Makarov (2007) and Tokovinin (2014). Fig. 8. HD 91312 RV time series. The assumed linear fit is overplotted in red to the RV. stellar pulsations for this spectral type. This long-term RV drift is best fitted by a linear trend with a −566 ± 25 m s−1 yr−1 slope; once corrected from the drift, the RV dispersion decreases from 1.75 km s−1 to 415 m s−1. HD 91312 is a known wide (23′′ or ∼796 au) visual binary, which is dynamically linked according to Kiyaeva et al. (2008). The very large projected separation of this companion makes it unlikely to be at the origin of the RV drift we detect. This RV trend is then likely induced by an unknown companion. Assum- ing an orbital period larger than our time baseline, this compan- ion has then to be of stellar mass and has to orbit further than 4.7 au around the primary (Fig. 7). Interestingly, HD 91312 is still young (∼200 Myr) and shows an IR excess characteristic of the presence of a cold debris disk, according to Rhee et al. (2007). A recent SED analysis (Rodriguez & Zuckerman 2012) gives a dust radius of 179 au; this could then be a circumbinary disk. 3.3.4. HD 43318 HD 43318 (HIP 29716, F5V) shows a small (5 ± 0.7 m s−1 yr−1) long-term linear drift in its RV over our ∼1800-day timebase, along with a possibly composite (RV, BIS) diagram (Fig. 5). We thus classify this trend as a candidate companion-induced one. When corrected from the linear trend, the RV residuals show a A87, page 10 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. 3.3.9. HD 162003 ψ1 Dra A (HIP 86614, F5IV-V) shows high-amplitude (∼2 km s−1), long-term RV variations over our 2890-day time baseline, along with a flat (RV, BIS) diagram (Fig. 9) characteris- tic of a companion. Furthermore, the RV and FWHM variations are strongly correlated (Pearson’s coefficient of 0.75), which make it a slightly SB2 binary. g y y HD 162003 has previously been reported as a spectro- scopic binary. Based on ∼40 RV measurements spanning over ∼1550 days, Toyota et al. (2009) detected a long-term RV trend of quadratic shape that they attributed to an unseen companion of mp sin i ∼50 MJup, with a sma lower than 140 au (assuming a circular orbit) so that it remains on a stable orbit given the exis- tence of the wide binary ψ1 Dra B (HD 162004) at a projected separation of ∼30′′ (∼667 au) from HD 162003. Gullikson et al. (2015) detected this unseen companion (hereafter ψ1 Dra C) by looking for a secondary peak in the CCF of HD 162003 spectra, in a way similar to Bouchy et al. (2016). From this analysis, they estimated that ψ1 Dra C had a mass of ∼0.7 M⊙, making it of stel- lar nature, and a sma of ∼9 au. Finally, Endl et al. (2016) directly detected ψ1 Dra C with speckle imaging on the one hand, and 85 RV measurements spanning 15 yr (2000–2015) on the other hand, estimated the orbital parameters mp sin i ∼550 ± 5 MJup, e = 0.67, and P = 6650 ± 160 days, corresponding to a ∼8.7 ± 0.1 au sma. We combined our SOPHIE data with the RV from Endl et al. (2016) and fitted a single Keplerian model to the com- bined RV data set with yorbit. Our best solution corresponds to a companion with a 661 ± 107 MJup minimal mass, a sma of 24.3±3.8 au and an eccentricity e = 0.87±0.02 (Fig. 6). We con- clude that the RV variations that we detect in our SOPHIE data are induced by ψ1 Dra C. However, the differences between our Keplerian model and the one derived by Endl et al. (2016) show that the sampling of the companion orbit is still not com- plete enough to adequately cover the period, meaning that large uncertainties still remain on its orbital parameters. 3.3.12. HD 196524 β Del (HIP 101769, F5IV) shows high-amplitude (2 km s−1) RV variations over our 1179-day timebase, along with a flat (RV, BIS) diagram characteristic of a companion (Fig. 9). The RV are best fitted by a quadratic model. Assuming an orbital period larger than our time baseline, the companion responsible for this RV trend would have to be of stellar mass and would have to orbit further than 2.8 au from the primary (Fig. 7). 3.3.11. HD 191195 HD 191195 (F5V, 1.49 M⊙) exhibits complex RV variations, with a RV peak-to-peak amplitude of 272 m s−1 and a dispersion of 56.5 m s−1 (Fig. D.4). We consequently followed this tar- get intensively, acquiring 265 spectra over a 3191-day (∼8.7 yr) time baseline. The RV Lomb–Scargle shows multiple peaks between 100 and 103 days, but they are all aliases of a single ∼300-day periodicity, as shown by the RV CLEAN periodogram. The BIS does not show high-amplitude variations (dispersion of 18 m s−1), hence the flat (RV, BIS) diagram. As did the RV, the FWHM exhibits high-amplitude (266 m s−1 peak-to-peak) complex variations. The FWHM Lomb–Scargle and CLEAN periodograms are strikingly similar to those of the RV, with a clear, single peak at ∼300 days in the CLEAN periodogram. We emphasize that HD 191195 RV and FWHM are strongly anti-correlated, with a Pearson’s correlation coefficient of −0.65 (Fig. D.4). 3.3.9. HD 162003 ( g ) Given HD 191195 v sin i of 5.5 km s−1, the flat (RV, BIS) dia- gram points towards a companion as the source of the observed RV variability. We tried to fit single- or multi-companion Keplerian models to HD 191195 RV, including one to three planets and an additional linear or quadratic trend. However, none of them was able to reproduce convincingly the RV variations. We consider that this complex RV variability is induced by a faint stellar companion rather than by a plan- etary companion. This is strongly supported by the observed RV–FWHM anti-correlation, which can be explained by an unre- solved SB2 binary (see Santerne et al. 2015, and the discussion about θ Cyg in Sect. 3.2). Given the relatively small v sin i of HD 191195, we might be seeing this possible binary system under an inclined or even close to pole-on configuration, which would explain such a low RV amplitude for a companion of stellar nature. HD 191195 has not been reported as a binary before. 3.3.7. HD 143584 3.3.7. HD 143584 HD 143584 (HIP 78296, F0IV) exhibits high-amplitude (∼2 km s−1) RV variations over our 2596-day timebase on both Fig. 8. HD 91312 RV time series. The assumed linear fit is overplotted in red to the RV. A87, page 11 of 30 A&A 621, A87 (2019) θ Cyg. This specific RV long-term trend is induced by a distant companion, as shown by the flat (RV, BIS) diagram (Fig. D.3). To derive the best constraints on this companion, we combined our SOPHIE RV data set to the RV acquired with ELODIE before (Desort et al. 2009) and to the RV data from Howard & Fulton (2016). This allowed us to expand the timebase to 5422 days (14.8 yr). Over the combined RV data set, the RV quadratic trend induced by the distant companion has an amplitude of ∼370 m s−1 (Fig. 6). The companion responsible for this trend is then either of BD or stellar nature and has to orbit further than 7 au from the primary (Fig. 7). the short and long term, along with a composite (RV, BIS) dia- gram (Fig. 5). This is best explained by the presence of both a massive companion and stellar pulsations. The RV variations are best fitted with a long-term quadratic trend, even if peri- ods shorter than our time baseline could still be possible. The dispersion of the RV residuals is 291 m s−1, compared to the 592 m s−1 RV dispersion. Assuming an orbital period larger than our time baseline, the companion responsible for the RV long- term variations would have to be of stellar nature and would have to orbit further than 4.5 au from the primary (Fig. 7). HD 143584 has not been reported as a binary before. A distant stellar companion to θ Cyg was imaged by Desort et al. (2009) as a projected separation of 46.5 au from the pri- mary. Based on the measured contrast between the two compo- nents and on stellar evolutionary models, these authors deduced a mass of ∼0.35 M⊙for the companion. These estimated mass and projected separation are compatible with the constrains we derive from the RV long-term trend (Fig. 7). 3.3.8. HD 148048 η Umi (HIP 79822, F5V) exhibits long-term RV variations that are best fitted by a quadratic trend of 1600 m s−1 amplitude over our 2988-day timebase. The (RV, BIS) diagram is composite with a horizontal spread induced by a companion and a vertical spread induced by stellar pulsations (Fig. 9). Assuming an orbital period larger than our time baseline, the companion responsible for these RV variations would have to be of stellar nature and would have to orbit further than 4.8 au from the primary (Fig. 7). HD 148048 has not been reported as a binary before. 3.3.14. HD 210715 HD 210715 (HIP 109521, A5V) exhibits a clear RV linear drift of 1.2 km s−1 amplitude over the 1880-day timebase. Even if the (RV, BIS) diagram is dominated by a vertical spread induced by stellar pulsations characteristic of its stellar type (Fig. 9), we con- sider that the RV long-term trend is induced by a distant massive companion. 3.3.10. HD 185395 As presented in Sect. 3.2, we detect a long-term quadratic RV drift in addition to the RV mid-term complex variability of A87, page 12 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. Fig. 9. Candidate binary companions (second part). From top to bottom rows: HD 148048, HD 162003, HD 196524, HD 210715, HD 212754. From left to right columns: RV time series, BIS vs RV, FWHM vs RV, stacked CCF. The best yorbit fit of the RV variations is overplotted in red to the RV. On the RV–FWHM diagram, the RV–FWHM correlation is overplotted in red if the absolute Pearson correlation coefficient is ≥0.6. Fig. 9. Candidate binary companions (second part). From top to bottom rows: HD 148048, HD 162003, HD 196524, HD 210715, HD 212754. From left to right columns: RV time series, BIS vs RV, FWHM vs RV, stacked CCF. The best yorbit fit of the RV variations is overplotted in red to the RV. On the RV–FWHM diagram, the RV–FWHM correlation is overplotted in red if the absolute Pearson correlation coefficient is ≥0.6. with properties compatible with these constraints was imaged around HD 199254 by De Rosa et al. (2014), see details in Paper IX. HD 196524 was already known as a spectroscopic binary (Pourbaix et al. 2004). Based on astrometric data, Malkov et al. (2012) estimated its period to be of ∼26.7 yr, its eccentricity of 0.36, and its sma of ∼0.44′′ (∼13.6 au). At such a separation and given the mass constraints we bring, the companion would have a mass greater than ∼200 MJup (Fig. 7). 3.3.15. HD 212754 we selected the instrument for which we had the most RV data and the longest time span (i.e. we used the SOPHIE data for HD 102647 and HD 218396, and the HARPS data for the seven other targets) and use the corresponding data in the following. We acquired only 19 RV measurements over 68 days on HD 212754 (HIP 110785, F7V), but we are able to detect a high- amplitude (2 km s−1) RV drift of quadratic shape, along with a flat (RV, BIS) diagram and large CCF variations that are indica- tive of a spectroscopic binary (Fig. 9). Given our small timebase, we were not able to fully determine the orbital parameters of the companion. This companion has to orbit further than 0.4 au from the primary and is most probably of stellar nature (Fig. 7), given the CCF variations. We do not combine HARPS and SOPHIE RV data for these targets as it is not possible here to compute an accurate value of the zero-point between the two RV data sets, the SAFIR RV being relative RV. Furthermore, our detection limits are com- puted based on an analysis of the RV periodogram (Sect. 4.3). In this context, a HARPS + SOPHIE RV combination might lead to a biased combined periodogram and thus biased detection lim- its. Hence, using only the RV data set with the most data and the longest time span ensures an unbiased periodogram, but it will lead to slightly more conservative detection limits. Our analysis of the combined survey is then based on 107 targets observed with HARPS and 118 targets observed with SOPHIE. The com- bination of these two samples is possible because we use the same target selection process (Appendix B). Both samples are thus very similar in terms of stellar physical properties (Fig. 10). HD 212754 is a known SB1 (Griffin 2010) and astromet- ric binary (Goldin & Makarov 2007) with an orbital period of ∼931 days, an eccentricity of 0.3–0.4 and a secondary minimal mass of 0.34 M⊙(Tokovinin 2014) at a separation of ∼59 mas (∼2.3 au). This already known stellar companion is most likely at the origin of the detected SOPHIE RV variations. 4. Combined analysis of the SOPHIE + HARPS surveys The median time baseline of our SOPHIE survey is 1448 days (mean time baseline 1640 days), with a median spectrum number per target of 23 (average 36) acquired during a median number of 11 visits (average 17). These values are lower than those obtained for our HARPS sample (see Paper IX and Fig. 10). This differ- ence is mostly explained by the increased performance and better observing conditions of HARPS compared to SOPHIE. When combining the two surveys, we obtain a median timebase of 1888 days (average 1832) and a median number of 30 spectra acquired per target. Here, we combine our SOPHIE AF survey with the similar HARPS survey described in Paper IX to make a global analysis in terms of achieved companion detections, sensitivity and statis- tics in the 1- to 1000-day period range. In addition to the two GPs detected in this period range in this SOPHIE survey (HD 113337b and HD 16232b), we include the three confirmed GPs detected in our HARPS survey around two F6V targets (HD 60532b, c and HD 111998b) in the following analysis. 3.3.13. HD 199254 We have already detected HD 199254 (HIP 103298, A5V) as a spectroscopic binary thanks to our HARPS RV (see Paper IX). We have also observed this target with SOPHIE and we detected a long-term, high-amplitude RV drift. When combining the SOPHIE and HARPS RV data and fitting a trend model with yorbit, we found the best solution to be a slightly quadratic trend of 923 m s−1 amplitude over the total time baseline of 2400 days (Fig. 6). Given our combined RV data, the companion respon- sible for such a RV trend is of stellar nature and orbits further than 4.5 au from the primary (Fig. 7). A stellar companion HD 210715 has been reported as an astrometric binary (Makarov & Kaplan 2005; Frankowski et al. 2007), but no constraints on the companion parameters are available in the lit- erature. Assuming an orbital period larger than our time baseline, the unseen companion responsible for the RV long-term trend would have to be either of BD or stellar nature and would have to orbit further than 3.9 au from the primary (Fig. 7). A87, page 13 of 30 A&A 621, A87 (2019) A&A 621, A87 (2019) HD l d h i Fig. 10. Properties of our combined sample (SOPHIE +HARPS). Top left panel: position of our targets in an H–R diagram. Blue diamonds: SOPHIE targets; orange crosses: HARPS tar- gets. The Sun is displayed (red star) for com- parison. Top right panel: stellar mass histogram of our combined sample. Blue: SOPHIE tar- gets; orange: HARPS targets. Bottom left panel: spectrum number per target. Bottom right panel: timebase histogram. 3 3 15 HD 212754 l d h i f hi h h d h RV Fig. 10. Properties of our combined sample (SOPHIE +HARPS). Top left panel: position of our targets in an H–R diagram. Blue diamonds: SOPHIE targets; orange crosses: HARPS tar- gets. The Sun is displayed (red star) for com- parison. Top right panel: stellar mass histogram of our combined sample. Blue: SOPHIE tar- gets; orange: HARPS targets. Bottom left panel: spectrum number per target. Bottom right panel: timebase histogram. 3.3.15. HD 212754 4.2. Stellar intrinsic variability 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 10 0 10 1 10 2 vsini (km/s) 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 0 10 1 10 2 10 3 10 4 σRV (m/s) vsini (km/s) 10 0 10 1 10 2 vsini (km/s) 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) B V 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) ⊙ 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) Fig. 11. Stellar intrinsic RV variability vs stellar properties. First row: stellar intrinsic RV jitter vs B −V, vs v sin i, and vs mass (from left to right). Blue diamonds: SOPHIE targets; orange crosses: HARPS targets. Second row: averaged RV uncertainty (accounting for the photon noise only) vs B −V, vs v sin i, and vs mass. 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 0 5 10 15 20 25 30 35 40 Number of stars 10 0 10 1 10 2 10 3 10 4 10 5 σBIS (m/s) 0 5 10 15 20 25 30 35 40 Number of stars 10 0 10 1 10 2 10 3 σRV (m/s) 10 0 10 1 10 2 10 3 10 4 10 5 σBIS (m/s) Fig. 12. Stellar intrinsic variability in our observables. Left panel: histogram of the stellar intrinsic RV jitter (blue: SOPHIE targets; orange: HARPS targets). Middle panel: histogram of the BIS dispersion. Right panel: BIS dispersion vs intrinsic RV jitter (SOPHIE targets are shown as blue diamonds and HARPS targets as orange crosses). The best power-law fits to the BIS rms vs RV rms distribution in the two variability regimes are shown as red solid lines. 4.2. Stellar intrinsic variability 10 0 10 1 10 2 10 3 10 4 10 5 σBIS (m/s) 0 5 10 15 20 25 30 35 40 Number of stars (m/s) 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 0 5 10 15 20 25 30 35 40 Number of stars 10 0 10 1 10 2 10 3 σRV (m/s) 0 1 3 4 5 Fig. 12. Stellar intrinsic variability in our observables. Left panel: histogram of the stellar intrinsic RV jitter (blue: SOPHIE targets; orange: HARPS targets). Middle panel: histogram of the BIS dispersion. Right panel: BIS dispersion vs intrinsic RV jitter (SOPHIE targets are shown as blue diamonds and HARPS targets as orange crosses). The best power-law fits to the BIS rms vs RV rms distribution in the two variability regimes are shown as red solid lines. targets, and by high-frequency pulsations for our earlier-type (A) targets. These two variability regimes are easily seen on a BIS dispersion versus RV dispersion plot (Fig. 12). For our targets with a BIS dispersion below ∼20 m s−1, there is a correlation of 0.7 (Pearson) between the logarithms of the RV and BIS disper- sions, and the BIS dispersion increases with the RV dispersion as targets, and by high-frequency pulsations for our earlier-type (A) targets. These two variability regimes are easily seen on a BIS dispersion versus RV dispersion plot (Fig. 12). For our targets with a BIS dispersion below ∼20 m s−1, there is a correlation of 0.7 (Pearson) between the logarithms of the RV and BIS disper- sions, and the BIS dispersion increases with the RV dispersion as RV uncertainties, and to a lesser degree the RV dispersion, are correlated with the v sin i and anti-correlated with the B −V, and consequently are loosely correlated with the stellar mass. In terms of RV and BIS dispersion, the SOPHIE and HARPS targets show the same behaviour (Fig. 12). The only remarkable differ- ences between the two surveys are the larger mean SOPHIE RV uncertainty compared to HARPS (Fig. 11) and the relative lack of small RV and BIS dispersion values (<10 m s−1) for the SOPHIE survey compared to HARPS (Fig. 12). This is not induced by a difference between the two stellar samples, but rather by the decreased spectral resolution (and thus decreased RV accuracy) of SOPHIE with respect to HARPS. 4.2. Stellar intrinsic variability We combine the 125 AF dwarf stars of our SOPHIE sample to the 109 targets of our HARPS survey (Paper IX). We note that nine of our SOPHIE targets were also part of our HARPS sam- ple: HD 13555, HD 25490, HD 29488, HD 102647, HD 197890, HD 199254, HD 211976, HD 218396, and HD 222368. Our com- bined AF sample is thus made of N = 225 distinct targets. In the case of the targets with both HARPS and SOPHIE observations, To characterize the stellar intrinsic variability of our targets, we display in Fig. 11 the RV dispersion (after having removed the companion-induced RV variations) and the mean RV uncertain- ties (accounting only for the photon noise) of our targets versus their main physical properties (B −V, v sin i, M⋆), as we did in Paper IX. Our results are similar to those in Paper IX: the A87, page 14 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 10 0 10 1 10 2 vsini (km/s) 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 0 10 1 10 2 10 3 10 4 σRV (m/s) 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) 10 0 10 1 10 2 vsini (km/s) 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -1 10 0 10 1 10 2 10 3 < RV unc> (m/s) Fig. 11. Stellar intrinsic RV variability vs stellar properties. First row: stellar intrinsic RV jitter vs B −V, vs v sin i, and vs mass (from left to right). Blue diamonds: SOPHIE targets; orange crosses: HARPS targets. Second row: averaged RV uncertainty (accounting for the photon noise only) vs B −V, vs v sin i, and vs mass. 4.2. Stellar intrinsic variability RV uncertainties, and to a lesser degree the RV dispersion, are correlated with the v sin i and anti-correlated with the B −V, and consequently are loosely correlated with the stellar mass. In terms of RV and BIS dispersion, the SOPHIE and HARPS targets show the same behaviour (Fig. 12). The only remarkable differ- ences between the two surveys are the larger mean SOPHIE RV uncertainty compared to HARPS (Fig. 11) and the relative lack of small RV and BIS dispersion values (<10 m s−1) for the SOPHIE survey compared to HARPS (Fig. 12). This is not induced by a difference between the two stellar samples, but rather by the decreased spectral resolution (and thus decreased RV accuracy) of SOPHIE with respect to HARPS. as σBIS = 2.1 (σRV)0.78±0.10. (1) (1) In contrast, for our targets with a BIS dispersion above ∼20 m s−1 (mostly pulsation-dominated jitter), the correlation is 0.73 and the BIS dispersion increases with the RV dispersion as The median RV dispersion is 61 m s−1 for the combined sam- ple (191 m s−1 on average), and the median RV uncertainty (not accounting for the instrumental error) is 14 m s−1 (49 m s−1 on average). As we detailed in Lagrange et al. (2009) and Paper IX, the stellar jitter of AF-type MS dwarfs is mainly induced by stel- lar magnetic activity (spots and faculae) for our later-type (F) σBIS = 0.5 (σRV)1.56±0.14 (2) (2) where the exponent to σRV happens to be twice that in Eq. (1). 4.2. Stellar intrinsic variability B-V 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 3 10 0 10 1 10 2 vsini (km/s) 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA vsini (km/s) 10 1 10 2 vsini (km/s) 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0. B-V 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 7 10 0 10 1 10 2 vsini (km/s) 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA Fig. 13. Detection limits vs stellar properties. From top to bottom rows: 10-day, 102-day, and 103-day LPA detection limits; vs (from left to right columns) B −V, v sin i, and mass. SOPHIE targets are shown as blue diamonds and HARPS targets as orange crosses. the periodogram of the observed RV within a localized period range to compute the mp sin i detection limits at each period. Our method as well as the LPA process are fully described in Paper IX. stellar activity, we partially corrected the RV stellar variability by using the RV–BIS anti-correlation, as we did in Paper IX. We used the same criterion: we made a linear fit of the BIS versus RV data and removed it from the RV if the corresponding Pear- son’s coefficient was above 0.7 in absolute. This was the case for six of our SOPHIE targets (Appendix A). On average, this RV- BIS correction decreases the RV variation amplitude by a factor of 1.6 and the RV dispersion (rms) by a factor of 1.7, similarly to the value we obtained in Paper IX. We display our detection limits at meaningful ranges (10, 102, and 103 days) versus the main stellar properties (B −V, v sin i, mass) in Fig. 13. 4.2. Stellar intrinsic variability For a few of our SOPHIE targets with a RV jitter dominated by A87, page 15 of 30 A&A 621, A87 (2019) 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 10 0 10 1 10 2 vsini (km/s) 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 10 0 10 1 10 2 vsini (km/s) 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 B-V 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 10 0 10 1 10 2 vsini (km/s) 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 1.0 1.5 2.0 2.5 3.0 Stellar mass (M ⊙) 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA Fig. 13. Detection limits vs stellar properties. From top to bottom rows: 10-day, 102-day, and 103-day LPA detection limits; vs (from left to right columns) B −V, v sin i, and mass. SOPHIE targets are shown as blue diamonds and HARPS targets as orange crosses. 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0. 4.2. Stellar intrinsic variability On the whole, our results are the same as in Paper IX, i.e. the detection limits are correlated with the v sin i and anti-correlated with the B −V, in agreement with our results on the intrinsic variability. Overall, the SOPHIE targets show slightly higher detection limits than the HARPS targets (Fig. 14), which is again in agreement with the slightly decreased RV accuracy of SOPHIE compared to HARPS. Companion frequency versus mp sin i. Companion frequency versus mp sin i. 1. We first consider companions with masses in the 13–80 MJup range. We do not detect any companions in the 1–103 day range. Our completeness in this mp sin i domain is close to 100%. We thus obtain an upper limit on the BD occurrence rate of ≲3.5–4% (at 1σ uncertainty, see below) for both our A- and F-type samples for periods below 1000 days. The differences in occurrence rates between our stellar mass subsamples or between the different considered period ranges are negligible (Fig. 16). Given that these estimations are still upper limits on the occurrence rates (no actual detection), they are compatible with our previous statistical analysis on HARPS targets only (Paper IX). These upper limits on the BD-mass companion occurrence rates are roughly twice as small as in Paper IX (which is in agreement with a sample two times larger). 4.3. Detection limits We compute the minimal mass (mp sin i) versus period (P) detection limits for each target with no confirmed GPs in our combined survey, except for two targets with too few data points (<10), for which our periodogram-based detection limits are not effective, HD 196724 and HD 216627 from the HARPS sam- ple (see Paper IX), i.e. 219 targets in total. We use the local power analysis (LPA) method (Meunier et al. 2012) to com- pute the detection limits in the 1- to 2 × 103-day range on a log-spaced grid of 210 periods. Briefly, the LPA method com- pares the maximum power of the Lomb–Scargle periodogram of a synthetic planet on a circular orbit to the maximum power of y p We compute the search completeness C(P, mp sin i) of our combined survey by combining the detection limits of all our targets, as we did in Paper IX for the HARPS targets only. For a given couple (P, mp sin i), C gives the fraction of stars in the combined sample for which a companion with a minimal mass mp sin i and an orbital period P (on a circular orbit) would be detected if present, given our observations. We refer to Paper IX for details. We display our combined survey completeness in Fig. 15, along with the confirmed GPs detected in the combined survey. A87, page 16 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 0 5 10 15 20 25 30 35 40 Number of stars 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 0 5 10 15 20 25 30 35 40 Number of stars 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 0 5 10 15 20 25 30 35 40 Number of stars Fig. 14. Distribution of the achieved detection limits. From left to right panels: histograms of the 10-day, 102-day, and 103-day LPA detection limits. Blue: SOPHIE targets; orange: HARPS targets. 4.3. Detection limits 10 -2 10 -1 10 0 10 1 10 2 10-day LPA 0 5 10 15 20 25 30 35 40 Number of stars 10 -2 10 -1 10 0 10 1 10 2 10 3 100-day LPA 0 5 10 15 20 25 30 35 40 Number of stars 10 -2 10 -1 10 0 10 1 10 2 10 3 1000-day LPA 0 5 10 15 20 25 30 35 40 Number of stars Fig. 14. Distribution of the achieved detection limits. From left to right panels: histograms of the 10-day, 102-day, and 103-day LPA detection limits. Blue: SOPHIE targets; orange: HARPS targets. Fig. 15. Search completeness of our combined survey. From bottom to top: 0 to 100% search completeness (i.e. the fraction of stars with good enough detection limits to rule out a companion of a given mp sin i at a given orbital period P). Black dots: confirmed GPs detected in the combined survey. HD 16232b, whose detection was confirmed by Kane et al. (2015), is included here (see Sect. 3.1.2). targets (respectively 102 and 117 targets for the high and low stellar mass subsamples). 4. For each (P, mp sin i) domain, we estimate the number of systems with missed companions nmiss that we potentially did not detect in our survey, based on the search completeness CD and the number ndet of systems with at least one detected GP. 5. We derive the companion occurrence rate in each domain and for our different mass subsamples using binomial statistics, i.e. by computing the probability distribution function (PDF) of drawing ndet systems with detected GPs among N = 225 stars, and then computing the product of this PDF by (ndet + nmiss)/ndet to correct for our search incompleteness (see details in Paper IX). The companion occurrence rate corresponds to the companion frequency value with the maximum probability (i.e. to the mode of the PDF). If ndet = 0, we consider n ′ det = 1 (as an upper limit) and compute the corresponding upper limit on the occurrence rate. 5. We derive the companion occurrence rate in each domain and for our different mass subsamples using binomial statistics, i.e. 4.3. Detection limits by computing the probability distribution function (PDF) of drawing ndet systems with detected GPs among N = 225 stars, and then computing the product of this PDF by (ndet + nmiss)/ndet to correct for our search incompleteness (see details in Paper IX). The companion occurrence rate corresponds to the companion frequency value with the maximum probability (i.e. to the mode of the PDF). If ndet = 0, we consider n ′ det = 1 (as an upper limit) and compute the corresponding upper limit on the occurrence rate. Fig. 15. Search completeness of our combined survey. From bottom to top: 0 to 100% search completeness (i.e. the fraction of stars with good enough detection limits to rule out a companion of a given mp sin i at a given orbital period P). Black dots: confirmed GPs detected in the combined survey. HD 16232b, whose detection was confirmed by Kane et al. (2015), is included here (see Sect. 3.1.2). 4.4.1. Method summary We compute the companion frequency around AF MS stars based on the detected GPs and on the detection limits of our 225- target combined sample. Our method is fully detailed in Paper IX and in Appendix C. Briefly: 1. We consider different mp sin i ranges (0.3–1 MJup for Saturn-mass GPs, 1–13 MJup for Jupiter-mass GPs, 13–80 MJup for BDs) and different period ranges (1–10, 10–102 and 102–103 days) to define the (mp sin i, P) domains (D) where the companion frequency is computed. p q y p 2. We consider two target subsamples depending on the stellar mass: 104 targets with masses above 1.5 M⊙, and 121 targets with masses ≤1.5 M⊙. Our upper limits on the BD occurrence rate around AF stars (≤4% at 1σ) are compatible with the results obtained for solar- mass stars. The close BD occurrence rate for solar-mass stars is estimated to be <1% for P < 5 yr (or sma ≲3 au; see Grether & Lineweaver 2006) and around 0.6% for P ≲1 yr (Sahlmann et al. 2011; Grieves et al. 2017). Jones et al. (2017) have found an occurrence rate of 1.21.5 −0.4% for BD companions within 5 au around evolved intermediate-mass giant stars off the main-sequence (1.62 −0.5% when restricted to stars ≥1.5 M⊙), 3. We sum the search completeness C(P, mp sin i) over mp sin i and over P in each defined domain D to get the search completeness function CD over D. Excluding the four targets with detected GPs (HD 16232, HD 113337 from the SOPHIE sur- vey; HD 60532 and HD 111998 from the HARPS survey) and two targets with too few spectra from the HARPS survey (see above), we compute the search completeness for each domain over 219 A87, page 17 of 30 A87, page 17 of 30 A&A 621, A87 (2019) Table 3. GP occurrence rate around AF dwarf stars. Table 3. GP occurrence rate around AF dwarf stars. mp sin i Orbital period Search Detected Missed GP occ. rate Confidence intervals GP occ. 4.4.1. Method summary rate interval interval completeness GP systems GP systems (computed value) 1σ 2σ (MJup) (day) C (%) ndet nmiss (%) (%) (%) (%) 13–80 1–10 99 0 ∼0 0.5 0–1.5 0–2.5 ≤1.5 (BD) 98 0 ∼0 1 0–3.2 0–5.5 ≤3.2 ∼99 0 ∼0 0.8 0–2.7 0–4.6 ≤2.7 10–100 95 0 ∼0 0.5 0–1.5 0–2.6 ≤1.5 92 0 0.1 1.1 0–3.4 0–5.9 ≤3.4 98 0 ∼0 0.8 0–2.7 0–4.7 ≤2.7 100–1000 87 0 0.2 0.5 0–1.7 0–2.9 ≤1.7 78 0 0.3 1.3 0–4.1 0–7 ≤4.1 95 0 0.1 0.9 0–2.8 0–4.8 ≤2.8 1–100 97 0 ∼0 0.5 0–1.5 0–2.6 ≤1.5 95 0 0.1 1 0–3.3 0–5.7 ≤3.3 99 0 ∼0 0.8 0–2.7 0–4.6 ≤2.7 1–1000 94 0 0.1 0.5 0–1.6 0–2.7 ≤1.6 89 0 0.1 1.1 0–3.6 0–6 ≤3.6 97 0 ∼0 0.9 0–2.8 0–4.7 ≤2.8 1–13 1–10 84 0 0.2 0.5 0–1.8 0–3 ≤1.8 (Jupiters) 71 0 0.4 1.4 0–4.5 0–7.6 ≤4.5 95 0 0.1 0.9 0–2.8 0–4.8 ≤2.8 10–100 72 0 0.4 0.6 0–2.1 0–3.5 ≤2.1 49 0 1 2 0–6.5 0–11 ≤6.5 91 0 0.1 0.9 0–2.9 0–5 ≤2.9 100–1000 58 4 2.9 3.1 2.2–5.5 1.2–7.9 3.1+2.4 −0.9 30 0 2.3 3.2 0–10.4 0–17.7 ≤10.4 82 4 0.9 4.1 2.9–7.1 1.6–10.2 4.1+3 −1.2 1–100 78 0 0.3 0.6 0–1.9 0–3.2 ≤1.9 60 0 0.7 1.6 0–5.3 0–9 ≤5.3 93 0 0.1 0.9 0–2.9 0–4.9 ≤2.9 1–1000 71 4 1.6 2.5 1.8–4.5 1–6.4 2.5+2 −0.7 50 0 1 2 0–6.3 0–10.8 ≤6.3 89 4 0.5 3.7 2.6–6.5 1.5–9.3 3.7+2.8 −1.1 0.3–1 (Saturns) 1–10 76 0 0.3 1.1 0–3.5 0–6 ≤3.5 10–100 56 0 0.8 1.5 0–4.8 0–8.2 ≤4.8 100–1000 31 0 2.2 2.7 0–8.6 0–14.7 ≤8.6 1–100 66 0 0.5 1.3 0–4.1 0–6.9 ≤4.1 1–1000 54 0 0.8 1.5 0–4.9 0–8.4 ≤4.9 Notes. The parameters are displayed in normal, bold, or italic fonts when considering the full star sample, the most massive (>1.5 M⊙) stars only or the least massive (≤1.5 M⊙) stars only, respectively. For mp sin i in the 0.3–1 MJup range, we display the GP occurrence rate and other parameters only for our low-M⋆subsample, as our completeness is almost null in this domain for our higher mass targets. Column 6 gives the value derived from the binomial statistics based on ndet or n ′ det, while Col. 9 gives the corresponding GP occurrence rate or upper limit at a 1σ uncertainty. Notes. 4.4.1. Method summary Thus, this apparent concentration of GPs at the largest considered period ranges is most probably not induced by a bias in our method. In terms of companion frequency, we constrain the hot Jupiter (P < 10 or <102 days) occurrence rate to be below ≤3% for our targets ≤1.5 M⊙and to be below ≤4.5–6.5% for our targets >1.5 M⊙. These upper limits on the occurrence rates and their 1σ uncertainties are a factor of ∼2 lower compared to Paper IX. In the case of our low-mass targets, this hot- Jupiter upper limit is smaller than (but still compatible with) the 4.1+3 −1.2% value we get in the 102–103-day range (where most of our detected GPs are located). This result is compatible with a possible increase in the GP frequency with the orbital period, at least for our low-mass subsample. Based on the core-accretion theory, Kennedy & Kenyon (2009) predicted that GPs would be found at longer periods around more massive stars. That we do not detect any GPs around our high-mass targets in the 1- to 103-day range could be in agreement with such a trend, although it remains speculative as long as we are not able to constrain the occurrence rate of GPs on wider (∼3–5 au) orbits around A-type stars (with the help of direct imaging surveys and/or Gaia). Fig. 16. Constraints on companion occurrence rates from our com- bined SOPHIE + HARPS survey. From top to bottom panels: brown dwarf companions (13–80 MJup), Jupiter-mass companions (1–13 MJup), Saturn-mass companions (0.3–1 MJup). From left to right on each row: period ranges of 1–10, 10–100, and 100–1000 days. Cyan: stellar mass ≤1.5 M⊙subsample (with 1σ error bars in blue). Orange: stellar mass >1.5 M⊙subsample (with 1σ error bars in red). Actual occurrence rates are displayed in full colour (ndet ≥1) and upper limits (ndet = 0) are displayed in hatch. Since we do not detect any hot Jupiters in our sample, we only have an upper limit on the hot-Jupiter frequency, i.e. ≤3– 4.5% (1σ). Then, we cannot tell if there is a difference with the hot-Jupiter rates of 0.5–1.5% derived for FGK MS stars (Santerne et al. 2016). Since several hot Jupiters have recently been detected by transits and Doppler tomography around AF stars (see e.g. Bourrier et al. 2015; Stevens et al. 2017; Crouzet et al. 2017; McLeod et al. 2017; Zhou et al. 4.4.1. Method summary Borgniet et al.: Extrasolar planets a Fi 16 C t i t i t f Fig. 16. Constraints on companion occurrence rates from our com bined SOPHIE + HARPS survey. From top to bottom panels: brow dwarf companions (13–80 MJup), Jupiter-mass companions (1–13 MJup) Saturn-mass companions (0 3–1 MJ ) From left to right on each row or from the GP occurrence rate provided by Cumming et al. (2008) for FGK dwarf stars. We can only say that the occur- rence rate of super-Jupiter GPs does not rise steeply for A-type dwarfs compared to FGK dwarfs for P ≤103 days. Given the remaining uncertainty, it is still compatible with the increased global GP frequency versus stellar mass predicted by the core- accretion theory (Kennedy & Kenyon 2008, see also below). 3. Finally, we consider Saturn-mass (0.3 ≤mp sin i ≤1 MJup) companions around our lower mass targets (≤1.5 M⊙). We do not detect any such companions in our SOPHIE survey, which was already the case for our HARPS survey (Paper IX). With a completeness between 75% (1 ≤P ≤10 days) and 40% (102 ≤P ≤103 days), we constrain the occurrence rate to be ≤3.5 and ≤8.6% (1σ), respectively. These upper limits are roughly two times lower than those we obtained in Paper IX for the HARPS survey only. Companion frequency versus period. For BD-mass com- panions, our completeness is above 90% for all the considered period ranges between 1 and 103 days; thus, there is no really significant difference between the occurrence rate values we get for these period ranges. In the case of planetary-mass companions, five out of the six GPs we detected (with the exception of candidate HD 113337c with its longer period of ∼3260 days) belong to a somewhat lim- ited (P, mp sin i) domain, with orbital periods between 200 and 1000 days and minimal masses between 1 and 10 MJup. Our sen- sitivity to companions in this domain ranges from 20–30% to 70–80%; we achieve a better completeness closer to the star, yet we do not detect any GPs for these smaller periods. We note that SAFIR preliminary tests based on ELODIE RV were shown to be able to re-detect a known P ∼3-day hot Jupiter (Galland et al. 2005a) and to detect a P ∼28-day BD (Galland et al. 2006). 4.4.1. Method summary The parameters are displayed in normal, bold, or italic fonts when considering the full star sample, the most massive (>1.5 M⊙) stars only or the least massive (≤1.5 M⊙) stars only, respectively. For mp sin i in the 0.3–1 MJup range, we display the GP occurrence rate and other parameters only for our low-M⋆subsample, as our completeness is almost null in this domain for our higher mass targets. Column 6 gives the value derived from the binomial statistics based on ndet or n ′ det, while Col. 9 gives the corresponding GP occurrence rate or upper limit at a 1σ uncertainty. which is also compatible with our constraints. Interestingly, these authors did not detect any BD companions within 2 au of their targets, which is in agreement with our results. In short, we have not found any significant differences to date in terms of close BD companion frequency between AF dwarfs and FGK dwarfs, and between AF dwarfs and GK subgiants. periods within 103 days we considered in this analysis all hap- pen to orbit around stars <1.5 M⊙. For this low-M⋆subsample, our survey completeness is close to 90% (almost the same as in Paper IX); the occurrence rate is 3.7+2.8 −1.1% (P ≤103 days), which is very close to the value we found in Paper IX, but with a significantly lower uncertainty. This result is compatible with the ∼4 ± 1% GP frequency found by Cumming et al. (2008) for GPs above 1 MJup with sma below 2 au orbiting around FGK solar-like stars. 2. We then consider Jupiter-mass companions (1 ≤mp sin i ≤ 13 MJup). The GP system occurrence rate for our full AF sample in the 1–103 day range is 2.5+2 −0.7%, which is slightly lower than (but still compatible with) the 3+4.7 −0.6% value we obtained in Paper IX. If looking at our two stellar mass subsamples sep- arately, we find that the five planetary-mass companions with For our high-mass subsample, we do not detect any GPs and get a completeness of 50%. We thus constrain the 1–13 MJup GP system occurrence rate around A-type stars to be ≲6% (1σ) for A87, page 18 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. 4.4.1. Method summary 2017), it would be very interesting to compare our constraints on the hot-Jupiter P ≤103 days. This upper limit is two to three times lower than the upper limit we obtained in Paper IX (in agreement with a sample twice larger). Still, we cannot determine from this result whether the GP occurrence rate around A-type stars is signifi- cantly different from the value we derive for our F-type targets, A87, page 19 of 30 A87, page 19 of 30 A&A 621, A87 (2019) frequencies around AF stars with similar statistics derived from transit survey results. dwarfs and for evolved GK subgiants and giants. Our GP occur- rence rates for stars with masses in the 1–1.5 M⊙range are compatible with the GP frequencies reported for FGK dwarfs. However, our upper limits on the GP frequency around stars with masses in the 1.5–2.5 M⊙range are somewhat smaller than the GP frequencies reported for evolved GK stars, which are considered to be their descendants. Comparison to other stars. The GP occurrence rate (3.7+2.8 −1.1%) we derive for our F-type (1–1.5 M⊙) subsample shows no significant difference when compared to solar-like FGK stars. However, our upper limit on the GP occurrence rate (≤6.3% at 1σ) around A-type stars (1.5–2.5 M⊙) is significantly smaller than both the GP frequencies around GK subgiants (11 ± 2%) and giants (≃15+8 −3%) derived in approximately the same period ranges by Johnson et al. (2010) and Reffert et al. (2015), respec- tively. Since we compute our detection limits only for circular orbits (which is the common approach; see Paper IX for details), the uncertainties on our companion occurrence rates are slightly underestimated by not considering a variable companion eccen- tricity, and might still be compatible with the above results. Still, this discrepancy in the GP occurrence rate between our A-type subsample and the evolved star samples of Johnson et al. (2010) and Reffert et al. (2015) remains significant. A possible interpretation is that different stellar properties (e.g. stellar mass, metallicity) significantly impact the formation and evolution of giant planets (see e.g. Ghezzi et al. 2018). The fact that we do not detect any GPs around our A-type targets (compared to the six GPs detected or reported for our F-type targets) is compatible with the prediction that GPs form further away from the primary and migrate less with an increas- ing stellar mass, though it does not confirm it. 4.4.1. Method summary Our results can be of interest for planetary population synthesis studies (see e.g. Mordasini et al. 2009, 2012), especially when considering the influence of the stellar mass on the planetary formation and migration processes. In this context, the astrometry from Gaia will probe with an unprecedented sensitivity the GP population at a few au around A-type stars in the solar neighbourhood, thus allowing a promising synergy with our RV survey (Sozzetti 2015; Sahlmann et al. 2016). Finally, we did not detect any hot Jupiter in our 225-target sample, constraining their occurrence rate to be below 3–5% if considering A and F stars separately. While this result does not show a significant difference with the results reported for solar-mass stars, it would be of great interest to compare it to a statistical study of the hot Jupiters that have recently been detected by transits around more distant AF stars. g p g Finally, it is also interesting to compare the companion fre- quency around AF MS stars to the companion population around white dwarfs (WDs), as the latter typically have AF-type pro- genitors. Based on searches for an infrared excess in the WD spectral energy distribution, Farihi et al. (2005) and Girven et al. (2011) estimated the global BD occurrence rate around WDs to be ≲1%; this estimation should be robust, as WD are ideal tar- gets to search for low-mass stellar and substellar companions, due to their low luminosity. This result is consistent with the overall 3–4% upper limit we obtain on the BD occurrence rate in the 1- to 103-day period range. It is also in agreement with the absence of BD detections within 2 au of the primary reported by Jones et al. (2017) for subgiant stars. We note that our upper limit for the considered periods (P = 103 days, or sma ≃2.5-3 au) roughly corresponds to the closest orbits for which a substellar companion is expected to avoid engulfment during the red giant transition (Mustill & Villaver 2012; Nordhaus & Spiegel 2013). Acknowledgements. We acknowledge support from the French CNRS and from the Agence Nationale de la Recherche (ANR grant GIPSE ANR-14-CE33-0018). N.C.S. acknowledges support by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through national funds and by FEDER through COMPETE2020 by grants UID/FIS/04434/2013 & POCI-01-0145-FEDER-007672 and PTDC/FIS- AST/1526/2014 & POCI-01-0145-FEDER-016886, and through Investigador FCT contract no. IF/00169/2012/CP0150/CT0002. References Allen, C., Poveda, A., & Herrera, M. A. 2000, A&A, 356, 529 Allende Prieto, C., & Lambert, D. 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Stellar characteristics and detailed results for the 125 targets of our SOPHIE RV survey. Stellar characteristics Survey detailed results HD HIP ST B −V v sin i Mass TBL Nm RV BIS RV- ⟨FW⟩ V CL z }| { z }| { BIS A rms ⟨U⟩ A rms ⟨U⟩ corr. Appendix A: SOPHIE sample | {z } | {z } km s−1 M⊙ day m s−1 m s−1 km s−1 400 699 F8IV 0.456 3 1.15 1503 34 40.6 9.8 5.6 29.9 7.7 6.3 −0.37 11.1 C 1404 1473 A2V 0.05 110 2.21 1134 16 1280 338 259 C 3268 2832 F7V 0.52 5.8 1.37 1493 21 36.1 10.7 6.4 22.9 5.4 9.9 0.02 10.7 C 3440 3132 F6V 0.501 4.7 1.19 173 17 32.2 7.9 5.5 23.9 5.2 5.5 −0.12 9.4 C 6288 4979 A8IV 0.243 94 1.76 361 16 743 219 82.1 6114 1350 205 −0.17 134 V 6961 5542 A7V 0.18 91 1.8 1417 14 198 46.1 48.4 C 7193 5631 F5V 0.49 8.2 1.05 357 16 67.3 22.3 6.6 63.7 20.6 10.8 −0.66 14.3 V 8723 6706 F2V 0.343 60 1.38 1553 25 263 65.7 38.8 5293 1167 96.1 −0.37 88.6 C 8829 6748 F0.5V 0.283 18 1.49 102 16 251 89.6 12.5 123 28.9 28.6 −0.4 28.8 V 8907 6878 F8V 0.49 9.5 1.23 395 19 219 55.5 8.3 118 34.1 16.3 −0.56 20.7 V 9780 7447 F0IV 0.24 113 1.8 823 13 888 265 102 6261 2129 255 0.58 177 V 10453 7916 F5V 0.44 13 1.49 1589 17 2244 660 10.9 19133 4783 23.9 0.92 22.8 V a 71.9 21.6 C 11973 9153 F0V 0.271 97 1.77 648 21 926 236 59.9 3876 1173 149 −0.2 138 V 12111 9480 A3V 0.16 69 1.97 1399 14 202 48.8 35.5 3015 927 87.8 0.32 86.5 C 13555 10306 F5V 0.4 9 1.45 679 39 80.7 21.1 6.1 104 25 8.4 0.05 13.5 V 16232 12184 F6V 0.5 42 1.2 1892 27 568 161 21 700 160 50.9 −0.08 60.7 V a 262 72.5 V 16765 12530 F7V 0.52 32 1.2 318 30 276 60.1 27.6 556 129 67.8 −0.51 48.2 V 16895 12777 F8V 0.51 9 1.15 687 23 62.4 16.5 7.2 39 10 12.8 0.33 14.9 V 17948 13665 F5V 0.381 6.1 1.33 2102 24 62.1 12.5 6.6 45.9 11.4 10.5 −0.3 13.5 C 18404 13834 F5IV 0.41 26 1.35 1360 14 110 28.8 11.9 586 198 27 −0.73 37.7 V 20395 15244 F5V 0.364 6 1.36 1175 22 6444 2669 6.6 587 238 10.6 −0.97 13.2 V a 46.3 14.5 V 20677 15648 A3V 0.07 130 2 8 22 1445 299 178 C 25490 18907 A0.5V 0.045 65 2.27 1817 14 741 197 131 C 25621 18993 F6V 0.5 15 1.45 1433 15 65.5 21.1 7.8 137 32.8 14.9 −0.73 25.5 V b 51.8 14.5 0 C 27819 20542 A2V 0.15 45 1.96 1399 25 633 182 17.5 943 247 41.9 −0.05 75.3 V 27934 20635 A7IV-V 0.143 87 2.07 1883 18 1323 327 43.2 V 27946 20641 A7V 0.231 175 1.82 1750 17 1104 272 254 C 27962 20648 A2IV-V 0.049 8.3 2.29 1009 70 78.6 19 6.4 91.7 14.1 10 0.09 18 V 28355 20901 A5V 0.212 93 1.91 1535 20 408 89 42.7 36564 6306 106 0.36 115 V 29488 21683 A5V 0.157 115 2.04 1370 26 1085 271 79.2 V 31662 23380 F4V 0.391 28 1.44 2160 33 120 29.5 13 647 167 29.9 −0.22 42.5 V 31675 23484 F6V 0.48 8.8 1.23 645 32 110 31.4 6.6 150 32.7 10.6 −0.51 19.2 V 33608 24162 F5V 0.423 14 1.43 2216 14 116 32.4 8 195 67 15.5 −0.84 22.9 V b 68.2 17.4 0 V 43042 29650 F5IV-V 0.44 10 1.33 1018 49 65.8 13.6 7.2 40 7.5 12.9 −0.01 11.2 C 43318 29716 F5V 0.5 4.6 1.4 1850 21 33.7 10.2 5.8 32.8 8.1 7.2 0.42 10.6 C a 20.8 5.2 C 43386 29800 F5V 0.42 18 1.33 2247 21 90.3 23.8 9.8 258 66.3 21 −0.55 29.9 V 48737 32362 F5IV-V 0.43 68 1.63 1448 13 123 37.1 28.5 1163 393 70.1 −0.15 92 C 58461 35998 F5V 0.42 14 1.49 1861 22 371 114 10.1 837 252 21.6 −0.93 24.4 V b Notes. 546, A69 van Leeuwen F. 2007, A&A, 474, 653 White, T. R., Huber, D., Maestro, V., et al. 2013, MNRAS, 433, 1262 Wright, J. T. 2016, ArXiv e-prints [arXiv:1611.05398] Zh G B k G Á H t J D t l 2017 AJ 153 211 White, T. R., Huber, D., Maestro, V., et al. 2013, MNRAS, 433, 1262 Mayor, M., Lovis, C., & Santos, N. C. 2014, Nature, 513, 328 Wright, J. T. 2016, ArXiv e-prints [arXiv:1611.05398] Á McLeod, K. K., Rodriguez, J. E., Oelkers, R. J., et al. 2017, AJ, 153, 263 Wright, J. T. 2016, ArXiv e prints [arXiv:1611.05398] Zhou, G., Bakos, G. Á., Hartman, J. D., et al. 2017, AJ, 153, 211 Zhou, G., Bakos, G. Á., Hartman, J. D., et al. 2017, AJ, 153, 211 Meunier, N., Desort, M., & Lagrange, A.-M. 2010, A&A, 512, A39 A87, page 21 of 30 A&A 621, A87 (2019) Appendix A: SOPHIE sample Appendix A: SOPHIE sample Appendix A: SOPHIE sample Spectral type (ST) and B −V values and v sin i values are taken from the CDS database. Stellar masses are taken from Allende Prieto & Lambert (1999). The survey results include the observation time baseline (TBL), the number of computed spectra Nm, th peak-to-peak (i.e. maximum-to-minimum) amplitude A, rms and mean uncertainty ⟨U⟩on the RV and BIS measurements, the RV-BIS correlatio (Pearson’s coefficient), and the mean FWHM (⟨FW⟩). V stands for our RV variability criterion (Paper IX), with V for RV variable stars and C fo RV constant targets. The “CL” column reports the correction applied on the RV, if any: (a)correction from binary or planetary fit; (b)correction from RV BIS activity correlation Table A.1. Stellar characteristics and detailed results for the 125 targets of our SOPHIE RV survey. Table A.1. Stellar characteristics and detailed results for the 125 targets of our SOPHIE RV survey. Notes. Spectral type (ST) and B −V values and v sin i values are taken from the CDS database. Stellar masses are taken from Allende Prieto & Lambert (1999). The survey results include the observation time baseline (TBL), the number of computed spectra Nm, the peak-to-peak (i.e. maximum-to-minimum) amplitude A, rms and mean uncertainty ⟨U⟩on the RV and BIS measurements, the RV-BIS correlation (Pearson’s coefficient), and the mean FWHM (⟨FW⟩). V stands for our RV variability criterion (Paper IX), with V for RV variable stars and C for RV constant targets. The “CL” column reports the correction applied on the RV, if any: (a)correction from binary or planetary fit; (b)correction from RV-BIS activity correlation. A87, page 22 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. g p yp Table A.1. continued. Stellar characteristics Survey detailed results HD HIP ST B −V v sin i Mass TBL Nm RV BIS RV- ⟨FW⟩ V z }| { z }| { BIS A rms ⟨U⟩ A rms ⟨U⟩ corr. Appendix A: SOPHIE sample | {z } | {z } km s−1 M⊙ day m s−1 m s−1 km s−1 186 42.3 0 V 58855 36439 F6V 0.414 10 1.25 793 69 49.7 10.6 5.9 55.1 10 7.8 −0.02 14.9 C 58946 36366 F1V 0.32 63 1.5 456 18 372 86.7 35.8 V 63332 38325 F6V 0.441 7 1.13 1136 24 63.2 15 6.1 52.9 13.4 8.7 0.31 14.7 V 69548 40875 F4V 0.367 55 1.34 1500 24 298 62.9 29.1 1923 462 71.5 −0.33 76.2 V 69897 40843 F6V 0.51 5 1.25 1193 27 56.9 13.5 5.7 38.8 9.2 6.8 0.03 10.9 V 70313 41152 A3V 0.12 100 1.83 1004 13 650 221 143 77613 20230 358 −0.15 169 C 75332 43410 F7V 0.498 10 1.17 3412 68 125 33.4 6.1 95.5 20.6 8.6 −0.45 15.2 V 75616 43625 F5V 0.42 7.5 1.04 826 37 149 35.3 7 114 31.5 12.3 −0.49 16.5 V 76398 43932 A7IV 0.161 119 1.98 2246 28 503 143 85 77355 14173 212 −0.1 147 C 76582 44001 F0IV 0.207 92 1.71 754 16 457 123 64.9 9508 2930 162 −0.08 130 C 76644 44127 A7V 0.19 129 1.64 1163 20 1470 443 84 35467 8604 210 0.19 188 V 78154 45038 F7V 0.49 5.4 1.43 765 82 49.1 10.4 5.4 33 6.2 5.2 −0.01 12 C 79439 45493 A6V 0.175 145 1.89 742 34 1348 331 135 V 80290 45836 F3V 0.42 4.5 1.14 2670 40 40.2 9.6 5.8 32.2 7.4 7.4 −0.25 10.6 C 82328 46853 F7V 0.46 6.3 1.53 3367 75 102 17 5.7 106 13.3 14.3 0.04 13.9 V 66.5 11.7 V 89449 50564 F6IV-V 0.44 16 1.44 3375 47 112 25.1 9.1 372 75.1 22.6 −0.43 25.4 V 90277 51056 F0V 0.242 33 2.1 1171 30 169 38.4 12.7 800 168 29.1 −0.24 50.8 V 90470 51200 A3V 0.138 105 1.89 2157 24 2215 463 222 24058 5268 556 0.06 187 V 91312 51658 A7IV 0.197 128 1.83 2634 34 5202 1724 91.5 V 1809 417 V 95418 53910 A1IV −0.02 35 2.28 2619 28 187 47.3 28 C 97855 55044 F6V 0.413 5.7 1.32 2619 23 41.9 11.8 6.5 34.9 9.6 10.4 −0.42 12.8 C 99747 56035 F5V 0.39 8.4 1.41 1255 36 49.2 11 10.1 102 20.9 21.6 0.14 18.1 C 102647 57632 A3V 0.09 115 1.9 1145 102 700 134 111 C 102870 57757 F9V 0.55 4.4 1.36 3370 87 54 11.2 5.4 27 5.8 13.4 0.08 10.2 V 106591 59774 A2V 0.09 251 2.1 1990 21 1962 529 498 C 107113 59879 F4V 0.43 5.8 1.32 1154 36 71.8 16.1 6.6 52.3 14 10.5 −0.06 12.7 V 109141 61212 F2V 0.338 142 1.46 1392 16 845 279 152 8004 2462 380 0.87 195 C 111270 62402 A9V 0.2 93 1.89 3309 24 1031 223 84.9 V 113337 63584 F6V 0.372 6 1.41 3368 301 328 68.1 7.9 148 24.4 19.8 −0.32 13.7 V 132 22.1 V 115810 64979 A7IV 0.24 98 1.78 1433 27 739 169 83.9 8411 1766 209 −0.14 145 V 118232 66234 A4V 0.12 145 2.06 1435 19 1285 330 116 115938 25992 290 −0.17 221 V 119992 67103 F7IV-V 0.47 6.2 1.25 2621 53 49 12.2 11.5 61 11.6 28.8 −0.14 13.8 C 121164 67782 A8IV 0.187 65 1.9 3371 60 492 121 37.3 V 121560 68030 F6V 0.56 5 1 2680 27 40.9 10.6 7.6 27.4 6.2 19 0.11 9.9 C 124675 69483 A7IV 0.23 111 1.93 107 17 2448 810 77.5 36187 10492 193 −0.35 174 V 125040 69751 F8V 0.49 36 1.16 2677 17 171 45.1 21.6 437 133 54.1 −0.11 52.9 V 125451 69989 F5IV 0.364 40 1.4 1476 18 168 43.5 21.9 647 177 53.3 −0.63 58.5 C 126141 70310 F5V 0.358 7 1.36 2680 14 112 41.7 8.6 139 38.2 21.4 −0.67 15.1 V 129153 71759 F0V 0.216 105 1.68 48 22 1805 439 109 16573 3043 272 −0.17 159 V 130044 72066 F0V 0.261 15 1.55 519 36 245 63.9 9.5 338 99.8 20.1 −0.7 24.1 V 178 45.5 0 V 132254 73100 F8V 0.53 5 1.29 1462 19 34 9.3 6 29.8 9 8.2 −0.3 14 C 132375 73309 F8V 0.5 6.2 1.38 1520 14 25.3 9.3 6 37.3 9.4 8.1 −0.44 13.7 C 134083 73996 F5V 0.43 45 1.33 1505 20 110 28.4 19.8 372 85.6 47.7 −0.23 64 C 136729 75043 A4V 0.115 145 2.05 2956 32 1517 373 144 V 137391 75411 F0IV 0.291 89 1.8 668 21 1154 322 44.6 16935 3174 111 0.35 124 V 139389 76456 F5V 0.4 21 1.29 1224 16 287 68.3 14.7 177 49.7 34.6 0.57 34.1 V 142860 78072 F6IV 0.5 10 1.28 2071 23 49.8 14.2 6.2 69.4 15.5 8.8 −0.02 17.1 V 143584 78286 F0IV 0.258 68 1.58 2595 18 2137 575 75.8 2123 634 190 −0.21 103 V 1180 291 V Table A.1. Appendix A: SOPHIE sample continued. A87, page 23 of 30 A&A 621, A87 (2019) Table A.1. continued. Table A.1. continued. Stellar characteristics Survey detailed results HD HIP ST B −V v sin i Mass TBL Nm RV BIS RV- ⟨FW⟩ V CL z }| { z }| { BIS A rms ⟨U⟩ A rms ⟨U⟩ corr. Appendix A: SOPHIE sample | {z } | {z } km s−1 M⊙ day m s−1 m s−1 km s−1 147365 80008 F4V 0.4 77 1.38 2252 14 357 102 37.5 7755 1685 93 −0.05 104 V 148048 79822 F5V 0.37 80 1.52 2987 46 1678 571 56.5 9907 1582 141 −0.16 125 V a 272 71.2 C 149681 80480 F0V 0.24 118 1.62 2891 27 1942 417 119 6499 1707 297 −0.29 176 V 152303 81854 F4V 0.41 22 1.32 1803 24 278 73.7 13.2 531 138 30.4 −0.6 36.1 V 154431 83494 A6V 0.19 110 1.67 3195 23 561 150 109 C 156295 84183 A7V 0.192 103 1.66 3157 20 1184 331 80.3 V 159332 85912 F4V 0.45 7.2 1.41 3285 29 48.9 10.9 8.3 54.9 15.1 20.7 −0.26 15.7 C 162003 86614 F5IV-V 0.44 9 1.48 2890 22 1806 524 8.6 492 124 21.5 −0.28 19.2 V a 126 34.4 V 168151 89348 F5V 0.38 10 1.45 3197 23 79.5 19.5 11.5 89.3 18.1 28.7 −0.46 14.4 C 184960 96258 F7V 0.51 6.5 1.28 1900 29 66.4 14.8 6.1 44.9 9.9 8.7 −0.45 14.1 V 185395 96441 F3V 0.38 10 1.37 3482 326 405 82.3 7.5 82.4 12.5 18.6 0.02 11.1 V a 288 65.2 V 186689 97229 A3IV 0.18 33 1.71 3193 71 513 101 17.9 1738 358 44.7 −0.81 50 V b 270 59.4 0 V 187013 97295 F5IV-V 0.47 7.3 1.21 3234 27 92.1 17.7 8 81.8 18.9 20 0.15 16 V 191195 99026 F5V 0.39 5.5 1.49 3191 265 272 56.4 7.6 98 17.6 18.9 0.2 12.4 V 192985 99889 F5V 0.391 8.7 1.44 3236 19 94 22.3 10.6 167 41.6 26.6 −0.5 18.6 V 193369 100108 A2V 0.06 90 1.99 2795 37 1167 280 185 C 196524 101769 F5IV 0.44 48 1.92 1178 19 2075 601 18.3 934 269 43.8 −0.52 62.8 V a 197 45 V 197373 102011 F6IV 0.399 27 1.34 387 23 262 61.5 12.9 303 91.9 29.7 −0.56 42.9 V 197950 102253 A8V 0.21 160 1.67 783 21 1598 428 399 C 198390 102805 F5V 0.395 6.5 1.21 773 25 35.5 9.6 7.3 70.1 15.5 13.3 0.28 14.2 C 199254 103298 A5V 0.11 145 2.02 1382 15 1371 340 151 V a 597 158 C 204153 105769 F0V 0.32 115 1.49 340 15 814 255 88.3 3254 748 220 0.34 164 V 204414 105966 A1V 0.046 70 2 748 19 459 113 72.3 26815 5247 180 0.28 124 C 206677 107302 A7IV-V 0.204 113 1.67 1414 20 1412 338 92 8022 1992 230 −0.5 168 V 209369 108535 F5V 0.409 24 1.62 1056 24 369 93.8 11.7 728 204 26.2 −0.75 39.9 V b 229 62.5 0 V 210715 109521 A5V 0.142 130 1.95 1879 45 2152 609 111 105313 17072 277 −0.05 185 V a 1363 376 V 211976 110341 F5V 0.421 5 1.3 323 70 64.5 13 5.9 35.9 8.1 7.6 −0.15 12.5 V 212754 110785 F7V 0.52 6.8 1.37 67 19 2083 508 5.8 56.7 16.4 7.3 0.16 14.9 V a 39.4 11.8 V 214454 111674 A9V 0.24 96 2.02 1331 15 395 133 42.2 2256 717 105 −0.19 150 V 215588 112324 F5V 0.388 13 1.26 1829 57 82.6 19.5 7.8 132 27.7 14.9 −0.27 22.1 V 215648 112447 F6V 0.49 9 1.34 769 18 45.4 12.7 6.1 30.4 8.4 8.7 −0.16 14 V 216385 112935 F6V 0.48 4.7 1.48 760 74 76.9 13.9 5.5 40.3 7.7 5.8 −0.34 10.9 V 218261 114096 F6V 0.49 6 1.16 509 65 82 15.3 5.9 79.2 12.1 7.5 −0.29 13.4 V 218396 114189 F0V 0.257 49. Appendix A: SOPHIE sample 1.42 808 111 4508 1072 36.4 V 218470 114210 F5V 0.44 8.8 1.48 2213 27 93.1 26.2 6.9 221 51.5 11.6 0.23 18.8 V 220974 115770 A6IV 0.16 98 1.99 1592 27 200 61 52.1 28092 4746 130 0.22 153 C 222368 116771 F7V 0.5 7 1.38 3158 38 83.2 19.1 5.4 25.9 6.3 4.9 0.3 12.3 V 222603 116928 A7V 0.21 60 1.88 788 22 1556 380 26.3 4069 1336 64.5 −0.49 85.5 V 223731 117681 F5V 0.44 31 1.33 778 21 188 48.5 17.7 500 151 42.5 −0.21 46.9 V Appendix B: Sample selection F7–F9 limit roughly corresponds to the earliest spectral types for which the traditional cross-correlation technique with a binary mask gives accurate RV measurements. In terms of dis- tance to the Sun (d, taken from the HIPPARCOS catalogue), we set two cutoffs in order to obtain a statistically significant sample of nearby stars and to keep roughly the same propor- tion of A- and F-type targets: d ≤56 and d ≤33 parsecs for A- and F-type stars, respectively. To keep only objects of dwarf nature, we selected stars with an absolute magnitude We selected the targets in our sample to try to keep it as rep- resentative as possible of AF MS dwarfs more massive than the Sun, while making it appropriate for accurate RV measure- ments with SAFIR. In terms of spectral type, we selected stars between A0 and ∼F8 types. The A0 limit corresponds roughly to the earliest spectral type for which close (sma ≲2.5 au) GP and/or BD companions are detectable with SAFIR RV, while the A87, page 24 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. within 2.5 mag from the main sequence (Lagrange et al. 2009). For a given domain D with a search completeness CD and ndet systems with planets, the search completeness may be far from 100%. It is thus necessary to estimate the number nmiss of poten- tially missed planets in order to correct for this incompleteness (Howard et al. 2011). We define nmiss as follows: Then we removed confirmed spectroscopic binaries of SB2 nature, close (≤5 arcsec) visual binaries, and targets with highly variable luminosity to keep targets observable in RV. Finally, we also removed confirmed δ Scuti and γ Doradus pulsators, and chemically peculiar Am/Ap stars that are often associated with spectroscopic binaries (Lagrange et al. 2009). This selec- tion led to a visible dichotomy between our A- and F-type targets (as seen in Fig. 1), due to the removal of a significant num- ber of late A and early F stars belonging to the instability strip. We initially ended up with ∼320 stars. We then restrained our effective SOPHIE observing sample to 125 of these more than 300 targets (without any selection prior) to be able to fully cover the 1- to 1000-day periodicity space for all targets dur- ing the SOPHIE observations. (C.1) (C.1) Here, for each target (i) of our sample without a detected planet, we have δi = 1 if the computed detection limit at this (P, mp sin i) node is < mp sin i and δi = 0 otherwise. We compute C(P, mp sin i) for all the targets of our SOPHIE +HARPS com- bined sample without a detected planet and with more than ten spectra (219 targets overall). We compute the search com- pleteness function using the detection limits computed from RV corrected from binary trends or RV-BIS correlations where appropriate. Here, for each target (i) of our sample without a detected planet, we have δi = 1 if the computed detection limit at this (P, mp sin i) node is < mp sin i and δi = 0 otherwise. We compute C(P, mp sin i) for all the targets of our SOPHIE +HARPS com- bined sample without a detected planet and with more than ten spectra (219 targets overall). We compute the search com- pleteness function using the detection limits computed from RV corrected from binary trends or RV-BIS correlations where appropriate. Appendix B: Sample selection These 125 targets can be divided into 72 stars with a mass ≤1.5 M⊙and 53 stars with a mass >1.5 M⊙. nmiss = ndet × ( 1 CD −1). (C.3) (C.3) Now we can derive the GP occurrence rate and its 1σ and 2σ uncertainties. We use binomial statistics, computing the bino- mial distribution of ndet GP systems among the N targets. Given the probability p of having one or more GPs around one given star, we compute the probability f of drawing ndet systems among the N stellar targets of our sample as follows: f(ndet, N, p) = N ndet ! × pndet × (1 −p)N−ndet. (C.4) (C.4) The result is the probability density function (PDF) of the GP occurrence rate (i.e. probability density versus f) in the given (P, mp sin i) domain. The GP occurrence rate is thus equal to the probability f for which the PDF is the highest, multiplied by (ndet + nmiss)/ndet to account for the missed GPs (see Fig. 14 in Paper IX). To derive the 1σ and 2σ uncertainties, we integrate the PDF and compute its standard deviations at ∼68 and ∼95%, respectively. The result is the probability density function (PDF) of the GP occurrence rate (i.e. probability density versus f) in the given (P, mp sin i) domain. The GP occurrence rate is thus equal to the probability f for which the PDF is the highest, multiplied by (ndet + nmiss)/ndet to account for the missed GPs (see Fig. 14 in Paper IX). To derive the 1σ and 2σ uncertainties, we integrate the PDF and compute its standard deviations at ∼68 and ∼95%, respectively. Appendix D: Targets of peculiar interest: detailed plots Here, we display in detail the spectroscopic data of our targets either with planetary companions (HD 113337, HD 16232) or that exhibit complex RV variations (HD 185395, HD 191195). These plots include specifically the Lomb–Scargle and CLEAN periodograms of the different observables. Regarding θ Cyg, we explored in more detail the ∼150-day RV periodicity to try to understand why no Keplerian model can be correctly fitted. We separated the SOPHIE RV data set over four 600-day slices (with approximately the same amount of data) and computed the corre- sponding Lomb–Scargle and CLEAN periodograms (Fig. D.5). We find, quite astonishingly, that the main mid-term RV peri- odicity is not stable at around 150 days, but decreases from ∼180 to ∼120 days. Furthermore, if looking at the ELODIE RV data set (which extends from BJD −2453000 ≃−100 to ≃1000, i.e. before the SOPHIE observations), we find instead a period- icity of ∼130 days. A similar process applied to the Howard & Fulton (2016) RV data set (two ∼2000-day slices) gives a main mid-term RV periodicity of 260 or 150 days, depending on the selected time slice. We thus conclude that the observed θ Cyg RV variations around ∼150 days are probably not really periodic, but rather quasi-periodic. This does not favour the hypothesis of a substellar companion for which the RV periodicity would have to be more stable. Then, we consider a (P, mp sin i) domain ranging from P1 to P2 in periods and from mp1 sin i to mp2 sin i in min- imal masses, the (P, mp sin i) grid being the same as used for the detection limit computation. The number of systems with detected planets within this domain is ndet (i.e. a sys- tem with two planets within the same given domain counts as one system). To derive the search completeness of our sample over D, we have to sum the search completeness func- tion C(P, mp sin i) over the period and the minimal mass ranges: CD = P2 X P1 mp2 sin i X mp1 sin i ( 1 N N X i=1 δi) dP d(mp sin i) P2 X P1 mp2 sin i X mp1 sin i 1 dP d(mp sin i) . Appendix C: Occurrence rate computation For a given sample with N stars, the search completeness func- tion C(P, mp sin i) gives the fraction of targets for which we can rule out a planet with a period P and a minimal mass mp sin i given the computed detection limits (see e.g. Howard et al. 2011). At a given (P, mp sin i) node, the completeness C is defined as p y In the case of a domain with no detected GPs (i.e. ndet = 0), we (re-)define n ′ det = 1, nmiss = 1/CD −1, and compute the corre- sponding occurrence rate. The obtained value is then an upper limit on the real GP occurrence rate. C = 1 N N X i=1 δi. (C.1) C = 1 N N X i=1 δi. Appendix D: Targets of peculiar interest: detailed plots (C.2) CD = P2 X P1 mp2 sin i X mp1 sin i ( 1 N N X i=1 δi) dP d(mp sin i) P2 X P1 mp2 sin i X mp1 sin i 1 dP d(mp sin i) . (C.2) A87, page 25 of 30 A&A 621, A87 (2019) A&A 621, A87 (2019) Fig. D.1. HD 113337 spectroscopic data. On all Lomb–Scargle periodograms, the 1% false-alarm probability (FAP) is plotted in red. On all periodograms, the periods of planet b and/or candidate planet c are plotted in blue. Top row, from left to right: RV time series, Lomb–Scargle RV periodogram, CLEAN RV periodogram, and observational window function. Overplotted on the RV is the two-planet Keplerian fit (red curve) and the Keplerian fit of the second candidate planet only (blue curve). Second and third rows: BIS and FWHM time series, corresponding Lomb– Scargle and CLEAN periodograms, and correlations with the RV data. Fourth row: RV residuals from planet b, corresponding Lomb–Scargle and CLEAN periodograms, and correlation with the BIS. The Keplerian fit of the candidate second planet is overplotted on the RV residuals (red curve). Fifth row: same, for the RV residuals of the two-planet Keplerian fit. Fig. D.1. HD 113337 spectroscopic data. On all Lomb–Scargle periodograms, the 1% false-alarm probability (FAP) is plotted in red. On all periodograms, the periods of planet b and/or candidate planet c are plotted in blue. Top row, from left to right: RV time series, Lomb–Scargle RV periodogram, CLEAN RV periodogram, and observational window function. Overplotted on the RV is the two-planet Keplerian fit (red curve) and the Keplerian fit of the second candidate planet only (blue curve). Second and third rows: BIS and FWHM time series, corresponding Lomb– Scargle and CLEAN periodograms, and correlations with the RV data. Fourth row: RV residuals from planet b, corresponding Lomb–Scargle and CLEAN periodograms, and correlation with the BIS. The Keplerian fit of the candidate second planet is overplotted on the RV residuals (red curve). Fifth row: same, for the RV residuals of the two-planet Keplerian fit. A87, page 26 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. Fig. D.2. HD 16232 spectroscopic data. On the RV periodograms, HD 16232b period is overplotted in blue. Appendix D: Targets of peculiar interest: detailed plots First row, from left to right: HD 16232 RV time series from K15, corresponding Lomb–Scargle and CLEAN periodograms, and window function. Overplotted on the RV is the Keplerian fit of HD 16232b we obtained with yorbit based on the K15 data (red) and the linear drift (blue). Second row: same for the RV expected from the Keplerian+linear fit of K15 data extrapolated at the epochs of our SOPHIE observations, plus added noise. Third row: same for the SOPHIE RV time series. Overplotted on the RV is the linear fit we computed with yorbit based on the SOPHIE RV (red solid line). Each RV data set (SOPHIE and K15 data) was independently centred on 0, hence the global RV offset between the two. Fourth and fifth rows: SOPHIE BIS and FWHM time series, corresponding periodograms and correlations with SOPHIE RV. Sixth row: residuals from the linear fit to the SOPHIE RV, corresponding periodograms and correlation to SOPHIE BIS. RV periodograms, HD 16232b period is overplotted in blue. First row, from left to right: HD 16232 Fig. D.2. HD 16232 spectroscopic data. On the RV periodograms, HD 16232b period is overplotted in blue. First ro Fig. D.2. HD 16232 spectroscopic data. On the RV periodograms, HD 16232b period is overplotted in blue. First row, from left to right: HD 16232 RV time series from K15, corresponding Lomb–Scargle and CLEAN periodograms, and window function. Overplotted on the RV is the Keplerian fit of HD 16232b we obtained with yorbit based on the K15 data (red) and the linear drift (blue). Second row: same for the RV expected from the Keplerian+linear fit of K15 data extrapolated at the epochs of our SOPHIE observations, plus added noise. Third row: same for the SOPHIE RV time series. Overplotted on the RV is the linear fit we computed with yorbit based on the SOPHIE RV (red solid line). Each RV data set (SOPHIE and K15 data) was independently centred on 0, hence the global RV offset between the two. Fourth and fifth rows: SOPHIE BIS and FWHM time series, corresponding periodograms and correlations with SOPHIE RV. Sixth row: residuals from the linear fit to the SOPHIE RV, corresponding periodograms and correlation to SOPHIE BIS. Fig. D.2. HD 16232 spectroscopic data. On the RV periodograms, HD 16232b period is overplotted in blue. Appendix D: Targets of peculiar interest: detailed plots First row, from left to right: HD 16232 RV time series from K15, corresponding Lomb–Scargle and CLEAN periodograms, and window function. Overplotted on the RV is the Keplerian fit of HD 16232b we obtained with yorbit based on the K15 data (red) and the linear drift (blue). Second row: same for the RV expected from the Keplerian+linear fit of K15 data extrapolated at the epochs of our SOPHIE observations, plus added noise. Third row: same for the SOPHIE RV time series. Overplotted on the RV is the linear fit we computed with yorbit based on the SOPHIE RV (red solid line). Each RV data set (SOPHIE and K15 data) was independently centred on 0, hence the global RV offset between the two. Fourth and fifth rows: SOPHIE BIS and FWHM time series, corresponding periodograms and correlations with SOPHIE RV. Sixth row: residuals from the linear fit to the SOPHIE RV, corresponding periodograms and correlation to SOPHIE BIS. A87, page 27 of 30 A&A 621, A87 (2019) A&A 621, A87 (2019) Fig. D.3. θ Cyg spectroscopic data. Top row, from left to right: RV time series, Lomb–Scargle and CLEAN periodograms of the RV, and window function of the observations. The RV long-term quadratic trend is overplotted on the RV (red solid curve). Second and third rows: BIS and FWHM time series, corresponding Lomb–Scargle and CLEAN periodograms, and correlations with the RV. Fourth row: RV residuals from the quadratic fit, Lomb–Scargle and CLEAN periodograms of the RV residuals, and correlation of the RV residuals with the BIS. Fig. D.3. θ Cyg spectroscopic data. Top row, from left to right: RV time series, Lomb–Scargle and CLEAN periodograms of the RV, and window function of the observations. The RV long-term quadratic trend is overplotted on the RV (red solid curve). Second and third rows: BIS and FWHM time series, corresponding Lomb–Scargle and CLEAN periodograms, and correlations with the RV. Fourth row: RV residuals from the quadratic fit, Lomb–Scargle and CLEAN periodograms of the RV residuals, and correlation of the RV residuals with the BIS. A87, page 28 of 30 S. Borgniet et al.: Extrasolar planets and brown dwarfs around AF-type stars. X. g p yp Fig. D.4. HD 191195 spectroscopic data. Top row, from left to right: RV time series, Lomb–Scargle and CLEAN periodograms of the RV, and win- dow function of the observations. Appendix D: Targets of peculiar interest: detailed plots Second and third rows: BIS and FWHM time series, corresponding Lomb–Scargle and CLEAN periodograms, and correlations with the RV. The RV–FWHM correlation is overplotted in red. Fig. D.4. HD 191195 spectroscopic data. Top row, from left to right: RV time series, Lomb–Scargle and CLEAN periodograms of the RV, and win- dow function of the observations. Second and third rows: BIS and FWHM time series, corresponding Lomb–Scargle and CLEAN periodograms, and correlations with the RV. The RV–FWHM correlation is overplotted in red. A87, page 29 of 30 A&A 621, A87 (2019) , ( ) Fig. D.5. Detail of θ Cyg SOPHIE RV variations (from top to bottom rows) over four 600-day slices. From left to right columns: RV variations over the time slice, RV Lomb–Scargle periodogram (with 1% FAP in red), RV CLEAN periodogram, detail of the CLEAN periodogram. For each time slice, the main RV periodicity deduced from the CLEAN periodogram is overplotted in red. Fig. D.5. Detail of θ Cyg SOPHIE RV variations (from top to bottom rows) over four 600-day slices. From left to right columns: RV variations over the time slice, RV Lomb–Scargle periodogram (with 1% FAP in red), RV CLEAN periodogram, detail of the CLEAN periodogram. For each time slice, the main RV periodicity deduced from the CLEAN periodogram is overplotted in red. A87, page 30 of 30
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Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury
Frontiers in pharmacology
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Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury Carmela Belardo1†, Monica Iannotta1†, Serena Boccella1†, Rosamaria Cristina Rubino2, Flavia Ricciardi1, Rosmara Infantino1, Gorizio Pieretti3, Luigi Stella4, Salvatore Paino1, Ida Marabese1, Rosa Maisto1, Livio Luongo1, Sabatino Maione1* and Francesca Guida1* Carmela Belardo1†, Monica Iannotta1†, Serena Boccella1†, Rosamaria Cristina Rubino2, Flavia Ricciardi1, Rosmara Infantino1, Gorizio Pieretti3, Luigi Stella4, Salvatore Paino1, Ida Marabese1, Rosa Maisto1, Livio Luongo1, Sabatino Maione1* and Francesca Guida1* 1 Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy, 2 Enecta s.r.l., Bologna, Italy, 3 Department of Plastic Surgery, University of Campania Luigi Vanvitelli, Naples, Italy, 4 Drug Addiction Unit (SerT), Naples, Italy Keywords: cannabidiol, traumatic brain injury, pain, behavior, microdialysis ORIGINAL RESEARCH published: 16 April 2019 doi: 10.3389/fphar.2019.00352 Edited by: Marialessandra Contino, University of Bari Aldo Moro, Italy Edited by: Marialessandra Contino, University of Bari Aldo Moro, Italy Neurological dysfunctions are the most impactful and persistent consequences of traumatic brain injury (TBI). Indeed, previous reports suggest that an association between TBI and chronic pain syndromes, as well anxio-depressive behaviors, tends to be more common in patients with mild forms of TBI. At present, no effective treatment options are available for these symptoms. In the present study, we used a weight drop mild TBI mouse model to investigate the effect of a commercially available 10% Cannabidiol (CBD) oil on both the sensorial and neuropsychiatric dysfunctions associated with mild TBI through behavioral and biomolecular approaches. TBI mice developed chronic pain associated with anxious and aggressive behavior, followed by a late depressive-like behavior and impaired social interaction. Such behaviors were related with specific changes in neurotransmitters release at cortical levels. CBD oral treatment restored the behavioral alterations and partially normalized the cortical biochemical changes. In conclusion, our data show some of the brain modifications probably responsible for the behavioral phenotype associated with TBI and suggest the CBD as a pharmacological tool to improve neurological dysfunctions caused by the trauma. Reviewed by: Faramarz Dehghani, Martin Luther University of Halle-Wittenberg, Germany Joseph T. McCabe, Uniformed Services University of the Health Sciences, United States *Correspondence: Sabatino Maione sabatino.maione@unicampania.it Francesca Guida franc.guida@gmail.com †These authors have contributed equally to this work Reviewed by: Faramarz Dehghani, Martin Luther University of Halle-Wittenberg, Germany Joseph T. McCabe, Uniformed Services University of the Health Sciences, United States *Correspondence: Sabatino Maione sabatino.maione@unicampania.it Francesca Guida franc.guida@gmail.com †These authors have contributed equally to this work Reviewed by: Faramarz Dehghani, Martin Luther University of Halle-Wittenberg, Germany Joseph T. McCabe, Uniformed Services University of the Health Sciences, United States *Correspondence: Sabatino Maione sabatino.maione@unicampania.it Francesca Guida franc.guida@gmail.com †These authors have contributed equally to this work Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 19 December 2018 Accepted: 21 March 2019 Published: 16 April 2019 Citation: Belardo C, Iannotta M, Boccella S, Rubino RC, Ricciardi F, Infantino R, Pieretti G, Stella L, Paino S, Marabese I, Maisto R, Luongo L, Maione S and Guida F (2019) Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury. Front. Pharmacol. 10:352. doi: 10.3389/fphar.2019.00352 The phytocannabinoid cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, exhibits a broad spectrum of potential therapeutic properties, including neuroprotective effects in Central Nervous System (CNS) disorders (Fernández-Ruiz et al., 2013; De Gregorio et al., 2018; Schonhofen et al., 2018). Through a multitarget mechanism, CBD shows potent anti-inflammatory and anti-oxidant properties which have been previously demonstrated in different models of neurodegenerative diseases and in acute episodes of brain damage (i.e., hypoxia-ischemia) (Hayakawa et al., 2007, 2010; Castillo et al., 2010). CBD has very low affinity for cannabinoid receptors type 1 (CB1) and type 2 (CB2), whereas different mechanisms, such as inhibition of anandamide uptake and enzymatic hydrolysis (Lastres-Becker et al., 2005), and April 2019 | Volume 10 | Article 352 1 Frontiers in Pharmacology | www.frontiersin.org Cannabidiol Effects in Traumatic Brain Injury Belardo et al. decrease of adenosine reuptake (Carrier et al., 2006), are believed to be responsible for its neuroprotective effects. not fixed. After a midline longitudinal incision, the skull was exposed to locate the area of impact and placed under a metal tube device where the opening was positioned directly over the animal’s head. The injury was induced by dropping a cylindrical metal weight (50 g), through a vertical metal guide tube from a height of 20 cm. The point of impact was between the anterior coronal suture (Bregma) and posterior coronal suture (Lambda). Immediately following injury, the skin was closed with surgical wound clips and mice were placed back in their cages to allow for recovery from the anesthesia and mTBI. Sham mice were submitted to the same procedure as described for mTBI, but without release of the weight. Sham and mTBI animals did not receive analgesic drugs after surgery. No animals have been excluded from the study. p p Traumatic brain injury (TBI) is a complex injury with a number of symptoms accompanied by inflammatory process and cell death (Arciniegas, 2011; Liu et al., 2019). Experimental Design Time points of evaluations were based on our previous study (Guida et al., 2017a). A total number of 80 mice were divided in four experimental groups: Sham/vehicle, mTBI/vehicle, Sham/CBD and mTBI/CBD. Behavioral tasks were performed at different time points and scheduled in order to avoid carry-over effects from prior testing experience. The number of animals for each experiment is represented in Table 1. Tactile Allodynia Tactile allodynia was evaluated at a series of calibrated nylon von Frey monofilaments (Semmes-Weinstein monofilaments, 2 Biological Instruments, Italy). Mice were allowed 7, 14 and 21 days after mTBI or sham surgery by to move freely in the compartment of the enclosure positioned on the metal mesh surface. Mice were adapted to the testing environment for 30 min before any measurement was taken. The monofilaments, starting from the 0.008 g monofilament, was applied perpendicularly to the plantar surface of each hind- paw in a series of ascending forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0, and 4.0 g). Each stimulus was applied for approximately 1 s with an interstimulus interval of 5 s. Withdrawal responses evoked by each monofilament was obtained from five consecutive trials. Data (gr) were expressed as a mean of right and left response hind paws. Drugs Cannabidiol and vehicle were kindly provided by Enecta Group, Bologna (BO), Italy https://www.enecta.eu/?lang CBD was dissolved in hemp seed oil and natural tocopherols, used as vehicle. CBD (30 µl, oil 10%) was administered via gavage from day 1 to day 14 and from day 50 to day 60. Those time points represent the pathological windows in which we previously observed the main features of the mTBI, such as aggressiveness, recklessness and/or sensorial changes in the first phase, and the depressive-like behavior in a late phase (Guida et al., 2017a). Animals Male C57BL/6 mice (Charles River, Italy) weighing 18–20 g were housed three per cage under controlled illumination (12 h light/dark cycle; light on 6:00 A.M.) and standard environmental conditions (ambient temperature 20– 22◦C, humidity 55–60%) for at least 1 week before the commencement of experiments. Mice chow and tap water were available ad libitum. The experimental procedures were approved by the Animal Ethics Committee of University of Campania “L. Vanvitelli,” Naples. Animal care was in compliance with Italian (D.L. 116/92) and European Commission (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals. All efforts were made to reduce both animal numbers and suffering during the experiments. Following behavioral testing, mice were scarified for microdialysis experiments at 14 or 60 day after mTBI induction. The timeline of mTBI induction, treatments and behavioral and biochemical characterization is given in the Figure 1. Observers were blind to the treatments in each experiment. Citation: It is characterized by an initial neuroinflammation, mediated by a rapid glia cells activation, peripheral immune cells recruitment and secretion of inflammatory cytokines, followed by the late appearance of psychologically debilitating symptoms and cognitive impairments (Woodcock and Morganti-Kossmann, 2013). Despite recent advances in the knowledge of TBI pathophysiology, no adequate pharmacotherapies are currently available (Loane and Faden, 2010). It is assumed that the secondary neuropsychiatric changes that occur as a consequence of trauma are associated with plastic rearrangements at hippocampal and cortical circuitry (Schwarzbold et al., 2008). In these brain regions endocannabinoid (EC) molecules induce pro-homeostatic and neuroprotective effects, by affecting neuroplasticity in cognitive and affective processes (Vigano et al., 2009; Boccella et al., 2019). A growing body of evidence suggests that the pharmacological manipulation of EC attenuates neuroinflammation and improve the recovery of neurobehavioral function during the early weeks after TBI (Shohami et al., 2011; Mayeux et al., 2017; Schurman and Lichtman, 2017). To our knowledge, no study has evaluated the effects of CBD on the neurological dysfunctions associated with the TBI. In particular, we coupled behavioral tasks and biochemical evaluations to assess the CBD effects on long-term cognitive and emotional responses induced by trauma. Frontiers in Pharmacology | www.frontiersin.org Mild TBI Induction Groups Days 0 7 13–15 21 34 59–61 Sham/vehicle N = 20 N = 12 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) Sham/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBl/vehicle N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBI/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 11 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive.) N = 13 (Depression) TABLE 1 | Numbers of animals used in each experiment. N = 5 (Pain) N = 6 (Rotarod) FIGURE 1 | Timeline of mTBI induction, treatments and behavioral and biochemical characterization. FIGURE 1 | Timeline of mTBI induction, treatments and behavioral and biochemical characterization. of permanence of the mouse on the cylinder was expressed as latency time (s). Voluntary movement, associated with the locomotion, was not counted as a withdrawal response. Tactile allodynia was defined as a significant decrease in the withdrawal threshold to the von Frey hair application. Each mouse served as its own control, the responses being measured both before and after surgical procedures. Rotarod Test Possible motor coordination impairment was evaluated at 7, 21, and 60 days after mTBI or sham surgery by Rotarod test (Ugo Basile). Mice was measured for the time (s) of equilibrium before falling on a rotary cylinder by a magnet that, activated from the fall of the mouse on the plate, allows to record the time of permanence on the cylinder. After a period of adaptation of 30 s, the spin speed gradually increases from 5 to 40 rpm for a maximum time of 5 min. The animals were analyzed by two separate tests at 1-hour interval in the same day. The experiment was performed for every group of animals the day before the surgical procedure and the days before the behavioral tests in order to avoid stress. The time Open Field Test Motor activity was also evaluated by open field test in sham and mTBI mice. Briefly, each mouse was individually monitored for 5 min in an open arena (l × w × h: 25 cm × 25 cm divided into 16 square grids). Parameters evaluated included: (1) number of transitions; and (2) number of rearings; and (3) time spent in the periphery or center (s). Mild TBI Induction Experimental mTBI was performed using a weight-drop device developed in our laboratory. Mice were anesthetized with intraperitoneal injection of Tribromoethanol (250 mg/kg) and placed in a prone position on a spongy support. The head was April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 2 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. TABLE 1 | Numbers of animals used in each experiment. Groups Days 0 7 13–15 21 34 59–61 Sham/vehicle N = 20 N = 12 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) Sham/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBl/vehicle N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBI/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 11 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive.) N = 13 (Depression) FIGURE 1 | Timeline of mTBI induction, treatments and behavioral and biochemical characterization. Voluntary movement, associated with the locomotion, was not of permanence of the mouse on the cylinder was expressed as TABLE 1 | Numbers of animals used in each experiment. Frontiers in Pharmacology | www.frontiersin.org Three Chambers Sociability Microdialysis experiments were performed in awake and freely moving mice. In brief, mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the mPFC using the coordinates: AP: 1.4–1.8 mm, L: 0.3–05 mm from bregma and V: 3.0 mm below the dura (Paxinos and Franklin, 2004). Dialysis probes, were constructed with 25G (0.3 mm inner diameter, 0.5 mm outer diameter) stainless steel tubing (A-M Systems). Inlet and outlet cannulae (0.04 mm inner diameter, 0.14 mm outer diameter) consisted of fused silica tubing (Scientific Glass Engineering). The probe had a tubular dialysis membrane (Enka AG, Wuppertal, Germany) 1.3 mm in length. Following a recovery period of 24 h, dialysis was commenced with aCSF (NaCl 147 mM, CaCl2 2.2, KCl 4 mM; pH 7.2) perfused at a rate of 1 µL/min by a Harvard Apparatus infusion pump. The neurotransmitters release was evaluated after chronic treatment performed with CBD or Vehicle in m-TBI or sham animals. Data were expressed as the average of six repeated measurements (each 30 min) to give a more accurate value. No appreciable differences were observed between the different six dyslisate samples collected during the single experiment. At the end of experiments, mice were anesthetized and their brains perfused fixed via the left cardiac ventricle with heparinized paraformaldehyde saline (4%). Brains were dissected out and fixed in a 10% formaldehyde solution for 2 days and included in OCT compound. The brain was cut in 40-µm thick slices and observed under a light microscope to identify Test at 60 days after mTBI or sham surgery, mice were tested for social interaction using a three-chambered social interaction apparatus. A plexi-glass three-chambered box was custom-built as follows: doorways in the two dividing walls had sliding covers to control access to the outer-side chambers. The test consisted of two consecutive stages of 5 and 10 min each. During the 5-minute first stage of habituation the mouse was allowed to freely explore the three chambers of the apparatus, detecting at this stage any innate side preference. After that the mouse was gently encouraged into the central chamber and confined there briefly by closing the side chamber doors. During the following 10-minute stage sessions, a custom made stainless-steel barred cup (6.5 cm × 15 cm) was placed upside down in one of the side chambers. Three Chambers Sociability A never before-met intruder, previously habituated, was placed into an upside-down identical cup in the other chamber. The time spent sniffing each upside-down cup, the time spent in each chamber and the number of entries into each chamber were recorded. Resident-Intruder At 14 and 60 days after mTBI or sham surgery, mice were tested for aggressive behavior using a resident intruder test. Mice were individually housed for 1 week in Plexiglas cages to establish a home territory and to increase the aggression of the resident experimental mice. To begin, food containers were removed and an intruder mouse of the same gender was placed in a resident home cage and resident–intruder interactions were analyzed for 10 min. The April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 3 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. of the 6-minute test by a time recorder. Immobility time was defined as the absence of escape-oriented behavior. Mice were considered to be immobile when they did not show any body movement, hung passively and completely motionless. aggressive behavior of resident socially-isolated mice was characterized by an initial pattern of exploratory activity around the intruder, which was followed by rearing and tail rattle, accompanied in a few seconds by wrestling and/or a violent biting attack. The number of these attacks and latency to the first attack during the 10 min observation period was recorded. CBD Effects on Motor Coordination and Anxiety in mTBI Mice the probe locations (Figure 2). The concentrations of D- Aspartate, L-glutamate and GABA contained in the dialysate were analyzed using by HPLC coupled with fluorimetric detection method. The system comprised two Gilson pumps (model no. 303), a C-18 reverse-phase column, and a Gilson fluorimetric detector (model no. 121). Dialysates were pre-column derivatized with o-phthaldialdehyde- N-acetylcysteine (OPA-NAC) (10 µl dialysate + 5 µl OPA-NAC + 10 µl borate buffer 10%). The mobile phase consisted of two components: (A) 0.2 M Na2HPO4, 0.2 M citric acid and 20% methanol and (B) 90% acetonitrile. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Mobile phase flow rate was maintained at 1.2 ml/min. Data were collected using a Dell Corporation PC system 310 interfaced to the detector via a Drew data-collection unit. Data were expressed as mean ± SEM of six samples for each mouse. the probe locations (Figure 2). The concentrations of D- Aspartate, L-glutamate and GABA contained in the dialysate were analyzed using by HPLC coupled with fluorimetric detection method. The system comprised two Gilson pumps (model no. 303), a C-18 reverse-phase column, and a Gilson fluorimetric detector (model no. 121). Dialysates were pre-column derivatized with o-phthaldialdehyde- N-acetylcysteine (OPA-NAC) (10 µl dialysate + 5 µl OPA-NAC + 10 µl borate buffer 10%). The mobile phase consisted of two components: (A) 0.2 M Na2HPO4, 0.2 M citric acid and 20% methanol and (B) 90% acetonitrile. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Mobile phase flow rate was maintained at 1.2 ml/min. Data were collected using a Dell Corporation PC system 310 interfaced to the detector via a Drew data-collection unit. Data were expressed as mean ± SEM of six samples for each mouse. In the rotarod test, no difference in riding time was observed between any of the treatment groups (Figure 3B), indicating no impairments in motor coordination. Statistical Analysis Data were represented as mean ± SEM. Behavioral data were analyzed by using one-way ANOVA, followed by Bonferroni’s multiple comparison. Newman–Keuls post hoc test was used as post hoc test in microdialysis analysis. P values < 0.05 were considered statistically significant. Statistical analysis was carried out using Prism/Graphpad (GraphPad Software, Inc.,) software. Numbers of animals used in each experiment is given in Table 1. RESULTS CBD Effects on Allodynia in mTBI Mice A significant decrease of TWT was observed in vehicle- treated mTBI mice at 7, 14, and 21 days after trauma induction [0.14 g ± 0.025, F(3,47) = 11.06, P < 0.0001; 0.11 g ± 0.04, F(3,37) = 12.40, P < 0.0001; 0.11 g ± 0.04, F(3,16) = 8.833, P = 0.0011, at 7, 14, and 21 days, respectively] compared to the sham group (0.63 g ± 0.12, 0.61 g ± 0.09, 0.62 g ± 0.1, at 7, 14, and 21 days, respectively) (Figure 3A). Moreover, a physiological re-establishment of normal pain response was observed 34 days after trauma induction [Sham/vehicle 0.62 g ± 0.06; Sham/CBD 0.65 g ± 0.1; mTBI/vehicle 0.64 g ± 0.22; mTBI/CBD 0.62 g ± 0.07 F(3,16) = 0.01345, P = 0.9978] (Figure 3A). No difference in pain threshold was observed between right and left paw (see Supplementary Table S1). Oral CBD treatment significantly reduced the tactile allodynia in mTBI mice at 14 and 21 days (0.28 g ± 0.04; 0.41 g ± 0.04; 0.46 ± 0.02, at 7, 14, and 21 days, respectively) as compared with vehicle (0.14 g ± 0.025; 0.11 g ± 0.04;0.11 g ± 0.04, at 7, 14, and 21 days, respectively) (Figure 3A). The CBD administration in sham mice did not change the pain response (0.71 g ± 0.17; 0.51g ± 0.07; 0.41g ± 0.09; 0.65 ± 0.1 at 7, 14 and 21, and 34 days, respectively) compared to sham/vehicle mice (0.63 g ± 0.12; 0.61 g ± 0.09; 0.62 g ± 0.1; 062 ± 0.06, at 7, 14, 21, and 34 days, respectively). Tail Suspension Test (TST) The Depression like behavior was evaluated at 14 days and 60 days after mTBI or sham surgery, mice were individually suspended by the tail on a horizontal bar (55 cm from floor) using adhesive tape placed approximately 4 cm from the tip of the tail. The duration of immobility, recorded in seconds, was monitored during the last 4 min FIGURE 2 | Microdialysis probe location. (A) Shows a panoramic picture of the pre-frontal cortex, the star indicates the prelimbic area. (B) Shows a high magnification of the microdialysis probe location for amino acid collection within the pre/infra-limbic cortex. FIGURE 2 | Microdialysis probe location. (A) Shows a panoramic picture of the pre-frontal cortex, the star indicates the prelimbic area. (B) Shows a high magnification of the microdialysis probe location for amino acid collection within the pre/infra-limbic cortex. April 2019 | Volume 10 | Article 352 4 Frontiers in Pharmacology | www.frontiersin.org Cannabidiol Effects in Traumatic Brain Injury Belardo et al. CBD Effects on Motor Coordination and Anxiety in mTBI Mice In the open field test, used to assay general locomotor activity levels, but also anxiety, one-way ANOVA, followed by Bonferroni post hoc test, revealed no significant changes in the time spent in the center [sham/vehicle 73.33 s ± 15.63; sham/CBD 56.83 s ± 5.31; mTBI/ vehicle 53.80 s ± 19.51; mTBI/CBD 46.67 s ± 10.05, F(3,19) = 0.7663, P = 0.5269) or in the periphery [sham/vehicle 241.7 s ± 9.86; sham/CBD 243.2 s ± 5.31; mTBI/ vehicle 268.8 s ± 12.89; mTBI/CBD 263.3 s ± 11.12, F(3,19) = 1.896, P = 0.1646] or for the number of transitions [sham/vehicle 204.5 ± 17.50; sham/CBD 239.8 ± 10.41; mTBI/ vehicle 226.8 ± 13.62; mTBI/CBD 238.8 ± 14.06, F(3,20) = 1.354, P = 0.2854] after trauma or any treatment (Figures 3C,E,F). However, mTBI/vehicle [52.5 ± 2.38; F(3,20) = 24.00, P < 0.0001] mice showed an increase in the number of rearing as compared to sham/vehicle (12.50 ± 3.17) and this effect was significantly reduced by CBD treatment (33.33 ± 4.10). CBD did not change the number of rearing in sham animals (25.33 ± 3.77) (Figure 3D). CBD Effects on Aggressive Behavior in mTBI Mice No difference in the latency to the first attack in all groups of mice at 14 and 60 days after brain injury was observed [sham/vehicle 470.3 s ± 38.56; sham/CBD 424.6 s ± 71.80; mTBI/ vehicle 463.4 s ± 37.26; mTBI/CBD 424.5 s ± 34.10, F(3,36) = 0.2630, P = 0.8516; sham/vehicle 227.4 s ± 48.53; sham/CBD 200.4 s ± 53.56; mTBI/ vehicle 186.4 s ± 53.51; mTBI/CBD 226.0 s ± 45.33, F(3,16) = 0.1588, P = 0.9225] (Figures 4A,C). However, 14 days after the trauma, mTBI mice showed an increased number of attacks [20.20 ± 2.99, F(3,36) = 5.353, P = 0.0037], as compared to the controls (10.60 ± 1.36) (Figure 4B). CBD treatment significantly reduced this effect (9.9 ± 1.84) as compared with vehicle (20.20 ± 2.99). At 60 days after trauma, no significant change was observed in the number of attacks [sham/vehicle 0.6 ± 0.4; sham/CBD 0.8 ± 0.37; mTBI/ vehicle 0.8 ± 0.37; mTBI/CBD 0.4 ± 0.24, F(3,16) = 0.2933, P = 0.8296]. Sham mice treated with CBD did not show any change in the latency to the first attack or number of attacks compared to sham/vehicle mice (Figures 4C,D). CBD Effects on Depressive-Like Behavior in mTBI Mice mTBI mice showed an increased immobility time, measured as the lack of escape-oriented activity (169.4 s ± 5.93) compared to the sham mice (132.4 s ± 4.15) 60 days post trauma (Figure 5A). CBD treatment significantly reduced the immobility in mTBI condition (125.1 s ± 8.95) compared to the vehicle (169.4 s ± 5.93) (Figure 5A). Sham mice treated with CBD did not show any change in the duration of immobility compared to vehicle-treated April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 5 Belardo et al. Cannabidiol Effects in Traumatic Brain Injury FIGURE 3 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monofilaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the center, in the open field test, respectively. Data are represented as mean ± SEM of 10–11 mice per group. ∗, # and ◦indicate significant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc tests. Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 3 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monofilaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the center, in the open field test, respectively. Data are represented as mean ± SEM of 10–11 mice per group. ∗, # and ◦indicate significant differences compared to FIGURE 3 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monofilaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the center, in the open field test, respectively. Data are represented as mean ± SEM of 10–11 mice per group. ∗, # and ◦indicate significant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc tests. CBD Effects on Depressive-Like Behavior in mTBI Mice mice [110.4 s ± 10.53, F(3,45) = 13.64, P < 0.0001] (Figure 5A). At 14 days mTBI mice did not shown any change in the immobility time as compared with sham animals (Supplementary Figure S1). In the three chambers sociability test, no difference in the time spent in each chamber or in the number of transitions was observed in mTBI and Sham mice treated with vehicle or CBD (Supplementary Table S2). However, mTBI mice had reduced sociability level, spending a higher time in interacting with the object during the recorded time [27.33 s ± 4.1; F(3,8) = 11.40, P < 0.0029], compared to sham animals (4.67 s ± 0.88) (Figure 5B, session I). This effect was significantly improved in mTBI CBD-treated animals (5.0 s ± 1.0). Moreover, mTBI mice [interaction CBD Effects on Social Behavior in mTBI Mice At 60 days post mTBI, while GABA and D-Asp dialysate were not changed [GABA: sham/vehicle 2.21 pmol/µl ± 0.01; sham/CBD 3.65 pmol/µl ± 1.08; mTBI/vehicle 1.51 pmol/µl ± 0.36; mTBI/CBD 2.11 pmol/µl ± 0.36, F(3,7) = 2.025 P = 0.1990; D-Asp sham/vehicle 1.08 pmol/µl ± 0.53; sham/CBD 0.61 pmol/µl ± 0.18; mTBI/vehicle 0.47 pmol/µl ± 0.10; mTBI/CBD 0.29 pmol/µl ± 0.06, F(3,7) = 2.176 P = 0.1789] (Figures 6D,F), Glu levels were still high, but CBD did not revert this effect [Glu: sham/vehicle 10.81 pmol/µl ± 5.33; mTBI/vehicle 47.29 pmol/µl ± 11.14; mTBI/CBD 73.62 pmol/µl ± 4.80, F(3,5) = 15.26 P = 0.0060] (Figure 6E). Finally, we found that CBD increased per sè Glu levels in the mPFC of sham mice at both 14- and 60-days post trauma (sham/CBD: 17.12 pmol/µl ± 1.40; 47.02 pmol/µl ± 6.09, 14 and 60 days, respectively), as compared with vehicle (sham/Vehicle: 7.12 pmol/µl ± 0.26; 10.81 pmol/µl ± 5.33, 14 and 60 days, respectively) (Figures 6B,E). (Figures 6A,B). On the contrary, GABA levels were decreased by TBI, and CBD significantly reverted this effect (sham/vehicle 3.25 pmol/µl ± 0.6; sham/CBD 2.62 pmol/µl ± 0.31; mTBI/vehicle 0.191 pmol/µl ± 0.01; mTBI/CBD 1.38 pmol/µl ± 0.13, F(3,12) = 15.74 P = 0.0002) (Figure 6C). At 60 days post mTBI, while GABA and D-Asp dialysate were not changed [GABA: sham/vehicle 2.21 pmol/µl ± 0.01; sham/CBD 3.65 pmol/µl ± 1.08; mTBI/vehicle 1.51 pmol/µl ± 0.36; mTBI/CBD 2.11 pmol/µl ± 0.36, F(3,7) = 2.025 P = 0.1990; D-Asp sham/vehicle 1.08 pmol/µl ± 0.53; sham/CBD 0.61 pmol/µl ± 0.18; mTBI/vehicle 0.47 pmol/µl ± 0.10; mTBI/CBD 0.29 pmol/µl ± 0.06, F(3,7) = 2.176 P = 0.1789] (Figures 6D,F), Glu levels were still high, but CBD did not revert this effect [Glu: sham/vehicle 10.81 pmol/µl ± 5.33; mTBI/vehicle 47.29 pmol/µl ± 11.14; mTBI/CBD 73.62 pmol/µl ± 4.80, F(3,5) = 15.26 P = 0.0060] (Figure 6E). Finally, we found that CBD increased per sè Glu levels in the mPFC of sham mice at both 14- and 60-days post trauma (sham/CBD: 17.12 pmol/µl ± 1.40; 47.02 pmol/µl ± 6.09, 14 and 60 days, respectively), as compared with vehicle (sham/Vehicle: 7.12 pmol/µl ± 0.26; 10.81 pmol/µl ± 5.33, 14 and 60 days, respectively) (Figures 6B,E). CBD Effects on Social Behavior in mTBI Mice with mouse II: 24.0 s ± 11.55 F(3,8) = 1.308, P = 0.3373] did not show significantly altered preference for social novelty compared with control mice (interaction with mouse II: 40.67 s ± 11.05) (Figure 5C, session II). The CBD treatment did not induce any change in sociability, in the time spent in the two chambers or in the number of transitions between the chambers compared to vehicle in sham or mTBI animals (Sham/CBD and mTBI/CBD: interaction with mouse II: 39.67 s ± 7.86 and 48.67 ± 2.91) (Figures 5A,B). CBD Effects on Social Behavior in mTBI Mice Analysis of the social preference revealed an impairment of social interaction which occurred 60 days post trauma. April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 6 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 4 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the first attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate significant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Tukey post hoc test. FIGURE 4 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the first attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate significant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Tukey post hoc test. FIGURE 4 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the first attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate significant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Tukey post hoc test. (Figures 6A,B). On the contrary, GABA levels were decreased by TBI, and CBD significantly reverted this effect (sham/vehicle 3.25 pmol/µl ± 0.6; sham/CBD 2.62 pmol/µl ± 0.31; mTBI/vehicle 0.191 pmol/µl ± 0.01; mTBI/CBD 1.38 pmol/µl ± 0.13, F(3,12) = 15.74 P = 0.0002) (Figure 6C). CBD Effects on Neurotransmitters Release mTBI Mice In vivo microdialysis was used to assess the amino acids contents in the m-PFC of mTBI mice. HPLC analysis revealed a notable increase of extracellular glutamate (Glu) and D-Aspartate (D-Asp) levels in the mPFC of 14 days mTBI animals [Glu: 32.05 pmol/µl ± 1.33 F(3,12) = 123.1, P < 0.0001; D-Asp: 2.29 pmol/µl ± 0.38; F(3,8) = 7.922, P = 0.0088], as compared with controls (Glu: 7.12 pmol/µl ± 0.26; D-Asp: 0.93 pmol/µl ± 0.42). Remarkably, CBD treatment normalized both Glu and D-Asp levels (Glu: 9.43 pmol/µl ± 0.55; D-Asp: 0.28 pmol/µl ± 0.06) April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 7 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 5 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows the duration of immobility in the tail suspension test. (B,C) Show the duration of the time spent with an object or mouse in the three chambers sociability apparatus. Data are expressed in seconds and represented as mean ± SEM of 10–12 mice per group. ∗and ◦indicate significant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc test. FIGURE 5 | Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows the duration of immobility in the tail suspension test. (B,C) Show the duration of the time spent with an object or mouse in the three chambers sociability apparatus. Data are expressed in seconds and represented as mean ± SEM of 10–12 mice per group. ∗and ◦indicate significant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc test. Frontiers in Pharmacology | www.frontiersin.org DISCUSSION Though brain trauma did not affect the motor coordination or the April 2019 | Volume 10 | Article 352 8 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 6 | Effect of CBD on the release of glutamate (B,E), GABA (C,F) and D-Aspartate (A,D) in sham or mTBI mice at 14 and 60 days after trauma. The values of extracellular amino acids in the mPFC were expressed as pmol in 10 µl of perfusate. Each point represents the mean ± SEM of 3–4 animals per group. ∗, # and ◦ indicate significant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One- Way ANOVA, post hoc test Newman-Keuls Multiple Comparison. FIGURE 6 | Effect of CBD on the release of glutamate (B,E), GABA (C,F) and D-Aspartate (A,D) in sham or mTBI mice at 14 and 60 days after trauma. The values of extracellular amino acids in the mPFC were expressed as pmol in 10 µl of perfusate. Each point represents the mean ± SEM of 3–4 animals per group. ∗, # and ◦ indicate significant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically significant. One- Way ANOVA, post hoc test Newman-Keuls Multiple Comparison. that mTBI may be responsible for hyper-functional glutamate/D- aspartate signaling at the supraspinal level and, possibly, of the trauma-associated negative state (aggressive phenotype), at least at this time point. Indeed, while CBD reduced the depression and the impaired sociability, it was not able to change glutamate levels that were still high 60 days after trauma. This suggests the involvement of other brain areas, including the hippocampus, and/or other neurotransmissions (i.e., serotoninergic) in the altered neuropsychiatric behavioral profile of mTBI mice. Moreover, we demonstrated that CBD treatment also increased glutamate in sham mice, at both 14 and 60 days. Indeed, although it does not alter behavior in selected tasks, we cannot exclude that CBD may play a physiological role in other neuropsychological functions regulated by cortical processing, such as cognition, memory and reward. after trauma, indicating an impaired recognition memory. A positive trend to the increase was given by CBD treatment. TBI strongly affects the cortical neuronal plasticity. DISCUSSION exploratory activity, we found an increased rearing activity in mTBI mice, which was counteracted by CBD treatment. The significance of rearing movements seems to be strongly related to the specific surrounding environment. Rearing may reflect attentive processes underlying the assembling of information in novel situations (Aspide et al., 1998), however, in some circumstances, it may simply represent an escape motivation (Lever et al., 2006). It is possible that the rearing activity in our model may reflect a kind of recklessness-like behavior, as previously reported in TBI mice (Guida et al., 2017a) and humans (DSM V). mTBI mice presented a typical phenotype, characterized by an aggressive behavior followed by a depressive-like behavior. Indeed, the aggressiveness of mTBI animals, revealed by an increased number of attacks on intruder mouse, was followed by a depressive-like behavior, manifested as enhanced immobility in the tail suspension test (14 and 60 days after trauma, respectively). The impaired social activity was also observed in the three-chamber sociability task, suggesting a general illness, often reported in patients with TBI. CBD significantly prevented all these effects. Interestingly, mTBI mice showed a reduced interest for social novelty, compared with controls. In fact, even if was not significant, we found that the time spent with the novel mouse (stranger) was reduced Cognitive and emotional dysfunctions are the most impactful and persistent consequences of TBI. Indeed, motor and sensory deficits and psychiatric disorders may endure for weeks, as a consequence of the traumatic damage to the underlying brain structures. We previously showed that mTBI induced late (up to 60 days) neurological dysfunctions in mice and identified electrophysiological changes at the cortical level possibly associated with symptomatology (Guida et al., 2017a). In the present study, we demonstrated that the repeated treatment with commercially available 10% CBD oil exerts beneficial effects on the behavioral dysfunctions associated with TBI. Moreover, at the dose tested, CBD does not change the normal attitude, in term of locomotion, nociception or emotional behavior, in not injured animals. As previously shown (Guida et al., 2017a), 2 weeks-mTBI mice displayed abnormal pain response after innocuous stimuli to the paw (mechanical allodynia), probably due to the overall inflammatory condition (Feliciano et al., 2014). The daily treatment with CBD significantly reduced pain behavior, which, in fact, spontaneously disappeared in 30 days. DISCUSSION Indeed, we have previously shown that the altered behaviors following mTBI correlated with the biphasic firing activity of the pyramidal neurons in the mPFC, considered a key area regulating chronic pain (Giordano et al., 2011; Luongo et al., 2013) and negative affective states, such as anxiety and depression (Vialou et al., 2014; Apps and Strata, 2015; Guida et al., 2015). Remarkably, microdialysis/HPLC analysis revealed that mTBI (14 days) induced an increase of extracellular glutamate levels in the mPFC, which strengthens, the concept that plastic changes and novel neural remodeling may occur after trauma. Conversely, GABA levels were decreased, possibly as a counterbalance of the glutamate-mediated excitation. Remarkable, CBD normalized both GABA and glutamate levels. These latter data are in line with previous reports showing the protective effects of cannabinoids on the excitoxicity and inflammation correlated with glutamatergic system dysregulation in diverse neurodegenerative diseases (Guida et al., 2015, 2017b; Palazzo et al., 2015). In particular, the neuroprotective and antioxidant properties of CBD have been shown in high glutamate induced- toxicity in rat cortical neurons (Hampson et al., 1998). Our data also indicated that the extracellular levels of D-aspartic acid (D-Asp), endogenous NMDA receptor agonist, involved in pain and synaptic plasticity (Guida et al., 2015; D’Aniello et al., 2017) enhanced in mTBI mice. This effect was decreased in CBD- treated mTBI mice. 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Brain 8:47. doi: 10.1186/s13041-015-0139-5 Arciniegas, D. B. (2011). Clinical electrophysiologic assessments and mild traumatic brain injury: state-of-the-science and implications for clinical practice. Int. J. Psychol. 82, 41–52. doi: 10.1016/j.ijpsycho.2011.03.004 Aspide, R., Carnevale, U. A. G., Sergeant, J. A., and Sadile, A. G. (1998). Non- selective attention and nitric oxide in putative animal models of attention- deficit hyperactivity disorder. Behav. Brain Res. 95, 123–133. doi: 10.1016/ S0166-4328(97)00217-9 Hampson, A., Grimaldi, M., Axelrod, J., and Wink, D. (1998). Cannabidiol and (- ) 19-tetrahydrocannabinol are neuroprotective antioxidants. Proc. Natl. Acad. Sci. U.S.A. 95, 8268–8273. doi: 10.1073/pnas.95.14.8268 Hayakawa, K., Mishima, K., and Fujiwara, M. (2010). Therapeutic potential of non- psychotropic cannabidiol in ischemic stroke. Pharmaceuticals 3, 2197–2212. doi: 10.3390/ph3072197 Boccella, S., Cristiano, C., Romano, R., Iannotta, M., Belardo, C., Farina, A., et al. (2019). Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway. Neurobiol. Dis. 121, 106–119. doi: 10.1016/j.nbd. 2018.09.023 Hayakawa, K., Mishima, K., Nozako, M., Hazekawa, M., Irie, K., Fujioka, M., et al. (2007). Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism. J. Neurochem. 102, 1488–1496. doi: 10.1111/j.1471-4159.2007.04565.x Carrier, E. J., Auchampach, J. A., and Hillard, C. J. (2006). Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc. Natl. Acad. Sci. U.S.A. 103, 7895–7900. doi: 10.1073/pnas.0511232103 urochem. 102, 1488–1496. doi: 10.1111/j.1471-4159.2007.04565.x Lastres-Becker, I., Molina-Holgado, F., Ramos, J. A., Mechoulam, R., and Fernández-Ruiz, J. (2005). Cannabinoids provide neuroprotection against 6- hydroxydopamine toxicity in vivo and in vitro: relevance to Parkinson’s disease. Neurobiol. Dis. 19, 96–107. doi: 10.1016/j.nbd.2004.11.009 Castillo, A., Tolón, M., Fernández-Ruiz, J., Romero, J., and Martinez- Orgado, J. (2010). The neuroprotective effect of cannabidiol in an in vitro model of newborn hypoxic–ischemic brain damage in mice is mediated by CB2 and adenosine receptors. Neurobiol. Dis. 37, 434–440. CONCLUSION In conclusion, our data demonstrate that mTBI causes late sensorial affective/cognitive deficiencies linked to altered neurotransmitter release at cortical level. Moreover, we showed that chronic CBD treatment reduces behavioral dysfunctions by restoring at least in part cortical biochemical processes. Taken together, our results suggest that CBD could represent a novel approach for the management of neuropsychiatric disorders associated with TBI. In conclusion, our data demonstrate that mTBI causes late sensorial affective/cognitive deficiencies linked to altered neurotransmitter release at cortical level. Moreover, we showed that chronic CBD treatment reduces behavioral dysfunctions by restoring at least in part cortical biochemical processes. Taken together, our results suggest that CBD could represent a novel approach for the management of neuropsychiatric disorders associated with TBI. April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 9 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. SUPPLEMENTARY MATERIAL CB, LL, and RR conceived and designed the experiments. CB, MI, SB, FR, RI, and RM performed the experiments. GP, LS, SP, RR, LL, and IM analyzed the data and contributed to materials and analysis tools. CB, FG, and SM wrote the manuscript. CB, LL, and RR conceived and designed the experiments. CB, MI, SB, FR, RI, and RM performed the experiments. GP, LS, SP, RR, LL, and IM analyzed the data and contributed to materials and analysis tools. CB, FG, and SM wrote the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2019.00352/full#supplementary-material FIGURE S1 | Effect of CBD on depressive-like behavior in sham and mTBI mice. The duration of immobility is measured in the tail suspension test at 14 days after mTBI induction. TABLE S1 | Effect of CBD on pain behavior in sham and mTBI mice. Left and right paw tactile withdrawal thresholds (TWT) are measured through Von Frey monofilaments at 7, 14, 21, and 34 days after mTBI induction. ACKNOWLEDGMENTS We thank Enecta Group for providing Cannabidiol. We also thank the Department of Experimental Medicine of Universisty of Campania for publication fees. We thank Enecta Group for providing Cannabidiol. We also thank the Department of Experimental Medicine of Universisty of Campania for publication fees. TABLE S2 | Effect of CBD on social behavior in sham and mTBI mice. The time spent in each chamber (A) or in the number of transitions (B) in the three chambers sociability apparatus. Frontiers in Pharmacology | www.frontiersin.org REFERENCES Guida, F., Boccella, S., Iannotta, M., De Gregorio, D., Giordano, C., Belardo, C., et al. (2017a). Palmitoylethanolamide reduces neuropsychiatric behaviors by restoring cortical electrophysiological activity in a mouse model of mild traumatic brain injury. Front. Pharmacol. 8:95. doi: 10.3389/fphar.2017.00095 Schonhofen, P., Bristot, I. J., Crippa, J. A., Hallak, J. E. C., Zuardi, A. W., Parsons, R. B., et al. (2018). Cannabinoid-based therapies and brain development: potential harmful effect of early modulation of the endocannabinoid system. CNS Drugs 32, 697–712. doi: 10.1007/s40263-018-0550-4 April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 352 Cannabidiol Effects in Traumatic Brain Injury Cannabidiol Effects in Traumatic Brain Injury Cannabidiol Effects in Traumatic Brain Injury Belardo et al. Schurman, L. D., and Lichtman, A. H. (2017). Endocannabinoids: a promising impact for traumatic brain injury. Front. Pharmacol. 8:69. doi: 10.3389/fphar. 2017.00069 Woodcock, T., and Morganti-Kossmann, C. (2013). The role of markers of inflammation in traumatic braininjury. Front. Neurol. 4:18. doi: 10.3389/fneur. 2013.00018 Schwarzbold, M., Diaz, A., Martins, E. T., Rufino, A., Amante, L. N., Thais, M. E., et al. (2008). Psychiatric disorders and traumatic brain injury. Neuropsychiatr. Dis. Treat. 4, 797–816. Conflict of Interest Statement: RR is Enecta Group staff. CB is supported by a grant provided by Enecta Group. The experiments were not supported by Enecta. Conflict of Interest Statement: RR is Enecta Group staff. CB is supported by a grant provided by Enecta Group. The experiments were not supported by Enecta. Shohami, E., Cohen-Yeshurun, A., Magid, L., Algali, M., and Mechoulam, R. (2011). Endocannabinoids and traumatic brain injury. Br. J. Pharmacol. 163, 1402–1410. doi: 10.1111/j.1476-5381.2011. 01343.x The remaining 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. Vialou, V., Bagot, R. C., Cahill, M. E., Ferguson, D., Robison, A. J., Dietz, D. M., et al. (2014). Prefrontal cortical circuit for depression- and anxiety- related behaviors mediated by cholecystokinin: role of deltafosb. J. Neurosci. 34, 3878–3887. doi: 10.1523/JNEUROSCI.1787-13.2014 Copyright © 2019 Belardo, Iannotta, Boccella, Rubino, Ricciardi, Infantino, Pieretti, Stella, Paino, Marabese, Maisto, Luongo, Maione and Guida. 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. Vigano, D., Guidali, C., Petrosino, S., Realini, N., Rubino, T., Di Marzo, V., et al. (2009). Involvement of the endocannabinoid system in phencyclidine-induced cognitive deficits modelling schizophrenia. Int. J. Neuropsychopharmacol. 12, 599–614. doi: 10.1017/S1461145708009371 April 2019 | Volume 10 | Article 352 Frontiers in Pharmacology | www.frontiersin.org 11
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Simulation of nitrate and potassium concentrations in soil solution using parametric models and Hydrus-2D
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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. ABSTRACT This study estimated the nitrate and potassium concentration in the soil solution of drainage lysimeter using the mathematical models developed by Vogeler et al. (1996) and Muñoz- Carpena et al. (2005) and the computational model Hydrus-2D, while comparing the simulated and observed data using statistical parameters. The cultivar used for the study was ‘Prata Gorutuba’. The experimental plots were six lysimeters of drainage. Fertigation was performed weekly. The mathematical models developed by Vogeler et al. (1996) and Muñoz-Carpena et al. (2005) were used to determine the specific concentration of a given ion (Ci). The Hydrus software was used to simulate the dynamics of nutrients. The concentrations of nitrate and potassium in the soil solution were estimated by the model of Vogeler et al. (1996), adapted to the linear type CEw-Ci ratio and simulated by the Hydrus model, resulting in an acceptable characterization of the distribution of these nutrients. Keywords: banana, fertigation, lysimeter, modeling, nutrients. Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Simulation of nitrate and potassium concentrations in soil solution using parametric models and Hydrus-2D ARTICLES doi:10.4136/ambi-agua.2606 Received: 25 Jun. 2020; Accepted: 15 Dec. 2020 Beatriz Santos Conceição1 ; Eugênio Ferreira Coelho2 ; João José da Silva Junior3* ; José Antonio do Vale Sant’Ana1 ; Mauro Aparecido Martinez4 1Departamento de Agronomia. Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso (IFMT), Avenida Vilmar Fernandes, n° 300, CEP: 78652-000, Confresa, MT, Brazil. E-mail: beatriz.conceicao@cfs.ifmt.edu.br, jose.santana@cfs.ifmt.edu.br 2Embrapa Mandioca e Fruticultura (EMBRAPA), Rua Embrapa, s/n, CEP: 44380-000, Cruz das Almas, BA, Brazil. E-mail: eugenio.coelho@embrapa.br 3Faculdade de Agronomia e Medicina Veterinária. Universidade de Brasília (UnB), Campos Universitário Darcy Ribeiro, Asa Norte, CEP: 70910-900, Brasília, DF, Brazil. 4Departamento de Ciência do Solo. Universidade Federal de Viçosa (UFV), Avenida Peter Henry Rolfs, s/n, CEP: 36570-000, Viçosa, MG, Brazil. E-mail: mmauro@ufv.br *Corresponding author. E-mail: jjsjunior@unb.br Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com 1. INTRODUCTION Water is one of the main factors limiting crop development, with special importance in certain phenological phases in which its deficiency can more or less significantly compromise productivity (Vanhove et al., 2012; Ravi et al., 2013; Muthusamy et al., 2014; Kissel et al., 2015). A few years ago, a change in the traditional form of fertilization of irrigated crops occurred with the application of fertilizer via irrigation water. According to Langoni et al. (2019), in order for the irrigated crop to be viable it is necessary to adopt management that contributes to the increase of productivity and profit, in which fertigation is a technology that offers easy benefits in the parceling of fertilizer, providing nutrients according to the phenological stages of the crop more responsive to absorption. In fertigated areas, the monitoring of the spatial and temporal variation of the water content (θ), the electrical conductivity of the soil (CEs), the electrical conductivity of the soil solution (CEss) and the ionic concentration (Ci), makes it possible to estimate the loss of water to deep percolation and evapotranspiration of the crop, in addition to determining the regions of nutrient extraction by the plants and the possible loss of nutrients to leaching (Santana et al., 2007). For monitoring the distribution of water and ionic solutes in the soil, time domain reflectometry (TDR) has been used successfully in several studies (Vogeler et al., 1996; Persson and Uvo, 2003; Santos et al., 2010), due to the relationship between the apparent electrical conductivity of the soil and the electrical conductivity of the soil solution (CEw), and between CEw and the ionic concentration (Ci). Considering the computational advances in recent years, a viable alternative is the use of analytical and numerical models that predict the processes of water and solute transfer between the soil surface and the groundwater table. Among the computational models available in the literature, the Hydrus model has been distinguished by the accuracy of the results (Nimmer et al., 2009; Silva et al., 2015; Kanzari et al., 2012; Pinho and Miranda 2014; Ramos et al., 2013), and also by its graphical interface that allows for a good interaction with different types of users. However, the approach for calibration and validation of the model can vary greatly, depending on the complexity of the experiment. RESUMO O objetivo deste trabalho foi estimar a concentração de nitrato e potássio na solução do solo, em lisímetro de drenagem com o uso dos modelos matemáticos desenvolvidos por Vogeler et al. (1996) e Muñoz-Carpena et al. (2005) e do modelo computacional Hydrus-2D e comparar os dados simulados e observados usando parâmetros estatísticos. A cultivar utilizada para o estudo foi a ‘Prata Gorutuba’. As parcelas experimentais foram seis lisímetros de drenagem. A fertirrigação foi realizada semanalmente. Os modelos matemáticos desenvolvidos por Vogeler et al. (1996) e Muñoz-Carpena et al. (2005), foram utilizados para determinar a concentração 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. Beatriz Santos Conceição et al. 2 específica de um determinado íon (Ci). O software Hydrus foi utilizado para simular a dinâmica de nutrientes. As concentrações de nitrato e potássio na solução do solo, estimados por meio do modelo de Vogeler et al. (1996) adaptado para a relação CEw-Ci do tipo linear e simulado pelo modelo Hydrus apresentou uma caracterização aceitável da distribuição desses nutrientes. específica de um determinado íon (Ci). O software Hydrus foi utilizado para simular a dinâmica de nutrientes. As concentrações de nitrato e potássio na solução do solo, estimados por meio do modelo de Vogeler et al. (1996) adaptado para a relação CEw-Ci do tipo linear e simulado pelo modelo Hydrus apresentou uma caracterização aceitável da distribuição desses nutrientes. Palavras-chave: bananeira, fertirrigação, lisimetro, modelagem, nutrientes. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 1. INTRODUCTION The main advantage of using models is that it saves time and capital, since field experiments and laboratories are usually laborious, expensive and time-consuming (Martinez et al., 2010). Nevertheless, it is worth noting that field measurements are indispensable, since mathematical models require local calibration and validation (Rivera et al., 2008). Techniques for solving equations and the available computational resources can predict the contamination risks and impacts that the chemical components might cause to the soil- water-plant system (Garcia et al., 2012). The use of multidimensional modeling is indispensable for developing fertigation practices, and optimized to achieve optimal nutritional efficiency for plants. Thus, in recent decades several analytical and numerical models have emerged to predict the processes of water and solute transfer between the soil surface and groundwater (Pinho and Miranda, 2014). According to Kunz et al. (2014), the use of simulation models describing the flux of water in the soil, such as Hydrus 2D/3D, can be useful to explain the behavior of water in porous media, provided they are properly calibrated and validated from field experiments. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Simulation of nitrate and potassium concentrations in … 3 Elmi et al. (2012) investigated the vertical distribution of water in a soil column using Hydrus and observed that the estimated soil moisture values did not correspond to the measured values. According to the authors, the result may be associated with heterogeneity in soil profile, which was not considered in the discretization of the model. This result differs from that of Silva et al. (2015) who, seeking to model soil moisture dynamics through the Hydrus Model, reported that the results ensure the possibility of using the model to estimate soil moisture at a daily scale for the type of soil and coverage conditions investigated. Kanzari et al. (2012) also characterized water movement by using TDR probes installed up to 4m deep, using Hydrus to model water movement in a semi-arid region of Tunisia, and found that the simulated data showed the same trend as the measured data. Pinho and Miranda (2014), evaluating the application of the Hydrus model as well as its performance in potassium and water displacement simulations (in Red-Yellow Latossoil and Red Nitossoil), concluded that the Hydrus model was efficient in potassium and water displacement simulations in the two soil materials studied. Ramos et al. 1. INTRODUCTION (2013) used the Hydrus model to simulate the dynamics of water and nitrogen forms in a soil cultivated with saccharine sorghum, bearing different salt contents and fertigation levels, and reported that simulations with Hydrus were useful to understanding the best strategies to increase nutrient uptake by plants and reduce losses to leaching. Thus, the present work aimed to estimate the concentration of nitrate and potassium in the soil solution in drainage lysimeter using the mathematical models developed by Vogeler et al. (1996) and Muñoz-Carpena et al. (2005) and the computational model Hydrus-2D, while comparing the simulated and observed data using statistical parameters. 2. MATERIAL AND METHODS The experiment was conducted at the National Center of Cassava and Fruticulture Research (CNPMF), belonging to the Brazilian Agricultural Research Corporation (EMBRAPA), located in the municipality of Cruz das Almas - BA (12º40'12" S; 39º06'07" W; 220 m altitude) The climate is Aw and Am, classified in the Koppen classification as tropical hot and humid. The cultivar used for the study was the ‘Prata Gorutuba’ (Musa AAB ‘Prata Anã’ clone: Gorutuba). The treatments used in this experiment were obtained by the combination of two soils of distinct texture (sandy clay and sandy frank sand) and three concentrations (3.0, 6.0 and 9.0 g L-1) for application of potassium nitrate in the irrigation water for the banana tree ‘Prata Gorutuba’, totaling six treatments (sandy clay soil with 3 g L-1 (T1), sandy clay soil with 6. 0 g L-1 (T2), sandy clay soil with 9.0 g L-1 (T3), free sand soil with 3.0 g L-1 (T4), free sand soil with 6.0 g L-1 (T5), and free sand soil with 9.0 g L-1 (T6), which were distributed in an experimental design entirely randomized with four repetitions. The repetitions were composed of the four radial planes, containing TDR probes and solution extractors (installed at 0.20, 0.40, 0.60 and 0.80 m distance and depth (r,z) on each lysimeter. The lysimeters have a capacity of 5.0 m3, measuring 2.0 m wide, 2.5 m long and 1.0 m deep. To create a free drainage system, the last 0.2 m of the profile was divided in two layers of 0.1 m, the lower one composed of a drainage system with perforated PVC tubes at 0.05 m and zero gravel, and the upper one with washed sand. Samples of the soils were taken at depths of 0.0 - 0.4 m before the plantation of the banana tree and sent to the soil and nutrition laboratory of Embrapa - CNPMF for chemical analysis (Table 1). Table 2 shows the physical characteristics of the two types of soil used in lysimeters. Table 2 shows the physical characteristics of the two types of soil used in lysimeters. The usual practices of management of a banana tree (thinning, defoliation, propping, heart pruning, control of diseases, pests and invaders) were carried out according to need, according to Borges and Souza (2004). 2. MATERIAL AND METHODS Fertigation was performed weekly, during which nitrogen (N) and potassium (K) were applied in the form of potassium nitrate, obeying the following distribution: Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Beatriz Santos Conceição et al. 4 10% in the first three months of planting, 75% from the third to the eighth month, in the bloom phase, and 15% from this until harvest, with applications in concentrations 3, 6 and 9g/L (Borges and Souza, 2004). Table 1. Chemical characterization of soils studied. Characteristics Sandy-clay soil Sandy soil pH 5.0 7.6 P (mg d-3) 2 28 K (cmolc dm-3) 0.06 0.06 Ca (cmolc dm-3) 0.68 1.41 Mg (cmolc dm-3) 0.46 0.30 Ca+Mg (cmolc dm-3) 1.14 1.71 Al (cmolc dm-3) 0.5 0.0 Na (cmolc dm-3) 0.33 0.02 H+Al (cmolc dm-3) 3.19 0.00 SB 1.53 1.84 CTC 4.72 1.84 V (%) 32.0 100 MO (g kg-3) 12.0 9.0 Table 2. Physical characteristic of the lysimeter soil. Characteristics Soil layers (m) 0.0-0.2 0.2-0.4 0.4-0.6 0.6-0.8 0.0-0.2 0.2-0.4 0.4-0.6 0.6-0.8 Sandy soil Sandy-clay soil Ps (%) 36.47 31.49 35.49 35.97 43.93 44.49 48.33 45.56 BD (g cm-3) 1.62 1.76 1.71 1.66 1.41 1.46 1.34 1.40 Sand (%) 84.65 84.80 84.90 83.90 51.40 47.60 51.40 47.00 Silt (%) 8.75 8.65 8.50 9.55 10.10 9.90 7.10 8.50 Clay (%) 6.60 6.55 6.60 6.55 38.50 42.50 41.50 44.50 Ps: porosity; BD: bulk density. Table 1. Chemical characterization of soils studied. Characteristics Sandy-clay soil Sandy soil pH 5.0 7.6 P (mg d-3) 2 28 K (cmolc dm-3) 0.06 0.06 Ca (cmolc dm-3) 0.68 1.41 Mg (cmolc dm-3) 0.46 0.30 Ca+Mg (cmolc dm-3) 1.14 1.71 Al (cmolc dm-3) 0.5 0.0 Na (cmolc dm-3) 0.33 0.02 H+Al (cmolc dm-3) 3.19 0.00 SB 1.53 1.84 CTC 4.72 1.84 V (%) 32.0 100 MO (g kg-3) 12.0 9.0 Table 1. Chemical characterization of soils studied. Table 2. Physical characteristic of the lysimeter soil. Table 2. Physical characteristic of the lysimeter soil. Characteristics Soil layers (m) 0.0-0.2 0.2-0.4 0.4-0.6 0.6-0.8 0.0-0.2 0.2-0.4 0.4-0.6 0.6-0.8 Sandy soil Sandy-clay soil Ps (%) 36.47 31.49 35.49 35.97 43.93 44.49 48.33 45.56 BD (g cm-3) 1.62 1.76 1.71 1.66 1.41 1.46 1.34 1.40 Sand (%) 84.65 84.80 84.90 83.90 51.40 47.60 51.40 47.00 Silt (%) 8.75 8.65 8.50 9.55 10.10 9.90 7.10 8.50 Clay (%) 6.60 6.55 6.60 6.55 38.50 42.50 41.50 44.50 Ps: porosity; BD: bulk density. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 CEw = CEa−(aθ+b) cθ−d 2. MATERIAL AND METHODS The development phases of the banana tree were: initial: 15/11/2014 to 04/01/2015 - 1 to 138 days after planting (DAP); vegetative growth: 02/04 to 07/20/2015 - 139 to 248 DAP; blooming: 21/07/2015 to 09/07/2015 - 249 to 297 DAP fruit growth: 07/09/2015 to 12/02/2015 - 298 to 383 DAP. The emission of the banana tree inflorescence planted on sandy clay soil occurred on 07/21/2015 and on sandy soil on 08/10/2015. The irrigation management was carried out based on the data of water content measured using TDR probes, while calculating the volume of water necessary to return the water content values to the field capacity. The irrigation was performed with a fixed irrigation shift and an interval of two days, therefore making the irrigation water depth variable, and adopting as reference the data of water availability in the soil. To determine the volumetric water content, from the apparent dielectric constant (Ka), the calibration of the TDR for the soils was previously performed according to (Equation 1) and (Equation 2). θAreia franca = 4. 10−5ε3 – 0.0012ε2 + 0.0277ε – 0.1238 (1) θArgiloarenoso = 6.10−5ε3 −0.0032ε2 + 0.0631ε −0.2422 (2) (1) (2) θArgiloarenoso = 6.10−5ε3 −0.0032ε2 + 0.0631ε −0.2422 Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Simulation of nitrate and potassium concentrations in … 5 Where, Where: ε - dielectric constant of the soil. After adjusting the parameters of the models that relate CEw, CEa and θ it was possible to relate CEw to the specific concentration of a certain ion (Ci) present in the soil solution using (Equation 8) and (Equation 9) (Santana et al., 2007; Muñoz-Carpena et al., 2005): Potential type CEw-Ci relationship: 𝐶𝐸𝑤=  𝛼𝐶𝑖𝜇 (8) CEw-Ci ratio of linear type: (9) 𝐶𝐸𝑤=  𝛼+ 𝜇𝐶𝑖 Where: Ci - is the nitrate concentration (mg L-1) Ci - is the nitrate concentration (mg L-1) Ci - is the nitrate concentration (mg L-1) Ci - is the nitrate concentration (mg L-1) CEw - is the electrical conductivity of the soil solution (dS m-1) and α e µ - are adjustment parameters of the equation. CEw - is the electrical conductivity of the soil solution (dS m-1) and α e µ - are adjustment parameters of the equation. The concentrations of nutrients were determined by means of (Equation 10), (Equation 11), (Equation 12) and (Equation 13). The concentrations of nutrients were determined by means of (Equation 10), (Equation 11), (Equation 12) and (Equation 13). Model de Vogeler et al. (1996) adapted for the potential-type CEw-Ci relationship: Model de Vogeler et al. (1996) adapted for the potential-type CEw-Ci relationship: 𝐶𝑖= { 1 𝜇 [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 } 1 𝜇 (10) 𝐶𝑖= { 1 𝜇 [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 } 1 𝜇 𝐶𝑖= { 1 𝜇 [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 } 1 𝜇 (10) (10) Model of Vogeler et al., (1996) adapted for the linear-type CEw-Ci relationship: Model of Vogeler et al., (1996) adapted for the linear-type CEw-Ci relationship: 𝐶𝑖= { [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 −𝛼} 1 𝜇 (11) 𝐶𝑖= { [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 −𝛼} 1 𝜇 (11) 𝐶𝑖= { [𝐶𝐸𝑎−(𝑎𝜃−𝑏)] 𝑐𝜃−𝑑 −𝛼} 1 𝜇 (11) Model of Muñoz-Carpena et al. (2005) adapted for the potential-type CEw-Ci relationship: Model of Muñoz-Carpena et al. (2005) adapted for the potential-type CEw-Ci relationship: 𝐶𝑖= { 1 𝜇( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃)} 1 𝜇 (12) 𝐶𝑖= { 1 𝜇( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃)} 1 𝜇 (12) 𝐶𝑖= { 1 𝜇( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃)} 1 𝜇 (12) Model of Muñoz-Carpena et al. (2005) adapted for the linear-type CEw-Ci relationsh Model of Muñoz-Carpena et al. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 ε - dielectric constant of the soil. ε - dielectric constant of the soil. The precipitation was measured by means of a rain gauge installed in the area near the lysimeter, and was included in the irrigation calculation. The water depth drained in the lysimeters were measured at the outlets of the drainage systems. This way the water storage in the soil was calculated for the four radial planes (0.20 m x 0.20 m), corresponding to the horizontal and vertical distances (0.20; 0.40; 0.60 and 0.80 m) using (Equation 3): A(z) = ∫θ(z) L 0 dz (3) Where: θ (z) = is the representative function of the moisture profile (L3 L-3); z = vertical coordinate (L); z = vertical coordinate (L); L = is the total depth (L); L = is the total depth (L); A(z) = water storage in the soil (L). A(z) = water storage in the soil (L). The Simpson rule was used to solve the numerical integral. The evapotranspiration of the crop was obtained by means of (Equation 4). The soil-water balance was carried out with the water depth infiltrated and extracted from the system in the times immediately before the day’s irrigation, after irrigation and before the next irrigation. ΔA = I + P −D −ET (4) In which: ΔA= storage variation (L); I= irrigation (L); I= irrigation (L); P = precipitation (L); P = precipitation (L); D= drainage (L); ET= Evapotranspiration (L). The mathematical models developed by Vogeler et al. (1996) and Muñoz-Carpena et al. (2005) related the electrical conductivity of the soil solution (CEw), apparent electrical conductivity (CEa) and soil moisture (θ), (Equation 5) and (Equation 6), respectively. CEw = CEa−(aθ+b) cθ−d (5) CEw = CEa−cθ2 aθ2−bθ (6) CEw = CEa−(aθ+b) cθ−d (5) CEw = CEa−cθ2 aθ2−bθ CEw = CEa−cθ2 aθ2−bθ (6) Where: a, b, c and d - equation adjustment parameters. 6 Beatriz Santos Conceição et al. The CEa conductivity values were corrected for a temperature of 25ºC by multiplying them by the correction factor (fT), which was calculated with (Equation 7) (Richards, 1954). fT = 1 + (25−T) 49.7 + (25−T)2 3728 (7) Where: T = Soil temperature, °C. ε - dielectric constant of the soil. (2005) adapted for the linear-type CEw-Ci relationship: 𝐶𝑖= {( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃) −𝛼} 1 𝜇 𝐶𝑖= {( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃) −𝛼} 1 𝜇 (13) 𝐶𝑖= {( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃) −𝛼} 1 𝜇 (13) 𝐶𝑖= {( 𝐶𝐸𝑎−𝑐𝜃2 𝑎𝜃2−𝑏𝜃) −𝛼} 1 𝜇 (13) (13) The calibration of Equations 10, 11, 12 and 13 was performed through a lysimeter test, in which different values of electrical conductivity and water content of the soil were established. y The values of electrical conductivity in the soil were obtained from the application of potassium Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Simulation of nitrate and potassium concentrations in … 7 nitrate solution in different concentrations (3, 6 and 9 g L-1) and the water content of the soil, which varied in values from borderline saturation to the minimum water content necessary for the extractor to remove the solution. nitrate solution in different concentrations (3, 6 and 9 g L-1) and the water content of the soil, which varied in values from borderline saturation to the minimum water content necessary for the extractor to remove the solution. The soil solution was extracted by applying a suction of 70 kPa four hours after the soil solution samples were collected. While the extractor removed the solution to determine the electrical conductivity of the soil solution (CEw), the CEa and θ readings were monitored every 10 minutes with the TDR, starting at the time of suction application and ending at the time of solution collection. The electrical conductivity of the soil solution and the leached volume of the lysimeters were evaluated with the bench conductivimeter. The concentrations of nitrate and potassium present in these samples were monitored using the rapid determination kit (Card Horiba), according to Coelho et al. (2014). The adjustment of the equations to the data and the evaluation of the models were based on the coefficient of determination (R2) (Equation 14), Willmott's concordance index (d) (Equation 15) and the root-mean-square error (RMSE) (Equation 16). ε - dielectric constant of the soil. R2 =  1 − SQR SQtot (14) R2 =  1 − SQR SQtot (14) d = 1 −[∑ (Ei −Oi) n i=1 2 / ∑ (|Ei −O| + |Oi + O| n i=1 )2] (15) 𝑅𝑀𝑆𝐸= √ 1 𝑛∑ (𝑂𝑖−𝐸𝑖)2 𝑛 𝑖=1 (16) R2 =  1 − SQR SQtot (14) d = 1 −[∑ (Ei −Oi) n i=1 2 / ∑ (|Ei −O| + |Oi + O| n i=1 )2] (15) 𝑅𝑀𝑆𝐸= √ 1 𝑛∑ (𝑂𝑖−𝐸𝑖)2 𝑛 𝑖=1 (16) (15) (16) Where: Where: SQR = residual sum of squares; SQR = residual sum of squares; SQtot = sum of total square; SQtot = sum of total square; n= number of data; Oi= observed value; 𝑂̅= average of estimated value; Ei= estimated value. Ei= estimated value. In addition to these statistical indicators, Pearson's correlation coefficient (r) (Figueiredo Filho and Silva Júnior, 2009) and the "c" index (according to the methodology presented by Ponciano, 2012) were also used to indicate the performance of the model. The “c” index brings together the correlation coefficient (r) and the agreement in (Equation 17), expressed as follows: c = r x d (17) (17) Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 c = r x d The criterion for interpreting the performance of the model according to index "c" (Equation 17) is: > 0.85 - Great; 0.76 to 0.85 - Very good; 0.66 to 0.75 - Good; 0.61 to 0.65 - Average; 0.51 to 0.60 - Sufferable; 0.41 to 0.50 - Bad and ≤ 0.41 - Very bad. After adjusting the parameters of the models and identifying the one that best explains the relation between CEw, CEa and θ in conjunction with the specific concentration of NO3- and K+ present in the soil solution, the model selected in the lysimeter can be gradually validated during fertigation by collecting weekly extractions of the soil solution in the phases of Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Beatriz Santos Conceição et al. 8 vegetative growth, flowering and fruit growth of the banana tree. vegetative growth, flowering and fruit growth of the banana tree. For the evaluation of the simulated and observed values, the following statistical indexes were used: the concordance coefficient (d) (Equation 15), proposed by Willmontt (1981); the root-mean-square error (RMSE) (Equation 16); the degree of adjustment of the dependent variable (Y) with the independent variable (X), by establishing an equation of the type Y = βX (“β’’ is the angular coefficient); and the correlation coefficient of Pearson (r). The modeling of water flow and solute transport was performed for all experimental plots with the Hydrus-2D software (Šimůnek et al., 2008), which simulated the three-dimensional axisymmetric and transient movement of water and nutrients in the soil. The transport domain was defined as a rectangle with a width of 100 cm (half the width of the lysimeter) and a depth of 100 cm, as the sides of the flow domain the boundary conditions were defined as without water or nutrient flow. At the upper boundary of the flow domain the boundary condition was defined as an atmospheric boundary condition (variable flow condition of daily irrigation events, rainfall, and evapotranspiration), while at the lower boundary the condition was defined as free drainage, with 4 observation points at depths of 20, 40, 60 and 80 cm inserted in the transport domain (Figure 1). Figure 1. Location of the observation points in the soil profile and flow domain geometry with the boundary conditions used in the Hydrus-2D simulations. Figure 1. c = r x d Parameters of nitrate and potassium transport: delay factor (R), diffusive dispersiv coefficient (D), number of Peclet (P), adsorption coefficient (KD) and (l). The methods of water absorption and root solute were determined by the model of Feddes et al. (1978) (Equation 18) for water absorption and for solute as a model without solute deficit: The methods of water absorption and root solute were determined by the model of Feddes et al. (1978) (Equation 18) for water absorption and for solute as a model without solute deficit: 𝑊𝑈(ℎ, 𝑟, 𝑧) = 𝛾(ℎ) ∗𝑅𝐷𝐹(𝑟, 𝑧) ∗𝑊∗𝑇𝑝𝑜𝑡 (18) (18) Where: γ(h) = water deficit function in the soil (adimensional); RDF = normalized distribution of water absorption by the roots (T); Tpot = potential perspiration rate (L T-1); Tpot = potential perspiration rate (L T-1); W= radius of soil surface associated with perspiration (L). c = r x d Location of the observation points in the soil profile and flow domain geometry with the boundary conditions used in the Hydrus-2D simulations. Figure 1. Location of the observation points in the soil profile and flow domain geometry with the boundary conditions used in the Hydrus-2D simulations. The Hydrus-2D numerically solves the Richards equation describing water flow variability in a radially symmetrical domain as well as the convection-dispersion equation for the transport of solutes. The hydraulic properties of soil used to describe water flow were described using the van Genuchten-Mualem model (van Genuchten, 1980), (Table 3). The distribution of solutes between the solid and liquid phases was described using a linear adsorption isothermal model. The parameters involved in the transport of the solute (coefficient of diffusion-dispersion D, coefficient of retardation, R and velocity of water in the pore) were obtained by the method of miscible displacement (Table 4). Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Simulation of nitrate and potassium concentrations in … 9 Table 3. Parameters of the van Genuchten model for the two soils (Sandy-soil and Clay-sand) used in lysimeters. Soil Soil layer θr (cm3 cm-3) θs (cm3 cm-3) 𝛼 (cm-1) N Ks (mm day-1) Sandy Soil 0.00-0.20 0.043 0.365 0.235 2.297 28.94 0.20-0.40 0.037 0.315 0.612 1.605 81.77 0.40-0.60 0.042 0.354 0.339 2.100 243.51 0.60-0.80 0.030 0.360 0.598 1.761 243.51 Clay sandy 0.00-0.20 0.174 0.439 0.133 1.982 20.88 0.20-0.40 0.198 0.483 0.105 2.338 58.72 0.40-0.60 0.179 0.483 0.201 1.811 85.37 0.60-0.80 0.189 0.456 0.128 1.841 85.37 Table 4. Parameters of nitrate and potassium transport: delay factor (R), diffusive dispersive coefficient (D), number of Peclet (P), adsorption coefficient (KD) and (l). Nitrate Conc. Sandy soil Sandy clay soil R KD D (cm² h-1) P(e) λ (cm) R KD D (cm² h-1) P(e) λ (cm) 3 1.33 0.07 59.48 13.02 1.72 1.63 0.20 92.19 5.02 4.47 6 1.40 0.08 73.70 10.93 2.05 1.68 0.22 76.18 6.34 3.54 9 1.68 0.14 43.80 17.36 1.29 1.71 0.23 64.23 7.16 3.13 Potassium 3 1.71 0.14 27.12 28.56 0.79 1.96 0.31 73.14 6.33 3.55 6 1.75 0.15 54.01 14.91 1.51 1.93 0.30 35.66 13.54 1.66 9 1.92 0.18 56.44 13.47 1.67 2.11 0.36 46.07 9.99 2.25 Table 3. Parameters of the van Genuchten model for the two soils (Sandy-soil and Clay-sand used in lysimeters. Parameters of the van Genuchten model for the two soils (Sandy-soil and Clay-sand) ysimeters Table 4. WU= function of water absorption and solute by the roots (T). WU= function of water absorption and solute by the roots (T). The parameters used by the model to simulate the potential of water extracted by the roots are presented in (Table 5). For such parameters, Hydrus assumes its nomenclature. The method of Feddes (1978) was applied. It relates the amount of water extracted from the soil as a function of root depth, with variables adopted by the software standard. Under optimal humidity conditions, the extraction of water by the root zone is equal to the potential perspiration rate. For the items in the (Table 5), consider that P2H = limit value for stress, below which roots cannot extract water at the maximum possible rate (assuming the potential rate of r2H); P2L = the same as above with potential perspiration rate of r2L; r2H = is the potential perspiration rate Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 10 Beatriz Santos Conceição et al. fixed at 0.5 cm day-1 and r2L = potential perspiration rate fixed at 0.1 cm per day-1. Table 5. Parameters used by the model to simulate the potential of water extracted by the root. Hydrus parameter P0 (cm) P0pt P2H P2L P3 r2H r2L cm cm.d-1 Adopted value -10 -25 -300 -1500 -8000 0.5 0.1 Table 5. Parameters used by the model to simulate the potential of water extracted by the root. In the Hydrus model, a database for different plants regarding the parameters of water absorption by the roots is provided based on studies by Taylor and Ashcroft (1972). In Hydrus, the model of solute absorption by the roots was also selected, for which it was assumed that the absorption of solutes was proportional to the product of the flow of water absorbed by the roots and the concentration of the solute dissolved in water. The simulations along the lysimeter drainage profile were performed for the different phases of the crop for a total of 372 days, and compared the nitrate and potassium concentrations simulated by the Hydrus model during fertigation through the data observed from the extraction of the soil solution in the phases of vegetative growth, flowering and fruit growth of the banana tree. The procedure for minimizing errors between measured and simulated data was outlined in the calibration using an iterative process in which the values of the model parameters were changed with each attempt. WU= function of water absorption and solute by the roots (T). The scenarios considered the variations of: the water content obtained from the TDR sensors installed in the lysimeter; the hydraulic properties; time; the solute transport parameters and the initial concentrations of the solute. These were used as a basis for changes and adjustments in the model to make it safer to allow a better understanding of the water and nutrient dynamics involved, as well as the prediction of future scenarios. The observation period used for the calibration was the initial phase of the banana tree. During this phase, manual calibration was used to estimate the values of the parameters that best represented the observed values. Once the parameters of the model had been calibrated, it was validated for the other periods of banana cultivation. After the simulations with the Hydrus-2D computer model, the following statistical indexes were used: Willmontt’s (1981) concordance coefficient (d) (Equation 15); the root- mean-square error (RMSE) (Equation 16); the degree of adjustment of the dependent variable (Y) with the independent variable (X), by establishing an equation of the type Y = βX ( “β’’ is the angular coefficient); and the correlation coefficient of Pearson (r) to compare the mean values of nitrate and potassium measured in the soil solution and simulated by the Hydrus-2D software. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 3. RESULTS AND DISCUSSION The models of Vogeler et al. (1996) and Muñoz-Carpena et al. (2005) presented low RMSE (<0.1920) and agreement index (d) values close to the unit (0.9999). The model of Vogeler et al. (1996) showed a higher coefficient of regression determination for the soils under study, which led to a better classification (Very good: sandy soil and sandy clay) as to the ability to estimate CEw from CEa and θ, when compared to the model of Muñoz- Carpena et al. (2005) (Good: sandy soil and sandy clay) (Table 6). These results are corroborated by those of Ponciano (2012), who observed that in CEw's estimate the model of Vogeler et al. (1996) adjusts better to soils with clay texture characteristics both by the application of KCl and by the application of KNO3 via irrigation water, and in sandy soil the models from Vogeler et al. (1996) and Munõz-Carpena et al. (2005) presented a similar quality of adjustments being classified as "Very good". Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 11 Simulation of nitrate and potassium concentrations in … Table 6. Models for CEw estimation of sandy clay and sandy soils. Table 6. Models for CEw estimation of sandy clay and sandy soils. 1 1 Soil Model R² r d RMSE Classif. of the Method (c = r x d) Sandy Soil Vogeler et al., (1996) CEw = [CEa + 2.5438θ −0.3730] -0.0871 θ + 0.7388 0.818 0.859 0.998 0.1677 Very good Muñoz-Carpena et al., (2005) CEw = CEa + 19.6472 θ2 −41.3941 θ2 + 19.4093 θ 0.754 0.871 0.999 0.1537 Good Sandy clay Vogeler et al., (1996) CEw = [CEa −0.0315θ −0.1111] -0.4152 θ + 0.4264 0.779 0.883 0.999 0.1538 Very good Muñoz-Carpena et al., (2005) CEw = CEa −1.0981 θ2 −10.0371 θ2 + 4.3187 θ 0.655 0.830 0.999 0.1920 Good 1 Model R² r d RMSE Classif. of the Method (c = r x d) il Vogeler et al., (1996) CEw = [CEa + 2.5438θ −0.3730] -0.0871 θ + 0.7388 0.818 0.859 0.998 0.1677 Very good Muñoz-Carpena et al., (2005) CEw = CEa + 19.6472 θ2 −41.3941 θ2 + 19.4093 θ 0.754 0.871 0.999 0.1537 Good y Vogeler et al., (1996) CEw = [CEa −0.0315θ −0.1111] -0.4152 θ + 0.4264 0.779 0.883 0.999 0.1538 Very good Muñoz-Carpena et al., (2005) CEw = CEa −1.0981 θ2 −10.0371 θ2 + 4.3187 θ 0.655 0.830 0.999 0.1920 Good h soils, there was a positive correlation (r+) for the estimation of nitrate and potassium concentration in the soil solution from water (θ) and apparent electrical conductivity (CEa) when a linear or power type relation is introduced to the adapted models (Vogeler et unõz-Carpena et al., 2005). This can be seen in the slight differences between the determination coefficients, thus presenting the same n for the "c" index for the CEw-NO3- ratio of the linear type and power type (Table 7), which was also observed in the CEw-K+ ratio type and power type (Table 8). ral, despite the concordance index (d) close to the unit (0.9999), the model of Vogeler et al. (1996) was superior to the model of pena et al. (2005), and always presented lower RMSE and higher coefficient of determination (R2). This led the model of Vogeler et o present a better classification according to the "c" index for the models estimating nitrate and potassium concentration (mg L-1) for nd sandy clay (Table 7 and 8). ults of this study agree with those obtained by Santana et al. Table 6. Models for CEw estimation of sandy clay and sandy soils. (2006), who reported that, regarding the models used, Rhoades et al., Vogeler et al. (1996) provided the best estimates of K concentration in soil solution. For both soils, the model of Vogeler et al. (1996) he linear type CEw-Ci ratio (Equation 9) was chosen to estimate the concentration of nitrate and potassium in the soil solution after le of banana crop in the drainage lysimeter. Model For both soils, there was a positive correlation (r+) for the estimation of nitrate and potassium concentration in the soil solution from water content data (θ) and apparent electrical conductivity (CEa) when a linear or power type relation is introduced to the adapted models (Vogeler et al., 1996; Munõz-Carpena et al., 2005). This can be seen in the slight differences between the determination coefficients, thus presenting the same classification for the "c" index for the CEw-NO3- ratio of the linear type and power type (Table 7), which was also observed in the CEw-K+ ratio of the linear type and power type (Table 8). In general, despite the concordance index (d) close to the unit (0.9999), the model of Vogeler et al. (1996) was superior to the model of Muñoz-Carpena et al. (2005), and always presented lower RMSE and higher coefficient of determination (R2). This led the model of Vogeler et al. (1996) to present a better classification according to the "c" index for the models estimating nitrate and potassium concentration (mg L-1) for sandy soil and sandy clay (Table 7 and 8). The results of this study agree with those obtained by Santana et al. (2006), who reported that, regarding the models used, Rhoades et al., (1976) and Vogeler et al. (1996) provided the best estimates of K concentration in soil solution. For both soils, the model of Vogeler et al. (1996) adapted to the linear type CEw-Ci ratio (Equation 9) was chosen to estimate the concentration of nitrate and potassium in the soil solution after the first cycle of banana crop in the drainage lysimeter. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 12 12 1 Beatriz Santos Conceição et al. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 1 1 1 Table 7. Models for estimation of nitrate concentration (mg L-1) for sandy soil and sandy clay. Soil Model R² r d RMSE Classif. Table 6. Models for CEw estimation of sandy clay and sandy soils. of the Method (c = r x d) Adapted for linear type CEw-NO3- ratio Sandy soil Vogeler et al., (1996) NO3 − = [CEw −0.0283] 1 0.0024 0.7581 0.867 0.9999 66.7749 Very good Sandy soil Muñoz-Carpena et al., (2005) NO3 − = [CEw −0.0860] 1 0.0024 0.6158 0.785 0.9999 90.0959 Average Sandy clay Vogeler et al., (1996) NO3 − = [CEw −0.0955] 1 0.0034 0.8568 0.926 0.9999 35.1413 Great Sandy clay Muñoz-Carpena et al., (2005) NO3 − = [CEw −0.2126] 1 0.0027 0.8352 0.917 0.9995 37.6901 Very good Adapted for power type CEw-NO3- ratio Sandy soil Vogeler et al., (1996) NO3 −= [ 1 0.0038 CEw] 1 0.9257 0.7596 0.868 0.9999 69.1431 Very good Sandy soil Muñoz-Carpena et al., (2005) NO3 −= [ 1 0.0074 CEw] 1 0.8242 0.6212 0.786 0.9998 90.0767 Average Sandy clay Vogeler et al., (1996) NO3 −= [ 1 0.0108 CEw] 1 0.7994 0.8658 0.940 0.9998 32.5036 Great Sandy clay Muñoz-Carpena et al., (2005) NO3 −= [ 1 0.0231 CEw] 1 0.6623 0.8202 0.909 0.9998 39.3716 Very good for estimation of nitrate concentration (mg L-1) for sandy soil and sandy clay. Table 7. Models for estimation of nitrate concentration (mg L-1) for sandy soil and sandy clay Model Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 13 Simulation of nitrate and potassium concentrations in … 1 1 1 Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté Table 8. Models for potassium concentration estimation (mg L-1) for sandy soil and sandy clay. Soil Model R² r d RMSE Classif. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Table 6. Models for CEw estimation of sandy clay and sandy soils. Soil Concentration g L-1 Nitrate Potassium β R2 d RMSE β R2 d RMSE sand and soil 3.0 0.98 0.72 0.99 27.20 0.96 0.69 0.99 6.638 6.0 0.87 0.67 0.99 32.17 0.88 0.72 0.99 7.89 9.0 0.89 0.70 0.99 18.63 0.91 0.74 0.99 9.201 sand and clay 3.0 0.87 0.85 0.99 31.98 0.92 0.67 0.99 10.20 6.0 0.94 0.72 0.99 23.54 0.84 0.71 0.99 16.24 9.0 0.96 0.69 0.99 22.78 0.91 0.74 0.99 15.01 Andrade Neto et al. (2012), while using a potential model combined with the model of Vogeler et al. (1996), reported a general variation of 20.0% (average of normalized errors) for the measured values compared to those estimated in Yellow Latosol with sandy soil texture. According to Andrade Neto and Coelho (2014), these variations in the values may be acceptable in soil ion management due to the spatial variability of soil chemical attributes. Thus, the results shown in (Table 9) validate the model used to estimate the nitrate and potassium ion in soil solution. These results corroborate those of Andrade Neto et al. (2012), who concluded that it is feasible to estimate the concentration of K+ in the soil solution from water content data and CEa, obtained using the time domain reflectometry (TDR) technique for field conditions, while using an equation from the combination of a linear model and a potential. Santos et al. (2009) concluded that the TDR technique, together with the use of adjusted models, proved to be able to monitor the transport of solutes in the soil, regardless of the type of soil in use and the nutrient dosage applied. The transport analysis of solutes contemplated the two-dimensional model. To evaluate the performance of the Hydrus-2D model, a joint analysis with statistical index "d" was performed to verify if there was an agreement between the simulated and experimental values. The value d = 0 shows that there is no agreement and the value d = 1 shows that there is total agreement. It has been noted that in general the simulations performed for the displacement of nitrate and potassium in the soil showed high rates of agreement (d) and the values of RMSE were lower (Table 10) than those observed using the parametric model (Table 9), indicating that the values simulated by the Hydrus-2D software showed less dispersion of data. Table 6. Models for CEw estimation of sandy clay and sandy soils. of the Method (c = r x d) Adapted for linear type CEw-K + ratio Sandy soil Vogeler et al., (1996) K+ = [CEw −0.0274] 1 0.0076 0.7134 0.840 0.9999 23.6440 Good Sandy soil Muñoz-Carpena et al., (2005) K+ = [CEw −0.0731] 1 0.0077 0.5580 0.747 0.9999 29.0023 Sufferable Sandy clay Vogeler et al., (1996) K+ = [CEw + 0.0191] 1 0.0059 0.6563 0.822 0.9994 41.4654 Good Sandy clay Muñoz-Carpena et al., (2005) K+ = [CEw −0.1637] 1 0.0039 0.6222 0.789 0.9999 43.4716 Average Adapted for power type CEw-K + ratio Sandy soil Vogeler et al., (1996) K+ = [ 1 0.0121 CEw] 1 0.9073 0.7165 0.843 0.9999 23.5022 Good Sandy soil Muñoz-Carpena et al., (2005) K+ = [ 1 0.0167 CEw] 1 0.8529 0.5614 0.749 0.9998 28.8929 Sufferable Sandy clay Vogeler et al., (1996) K+ = [ 1 0.0094 CEw] 1 0.9041 0.6430 0.827 0.9964 41.5647 Average Sandy clay Muñoz-Carpena et al., (2005) K+ = [ 1 0.0173 CEw] 1 0.7562 0.6098 0.782 0.9998 44.1795 Average Table 8. Models for potassium concentration estimation (mg L-1) for sandy soil and sandy clay. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 14 Beatriz Santos Conceição et al. During the validation of the model in the drainage lysimeter, there was an underestimation of the nitrate and potassium values predicted as a function of the ion measured in the soil solution (Table 9). In sandy soil, mean nitrate values were underestimated by 2%, 13%, and 11%, and mean potassium values by 4%, 12%, and 9% for concentrations of 3, 6, and 9 g L-1, respectively. In the sandy clay soil, there was also an underestimation of the mean values of nitrate (13%, 6% and 4% for 3, 6 and 9 g L-1) and potassium (8%, 6% and 9% for 3, 6 and 9 g L-1) (Table 9). Table 9. Statistical indications for comparison of mean values of nitrate and potassium measured in the soil solution and estimated by the Vogeler et al. (1996) model adapted to the linear type CEw-Ci ratio. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Table 6. Models for CEw estimation of sandy clay and sandy soils. The “β” indexes confirm the performance of Hydrus-2D software in simulating the concentration of nitrate and potassium ion in the soil solution throughout the crop cycle. All the values of “β” were close to the unit, presenting little difference with an overestimation of 6% of the nitrate values in the concentration of 3 g L-1 (sandy soil) and 7% and 2% of the nitrate values in the concentration of 3 g L-1 and 9 g L-1, respectively (sandy clay soil), while the other simulations in the Hydrus-2D software underestimated the nitrate and potassium values. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 15 Simulation of nitrate and potassium concentrations in … Table 10. Statistical indicators for comparison of mean nitrate and potassium values measured in the soil solution and simulated by Hydrus-2D software. Soil Concentration g L-1 Nitrate Potassium β R2 d RMSE β R2 d RMSE Sandy soil 3.0 1.06 0.65 0.99 18.36 0.96 0.64 0.99 6.99 6.0 0.97 0.57 0.99 14.83 0.92 0.75 0.99 8.85 9.0 0.85 0.68 0.99 22.81 0.95 0.56 0.99 8.01 Sandy clay 3.0 1.07 0.70 0.99 19.06 0.86 0.68 0.99 9.66 6.0 0.93 0.72 0.99 16.07 0.94 0.70 0.99 7.17 9.0 1.02 0.64 0.99 17.38 0.97 0.71 0.99 6.78 Montenegro et al. (2010), Silva et al. (2013), Montenegro et al. (2013), and Pinho and Miranda (2014) also used statistical indices to assess the quality of the fit between measured and simulated values Table 10. Statistical indicators for comparison of mean nitrate and potassium values measured in the soil solution and simulated by Hydrus-2D software. Table 10. Statistical indicators for comparison of mean nitrate and potassium values measured in the soil solution and simulated by Hydrus-2D software. Montenegro et al. (2010), Silva et al. (2013), Montenegro et al. (2013), and Pinho and Miranda (2014) also used statistical indices to assess the quality of the fit between measured and simulated values Montenegro et al. (2010), Silva et al. (2013), Montenegro et al. (2013), and Pinho and Miranda (2014) also used statistical indices to assess the quality of the fit between measured and simulated values. The correlations between the average values of nitrate and potassium measured in the soil solution with the values simulated by the Hydrus-2D software and the values estimated by the Vogeler et al. Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 Table 6. Models for CEw estimation of sandy clay and sandy soils. parameter values required by the model needs precise adjustments to reflect the quality of the simulations, as the amount of input data required increases with the complexity of the simulations, due to the need for larger amounts of input parameters. Table 6. Models for CEw estimation of sandy clay and sandy soils. (1996) model adapted to the linear type CEw-Ci ratio in loam sand and sandy clay soils, may be observed in Figure 2. Figure 2. Average values of nitrate and potassium for concentrations 3, 6 and 9 g L-1, measured in the soil solution with the values simulated by the Hydrus-2D software and the values determined by means of the TDR through the Vogeler et al. (1996) model adapted to the CEw-Ci ratio of the linear type in the sandy soil (a and c) and sandy clay (b and d). Figure 2. Average values of nitrate and potassium for concentrations 3, 6 and 9 g L-1, measured in the soil solution with the values simulated by the Hydrus-2D software and the values determined by means of the TDR through the Vogeler et al. (1996) model adapted to the CEw-Ci ratio of the linear type in the sandy soil (a and c) and sandy clay (b and d). It appears that the measured data differed little from the simulated data, in which both the Vogeler et al. (1996) adapted to the linear type CEw-Ci relationship and Hydrus-2D showed positive correlation (r +) with the measured data, which demonstrates the adequacy of these models for modeling the levels of nitrate and potassium in the soil solution. These results are in agreement with those obtained by Oliveira et al. (2010), Ribeiro et al. (2011) and Pinho and Miranda (2014) who stated that the Hydrus model was efficient in simulating the displacement of potassium and water. In this study, all r values were above 0.70, which shows a high correlation between measured and estimated data (Figure 2), according to the Cohen (1988) classification. Thus, it notes that the mathematical model used and the Hydrus-2D software have been proven as feasible tools that can be used satisfactorily to describe the nitrate and potassium concentration of the soil solution under field conditions. However, the possibility of variations in the Rev. Ambient. Água vol. 16 n. 1, e2606 - Taubaté 2021 16 Beatriz Santos Conceição et al. parameter values required by the model needs precise adjustments to reflect the quality of the simulations, as the amount of input data required increases with the complexity of the simulations, due to the need for larger amounts of input parameters. 4. CONCLUSIONS The mathematical models proved effective in estimating the concentration of nitrate and potassium in the soil solution during the banana crop cycle under field conditions, with greater accuracy and less dispersion for Vogeler et al. (1996) combined with the linear model. The Hydrus-2D computer program proved to be feasible as a good tool for describing the nitrate and potassium concentration of the soil solution under the conditions under which the work was performed. The concentrations of nitrate and potassium in the soil estimated by adapting the Vogeler et al. (1996) model to the linear type CEw-Ci ratio, and simulated by the Hydrus model, presented an acceptable characterization of the distribution of these nutrients and their oscillations in the soil solution during the banana crop cycle, demonstrating satisfactory performance and feasibility of both methods. 5. REFERENCES ANDRADE NETO, T. M.; COELHO, E. F. Concentração de potássio em função da condutividade elétrica da solução do solo. Water Resources and Irrigation Management , v. 3, p. 13-19, 2014. http://dx.doi.org/10.1590/S1415- 43662012000600005 ANDRADE NETO, T. M. de; COELHO, E. F.; SANTANA, J. A. do V.; SANTANA JÚNIOR, E. B.; ALVES, M. da S. Estimativa de potássio na solução do solo baseada na condutividade elétrica e umidade do solo. Revista Brasileira de Engenharia Agrícola e Ambiental, v. 16, n. 6, p. 618-623, 2012. BORGES, A. L.; SOUZA, L. da F. O Cultivo da bananeira. Cruz das Almas: Embrapa Mandioca e Fruticultura, 2004. 279 p. Rev. Ambient. 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Application of latin square design for the evaluation and screening of supplementary nitrogen source for L-asparaginase production by Aspergillus terreus MTCC 1782
Indian journal of science and technology
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cc-by
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50 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) ISSN: 0974- 6846 Application of latin square design for the evaluation and screening of supplementary nitrogen source for L-asparaginase production by Aspergillus terreus MTCC 1782 G. Baskar and S.Renganathan1 Department of Biotechnology, St. Joseph’s College of Engineering, Chennai–600 119, India. Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai - 600 025, India. 1 basg2004@gmail.com; rengsah@rediffmail.com Abstract: The effect of ammonium chloride, urea and sodium nitrate as supplementary nitrogen source on the production of extracellular L-asparaginase using Aspergillus terreus MTCC 1782 was investigated using 5level Latin square design. The statistical reliability and significance of the variables was studied by performing ANOVA for experimental L-asparaginase activity using Dataplot software. Among the supplementary nitrogen sources studied, ammonium chloride was found to be the best for maximum L-asparaginase activity (confidence level of 96.61%) with less biomass formation (confidence level of 21.93%). The maximum L-asparaginase activity obtained was of 26.47 IU/mL by A. terreus MTCC 1782 using groundnut oil cake powder as natural substrate in submerged fermentation. Keywords: Aspergillus terreus, L-asparaginase, latin square design. Introduction L-asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) catalyzes the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This has been a clinically acceptable anti tumor agent for the effective treatment of acute lymphoblastic leukemia and lymphosarcoma. Lasparagine is an essential amino acid for the growth of tumor cells, whereas the growth of normal cells is not dependent on its requirement as it can be synthesized in amounts sufficient for their metabolic needs using their own enzyme L-asparagine synthetase. The presence of L-asparaginase deprives the tumor cells of an important growth factor and they fail to survive (Broome, 1961; Broome, 1965; Berenbaum et al., 1970). Recombinant Lasparaginase of A. niger and A. oryzae used in processing of starchy food products. It converts the amino acid asparagine to aspartic acid then reduces acrylamide formation during processing of high starch food products (FAO/WHO, 2001; Pedreschi et al., 2008). The demand for this enzyme is expected to increase several fold in coming years due to its potential industrial application as food processing aid besides clinical applications. L-asparaginase produced by several bacterial sources leads to adverse side effects in human trials. Therefore, there is a search for the other sour ces for Lasparaginase production with less adverse effects. It has been observed that eukaryotic microorganisms like yeast and filamentous fungi are potential sources for Lasparaginase production. These studies suggest Lasparaginase production by filamentous fungi is under nitrogen regulation (Sarquis et al., 2004). The A. nidulans (Shaffer et al., 1988), A. terreus (Ali et al., 1994; Sarquis et al., 2004) using synthetic substrates and A. niger (Mishra, 2006) using agro-wastes from three leguminous crops were reported to produce L-asparaginase which was not toxic and appeared to have myelosuppressive and immunosuppressive activity. Mesophilic fungus Cylindrocarpon obtusisporum MB-10 was also reported to produce intracellular L-asparaginase (Raha et al., 1990), which was very specific for L-asparagine and did not hydrolyse D-asparagine or L-glutamine. However, only few researchers studied the production of L-asparaginase using naturally available cheaper substrate. It also has been observed from our preliminary study that A. terreus MTCC 1782 was found to be a potential fungal source for L-asparaginase and used for further investigation in this study. Statistical experimental designs have been used in several steps of optimization strategy and it is better acknowledged than traditional one variable at a time method (Kwak et al., 2006; Zheng et al., 2008). Statistical experimental design such as Latin Square Design (LSD) minimizes the error in determining the effect of parameters, which allows simultaneous, systematic, and efficient variation of all parameters than classical method. LSD was first used in agricultural research to adjust for fertility differences in two physical directions (Box et al., 1978; Torbjorn et al., 1998). Identifying a low cost substrate is important to develop an economically viable bioprocess for any product. Groundnut oil cake is used as animal feed, was obtained as waste after extraction oil form groundnut. In the present work the groundnut oil cake powder (particle size of 80/120 mesh) was used as alternate substrate to synthetic L-proline. LSD was used to evaluate and compare the effect of urea, ammonium chloride and sodium nitrate as independent supplementary nitrogen source on production of extracellular L-asparaginase by A. terreus MTCC 1782 in submerged fermentation using groundnut oil cake powder as substrate. Materials and methods Fungal strain The filamentous fungi A. terreus MTCC 1782 was obtained from Institute of Microbial Technology, Chandigarh, India. The spores and mycelial fragments of this fungus was cultivated in Czapek agar slants at 37°C for 4 days, stored at 4°C and periodically subcultured. Research article “L-asparaginase” Indian Society for Education and Environment (iSee) http://www.indjst.org Baskar & Renganathan Indian J.Sci.Technol. 51 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) Ground nut oil cake powder square design The ground nut oil cake used in this work was purchased from local market (Chennai, India), dried overnight at 65°C to remove moisture if any, powdered and sieved. The ground nut oil cake powder passed through 80 and retained by 120 meshes was used as natural substrate for production of L-asparaginase. The statistical experimental designs are used to evaluate, screen and optimize the carbon, nitrogen sources and other medium and operating conditions. In particular the LSD is used to find the best source by evaluating and comparing the effect of various carbon or nitrogen sources (Kwak et al., 2006; Zheng et al., 2008). The effect of independent variables on response follows a linear model and is given by equation 1. The residual standard deviation reflects the effect of variables, smaller the residual standard deviation of a variable, more the Inoculum culture Inoculum culture was cultivated in modified Czapek agar slants with the following ingredients in g. Solution-A: L-asparagine, 1.0; NaNO3, 4.0; KCl, 1.0; MgSO4.7H2O, 0.052; FeSO4.7H2O, 0.02; were dissolved in 100 mL of distilled water and stored in refrigerator. Solution-B: K2HPO4 2.0; was dissolved in 100 mL of distilled water and stored in refrigerator. Solution-C: ZnSO4. 7 H2O 1.0; was dissolved in 100 mL of distilled water. Solution-D: 0.5 gram of CuSO4. H2O 0.5; was dissolved in 100 mL of distilled water. For one litre of modified Czapek-Dox was prepared using 50 mL of solution-A, 50 mL of solution-B, 1 mL of solution-C, solution-D, 30 g of glucose and 20 g of agar mixed in 900 mL distilled water. The inoculum culture slants were incubated at 37°C for 4 days. Inoculum culture as conidial suspensions was prepared with concentrations of 107 to 108 conidia per mL. Table 2. Latin square experimental design with Lasparaginase activity and cell mass Std. Run Variables in coded unit X1 X2 1 1 1 2 2 1 3 3 1 4 4 1 5 5 1 6 3 2 7 4 2 8 5 2 9 1 2 10 2 2 11 5 3 12 1 3 13 2 3 14 3 3 15 4 3 16 2 4 17 3 4 18 4 4 19 5 4 20 1 4 21 2 5 22 5 5 23 1 5 24 2 5 25 3 5 effect on response. Production and isolation of crude enzyme Based on LSD experiment design, 100 mL of liquid Czapek-Dox media modified with different supplementary nitrogen source (Sodium nitrate, Urea and Ammonium chloride), 2% groundnut oil cake powder, 1% Lasparagine, 0.2% glucose, 0.152% K2HPO4, 0.052% KCl, 0.052% MgSO4.7H2O, traces of ZnSO4.7H2O and FeSO4.7H2O was prepared in 250 mL in Erlenmeyer flask. Erlenmeyer flask were inoculated with 1 mL of conidial suspension and submitted to orbital shaking at 160 rpm, 30°C and pH 6.2 for 4 days. Then culture suspension was filtered through whattman-2 filter paper and cell-free filtrate was used as crude enzyme solution for estimation of L-asparaginase activity. Assay of L-asparaginase activity L-asparaginase activity of the crude enzyme solution was determined by Nesslarization, the most commonly used method for estimation of L-asparaginase activity. The quantity of ammonia formed during the hydrolysis of 0.04 M L-asparagine was estimated using Nessler’s Reagent in spectrophotometric analysis at 480 nm. One unit (IU) of L-asparaginase activity is defined as the amount of enzyme which liberates 1 µmole of ammonia per minute under the standard assay conditions (Wriston & Yellin, 2001). Y = µ + RX1 + CX 2 + TX 3 Comparison of supplementary nitrogen sources by latin Table 1. Experimental variables in coded and actual units Experimental variables in actual unit ISSN: 0974- 6846 Coded unit (Level) Sodium nitrate (X1) (%, w/v) Urea (X2) (%, w/v) 1 0 0 2 0.3 0.4 3 0.6 0.8 4 0.9 1.2 5 1.2 1.6 Ammonium chloride (X3) (%, w/v) 0 0.4 0.8 1.2 1.6 X3 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Lasparaginase activity (IU/mL) 22.55 14.82 13.12 12.32 26.45 6.51 14.50 15.25 28.32 20.05 4.74 11.99 19.89 22.39 21.06 15.19 14.72 27.83 22.55 35.46 25.59 19.30 21.54 24.21 29.33 Cell mass (mg/mL) 7.60 29.00 34.00 11.11 9.70 9.20 12.70 8.90 5.31 9.80 7.90 7.40 3.73 12.00 12.30 2.85 2.76 14.40 16.60 6.65 13.50 3.62 8.100 11.50 5.50 (1) Where Y denotes any observation for which, X1 and X2 are blocking factors and X3 is the primary factor. µ denotes the general location parameter, R denotes the residual standard deviation for X1, C denotes the residual standard deviation for X2 and T denotes the residual standard deviation for X3 (Box et al., 1978; Torbjorn et al., 1998). In the present investigation three supplementary nitrogen sources such as urea, sodium nitrate and Research article “L-asparaginase” Indian Society for Education and Environment (iSee) http://www.indjst.org Baskar & Renganathan Indian J.Sci.Technol. 52 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) ISSN: 0974- 6846 sodium nitrate (confidence level of 62.46%). ANOVA in Table 4 shows that ammonium chloride has lesser influence (confidence level of 21.93%) on growth of A. terreus than urea (confidence level of 75.84%) and sodium nitrate (confidence level of 28.04%). Ammonium chloride was found to be the best supplementary nitrogen source for L-asparaginse production with less cell mass formation. The mean, effect and residual standard deviation for L-asparaginase production and cell mass formation in Table 5 and 6 gives the independent effect of supplementary nitrogen sources at different concentration. Urea and ammonium chloride have lesser effect on L-asparaginase production at their low to Table 3. ANOVA on LSD for L-asparaginase activity middle level (concentration) and increases the LDegree Mean Sum of asparaginase production (23.99 and 26.47 IU/mL FConfidence of Factor square squares value level (%) freedom respectively) at their higher level. Sodium nitrate has (MS) (SS) (DF) lesser effect (17.66 IU/mL) at its higher level, higher Sodium effect (23.97 IU/mL) at its lower level. The mean L4 148.108 37.027 1.162 62.46 nitrate asparaginase activity of 23.97 and 23.15 IU/mL were Urea 4 274.921 68.730 2.156 86.38 obtained for modified Czapek-Dox media supplemented with urea and sodium nitrate Ammonium 4 475.985 118.996 3.733 96.61 chloride respectively was comparatively low when ammonium chloride was used at high level. Urea has high influence Residual 12 382.532 31.877 (18.28 mg/mL) on growth at its low level (effect > 0) and Total 24 1281.546 53.397 the least effect (minimum of 8.44 mg/mL) at its middle Table 4. ANOVA on LSD for cell mass and higher level (effect < 0) with low and constant Degree Sum of Mean Confidence standard deviation among the variables and their Fof Factor squares square level levels. Ammonium chloride and sodium nitrate have the value freedom (SS) (MS) (%) least influence on cell mass formation (8.21 and 7.01 (DF) Sodium mg/mL respectively) at their low level and higher level 4 121.342 30.335 0.525 28.04 nitrate and they give higher growth at their middle level (13.83 and 12.69 mg/mL respectively). Among the Urea 4 365.985 91.496 1.583 75.84 supplementary nitrogen sources studied at different Ammonium 4 100.633 25.158 0.435 21.93 concentrations, modified Czapek-Dox media with chloride groundnut oil cake and supplemented with ammonium Residual 12 693.409 57.784 chloride gives maximum L-asparaginase production (26.47 IU/mL) with less cell mass formation (8.79 Total 24 1281.371 53.390 mg/mL). Results and discussion The linear model The experimental L-asparaginase Table 5. Effect of supplementary nitrogen represents the effect of activity and cell mass were analyzed source on L-asparaginase activity independent variables on Lto study and evaluate the independent SD Factor Level Mean Effect asparaginase activity and cell effect of urea, ammonium chloride and (Effect) mass were given in equation 2 1 23.97 4.385 2.258 sodium nitrate as supplementary and 3, and the Residual 2 18.83 -0.755 2.258 nitrogen source on L-asparaginase Sodium Standard Deviation (RSD) of 3 17.21 -2.376 2.258 synthesis by A. terreus MTCC 1782 nitrate model parameters were given 4 20.26 0.674 2.258 using groundnut oil cake powder using in Table 7. Lesser the RSD 5 17.66 -1.927 2.258 Data plot Software. ANOVA (a formal higher will be the significance 1 17.85 -1.736 2.258 F-test) in Table 3 gives the F-value of the variable for maximization 2 16.92 -2.664 2.258 and confidence level of independent problem (Box et al., 1978) and Urea 3 16.02 -3.571 2.258 variables. Higher the F-value higher 4 23.15 3.564 2.258 vice versa for minimization. will be the confidence level (Zheng et 5 23.99 4.406 2.258 Yactivity = 7.307 + 6.346X 3 (2) al., 2008). It was observed that 1 14.92 -4.669 2.258 ammonium chloride has higher 2 15.06 -4.519 2.258 Ybiomass = 7.306+ 7.683X 3 (3) influence (confidence level of 96.61%) Ammonium 3 19.53 -0.062 2.258 chloride The RSD of ammonium on L-asparaginase production than 4 21.95 2.369 2.258 chloride (X3) was low (6.346) urea (confidence level of 86.38 %) and 5 26.47 6.881 2.258 ammonium chloride were explored at 5-level to study their independent effect on L-asparaginase production by A. terreus MTCC 1782. Groundnut oil cake powder was used as substrate in shake culture fermentation. Table 1 gives the coded and actual values of the variables. 5-level LSD for three variables was developed using Data plot software (Standard Engineering Division, NIST, Gaithersburg, MD 20899-8980) is given in Table 2. Experiments were conducted for L-asparaginase production by A. terreus MTCC 1782 as mentioned in production and isolation step. The analysis of variance (ANOVA) is applied to find the significance of the factors by providing estimates of grand mean and factor effects. Research article “L-asparaginase” Indian Society for Education and Environment (iSee) http://www.indjst.org Baskar & Renganathan Indian J.Sci.Technol. 53 Indian Journal of Science and Technology for L-asparaginase production and high (7.683) for cell mass formation. Ammonium chloride was validated as the best supplementary nitrogen source for maximum L-asparaginase production with less cell mass formation by A. terreus MTCC 1782 using Czapek-Dox media modified by groundnut oil cake as substrate. The Dex-Mean plot is a Graphical ANOVA, used to evaluate the effect of independent variables on response. Fig.1 and 2 shows the effect of supplementary nitrogen sources (Sodium nitrate - X1, Urea - X2, Ammonium chloride - X3) on Lasparaginase production and Vol.2 No. 12 (Dec. 2009) Table 6. Effect of supplementary nitrogen source on cell mass formation Factor Level Mean Effect 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 7.01 11.37 12.69 12.80 9.34 18.28 9.18 8.66 8.65 8.44 8.21 11.09 13.83 11.31 8.79 -3.633 0.731 2.046 2.156 -1.301 7.636 -1.463 -1.979 -1.993 -2.201 -2.435 0.451 3.181 0.658 -1.855 Sodium nitrate Urea Ammonium chloride Table 7. RSD of model parameters for L-asparaginase activity and cell mass Model Parameter Constant (µ) Sodium nitrate (X1) Urea (X2) Ammonium chloride (X3) Constant and all factors 4. Residual standard deviation (RSD) 5. L-asparaginase Cell mass activity formation 7.307 7.306 7.528 7.615 7.094 6.765 6.346 7.683 5.646 7.602 6. cell mass formation. Ammonium chloride was found to be primary supplementary nitrogen source for maximum Lasparaginase production (Fig.1) with low cell mass formation (Fig.2) by A. terreus MTCC 1782 using CzapekDox media modified with groundnut oil cake powder. The residual was plotted against normal distribution and forms an approximate linear line for both Lasparaginase production (Fig.3) and cell mass formation (Fig. 4), which indicates that the model was well fitted with the experimental results. As the residuals from the fitted model are normally distributed, all the major assumptions of the model have been validated. 7. SD (Effect) 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 3.041 ISSN: 0974- 6846 the production of L-asparaginase by A. terreus MTCC 1782 using a a low-cost substrate groundnut oil cake. References 1. Ali SS, Rai V, Soni K, Kulshrestha P and Lai SK (1994) A fungal Lasparaginase with potential antitumor activity. Ind. J. Microbiol. 34, 73–76. 2. Berenbaum MC, Ginsburg H and Gilbert DM (1970) Effects of L-asparaginase on lymphocyte target cell reactions In vitro. Nature. 227, 1147–1148. 3. Box GEP, Hunder WG and Hunder SJ (1978) Statistics for experiments, John Wiley & Sons Inc., New York. Broome JD (1961) Evidence that the L-asparaginase activity of Guinea pig serum is responsible for its antilymphoma effects. Nature. 171, 1114–1115. Broome JD (1965) Antilymphoma activity of Lasparaginase in vivo: Clearance rates of enzyme preparations from Guinea pig serum and yeast in relation to their effect on tumor growth. J. Natl. Cancer Inst. 35, 967–974. Joint FAO/WHO Expert Committee on Food Additives (JECFA) Compendium of Food Additive Specifications (2001) General specifications and considerations for enzyme preparations used in food processing, FAO Food & Nutrition. 52(9), 37–39. Kwak KO, Jung SJ, Chumg SY, Kang CM, Huh YI and Bae S O (2006) Optimization of culture conditions for CO2 fixation by chemoautotrophic Conclusion LSD was successfully applied to find the best supplementary nitrogen source for L-asparaginase production by A. terreus MTCC 1782 using groundnut oil cake power as low-cost substrate. It was found that LSD is an effective statistical tool to evaluate and compare the various carbon or nitrogen sources and find the best for maximum Lasparaginase production. Among the supplementary nitrogen sources studied, ammonium chloride was found to be the best for Fig.1. Effect of supplementary nitrogen sources on L-asparaginase production Research article “L-asparaginase” Indian Society for Education and Environment (iSee) http://www.indjst.org Baskar & Renganathan Indian J.Sci.Technol. 54 Indian Journal of Science and Technology Vol.2 No. 12 (Dec. 2009) microorganism strain YN-1 using factorial design. Biochem. Eng. J. 31, 1–7. 8. Mishra A (2006) Production of L-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation. Appl. Biochem. Biotechnol. 135, 33–42. 9. Pedreschi F, Kaack K and Granby K (2008) The effect of Asparaginase on acrylamide formation in Fig.2. Effect of supplementary nitrogen sources on cell mass formation French fries. Food Chem. 109, 386-392. 10. Raha SK, Roy SK, Dey SK and Chakrabarty SL (1990) Purification and properties of an Lasparaginase from Cylindrocarpon obtusisporum MB10. Biochem. Int. 2, 987–1000. 11. Sarquis MIM, Oliviera EMM, Santos AS and DaCosta GL (2004) Production of L-asparaginase by filamentous Fungi. Mem. Inst. Oswaldo. Cruz. 99, 489–492. 12. Shaffer PM, Arst HN Jr, Estberg L, Fernando L, Tran Ly and Sitter M (1988) An asparaginase of ISSN: 0974- 6846 Fig.4. Normal probability plot for cell mass formation Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression. Mol. Gen. Genet. 212, 337–341. 13. Torbjorn L, Elisabeth S, Lisbeth A, Bernt T, Asa N, Jarle P and Rolf B (1998) Experimental design and optimization. Chemometr. Intell. Lab. 42, 3–40. 14. Wriston JC Jr and Yellin TO (2001) L-asparaginase a review. Adv. Enz. 39, 185–248. 15. Zheng ZM, Hu QL, Hao J, Xu F, Guo NN, Sun Y and Liu DH (2008) Statistical optimization of culture conditions for 1,3-propanediol by Klebsiella pneumoniae AC 15 via Central Composite Design. Bioresour. Technol. 99, 1052–1056. Fig.3. Normal probability plot for L-asparaginase production Research article “L-asparaginase” Indian Society for Education and Environment (iSee) http://www.indjst.org Baskar & Renganathan Indian J.Sci.Technol.
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Mapping soil carbon, particle-size fractions, and water retention in tropical dry forest in Brazil
Pesquisa Agropecuária Brasileira
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Mapeamento de carbono, frações granulométricas e água do solo em Floresta Tropical Seca no Brasil Resumo – O objetivo deste trabalho foi comparar krigagem ordinária com regressão-krigagem para mapear atributos do solo, em diferentes profundidades, em área de Floresta Tropical Seca no Brasil. Os 11 atributos do solo avaliados foram: conteúdo e estoque de carbono orgânico; densidade do solo; conteúdos de argila, areia e silte; capacidade de troca catiônica; pH; retenção de água na capacidade de campo e no ponto de murcha permanente; e água disponível. As amostras foram retiradas de 327 locais a 0,0–0,10, 0,10–0,20 e 0,20–0,40 m de profundidade, em área de Floresta Tropical Seca de 102 km2. Os modelos de regressão linear “stepwise” tiveram o melhor ajuste para as frações granulométricas e as propriedades de retenção de água. Foram selecionadas covariáveis de relevo e material de origem em 31 dos 33 modelos (11 atributos em três profundidades) e de vegetação em 29 modelos. Com base na validação externa, a krigagem ordinária obteve maior acurácia para 21 das 33 combinações atributo vs. profundidade, o que é indicativo de que a inclusão de um modelo linear de tendência antes da krigagem não necessariamente melhora as predições. Portanto, para estudos semelhantes, os métodos geoestatísticos empregados devem ser comparados caso a caso. Termos para indexação: caatinga, mapeamento digital de solos, levantamento gamarradiométrico, geoestatística, pedometria. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Mapping soil carbon, particle-size fractions, and water retention in tropical dry forest in Brazil Gustavo Mattos Vasques(1), Maurício Rizzato Coelho(1), Ricardo Oliveira Dart(1), Ronaldo Pereira Oliveira(1) and Wenceslau Geraldes Teixeira(1) )Embrapa Solos, Rua Jardim Botânico, no 1.024, Jardim Botânico, CEP 22460-000 Rio de Janeiro, RJ, Brazil. E-mail: gustavo.vasques@embrapa.br, mauricio.coelho@embrapa.br, ricardo.dart@embrapa.br, ronaldo.oliveira@embrapa.br, wenceslau.teixeira@embrapa.br Abstract – The objective of this work was to compare ordinary kriging with regression kriging to map soil properties at different depths in a tropical dry forest area in Brazil. The 11 soil properties evaluated were: organic carbon content and stock; bulk density; clay, sand, and silt contents; cation exchange capacity; pH; water retention at field capacity and at permanent wilting point; and available water. Samples were taken from 327 sites at 0.0–0.10, 0.10–0.20, and 0.20–0.40-m depths, in a tropical dry forest area of 102 km2. Stepwise linear regression models for particle-size fractions and water retention properties had the best fit. Relief and parent material covariates were selected in 31 of the 33 models (11 properties at three depths) and vegetation covariates in 29 models. Based on external validation, ordinary kriging obtained higher accuracy for 21 out of 33 property x depth combinations, indicating that the inclusion of a linear trend model before kriging does not necessarily improve predictions. Therefore, for similar studies, the geostatistical methods employed should be compared on a case-by-case basis. Index terms: caatinga, digital soil mapping, gamma radiometric survey, geostatistics, pedometrics. Materials and Methods and external factors, generating complex spatial patterns (Grunwald, 2009). According to Boucneau et al. (1998), soil spatial predictions can be obtained either by: soil property estimation, using mean and range values from a choropleth soil map, for example; or spatial interpolation, using kriging, for instance. While the former assumes uniformity within mapping units and abrupt changes at their boundaries, the latter assumes that all variation is gradual; however, in reality, soil properties may present both gradual and abrupt changes spatially. The study was conducted at Parque Estadual da Mata Seca, a dry forest state park, in the north of the state of Minas Gerais, Brazil (43°58'48"W, 14°52'12"S) (Figure 1). The park is located in the ecotone of the Brazilian Atlantic Forest (tropical coastal moist forest), Cerrado (savanna), and Caatinga (xeric shrubland). It was created in 2000 and has been preserved since then. Previously, the land was mainly used for agriculture, cattle ranching, and logging for charcoal production. The study area has 102 km2, encompassing the part of the park prior to its expansion in 2009. Mean annual temperature and precipitation are around 24°C and 827 mm, respectively. Among spatial interpolation methods, kriging constitutes a best linear unbiased estimator and has been extensively used to map soil properties. One of its variants – regression kriging (RK) – has received special attention as a means to incorporate the variation of soil-forming environmental factors (so-called environmental covariates) into the variation of the target soil property (Knotters et al., 1995; Kravchenko & Robertson, 2007; Vasques et al., 2010). Regression kriging integrates remote, field, and laboratory data, and statistical methods in a quantitative estimation framework for soil property or class mapping – digital soil mapping –, with applications ranging from precision agriculture (Grunwald et al., 2015) to global mapping (Hengl et al., 2014). Vegetation in the study area can be roughly divided into three main types, considering plant species, size, and seasonality: typical dry forest, comprised mostly of deciduous and thorny species that dominate the study area, covering about 69% of the area in the central part; riparian forest, consisting of evergreen species that occur in 25% of the area on richer and moister soils in the eastern part; and “carrasco” forest, characterized by a few thorny and contorted species adapted to poor, sandy, and excessively drained soils, occurring in less than 6% of the area. Introduction serves as a habitat for many endemic species, as well as a food and wood source for traditional peoples, but is still little studied. Soil maps can be used to provide information to support government and private sector decisions for promoting the use and conservation of this poorly-known, threatened ecosystem and for guiding future research (Miles et al., 2006). Tropical dry forests correspond to 42% of the world’s tropical and subtropical forests (Murphy & Lugo, 1986), of which about 54% occur in South America (Miles et al., 2006). In Brazil, and possibly other tropical countries, laws concerning tropical dry forests are ill-defined, grouping them with moister or drier forest types. This creates a conundrum, hindering the proper conservation of this important ecosystem that Soils vary both horizontally and vertically in the landscape, and are continuously modified by internal Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 G.M. Vasques et al. 1372 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Materials and Methods Elevations range from 434 to 523 m (Figure 1 B), and slope gradients from 0 to 75%. Three geologic units are present in the area, along with their associated soil classes according to Coelho et al. (2013) and to Soil Survey Staff (2014) (Figure 1 C). The “Urucuia” Group takes up about 9% of the area and is mainly composed of sandstone from the Cretaceous Period. It has the poorest soils of the park, which are classified as Latossolos Amarelos (Embrapa, 2006), that is, Haplustox (Soil Survey Staff, 2014), and also the poorest vegetation, the “carrasco”. The “Bambuí” Group was formed during the Neoproterozoic Era and is composed of siltstone, shale, marl, and limestone from the Serra de Santa Helena and Lagoa do Jacaré geologic formations. It covers about 66% of the area, where typical dry forest occurs, and encompasses the highest diversity of soils, including Latossolos (Oxisols), Cambissolos (Inceptisols), Chernossolos (Mollisols), and Vertissolos (Vertisols). Finally, the “Quaternary sediments” unit (25% of the area) comprises fluvial deposits from the São Francisco River, under riparian forest, where the predominant soils are Gleissolos Háplicos (Fluvaquents, Endoaqualfs), Cambissolos Ordinary kriging (OK) and RK are commonly used interpolation methods in digital soil mapping, and have been compared in some studies for mapping various soil properties. Knotters et al. (1995) obtained better results with RK using apparent electrical conductivity as a covariate than with OK or cokriging, when mapping the depth to soft layers (peat or unripe clay) in a 97-ha area in the Netherlands. Kravchenko & Robertson (2007), mapping total carbon using topographic and crop yield as covariates, observed little or no improvement with RK in comparison to OK in 12 sites of 0.36 ha in Michigan, in the United States. In the same country, in Florida, RK outperformed OK for total carbon in three out of five depth intervals (Vasques et al., 2010). As reported in previous studies, preference for RK over OK is not granted nor expected, and there is no clear indication of when to use OK, RK, or any other geostatistical method for more accurate results. The objective of this work was to compare ordinary kriging with regression kriging to map soil properties at different depths in a tropical dry forest area in Brazil. Mapping soil carbon, particle-size fractions 1373 Flúvicos (Fluventic Haplustepts), and Neossolos Flúvicos (Ustifluvents, Ustorthents). Materials and Methods At all sites, soils were described and classified to the fourth hierarchical level (subgroup) of the Brazilian Soil Classification System (Embrapa, 2013). Sampling was done by horizon in deep pits and by layer in shallow pits and boreholes, at 0.0–0.10, 0.10–0.20, 0.20–0.40, 0.40–0.60, 0.60–0.80, and 0.80–100‑m depths. Because soil sampling served different purposes in the experiment, in each field campaign, the shallow pits were sampled differently, with most samples collected at the first three layers to 0.40 m. Undisturbed samples for bulk density (BD) and water retention were obtained using 100-cm3 steel rings. Soil samples were analyzed in the laboratory according to the methods described by Donagema et al. (2011) for organic carbon (OC), clay, silt, and sand contents (g kg‑1); BD (kg dm‑3); pH; and water retention (volumetric percentage) at field capacity (WFC, -0.01 Soils were described and sampled at 327 sites, allocated according to three sampling designs, which were carried out in separate field campaigns. In the first design (“survey”), 222 locations were chosen by tacit knowledge for the purpose of soil survey, comprising 44 deep pits (>1 m), 83 shallow pits (<1 m), and 95 boreholes (<1 m). The second design (“cLHS”) consisted of 41 shallow pits allocated by conditioned Latin hypercube sampling (Minasny & McBratney, 2006), using elevation, normalized difference vegetation index, and land use as covariates. The third design (“random transect”) was composed of 64 shallow pits randomly distributed along transects in different directions, cutting across relatively undersampled areas (Figure 1). Figure 1. Location of study area, that is, of Parque Estadual da Mata Seca, a dry forest state park in Brazil (A), as well as digital elevation model (B), and sampling design, soil suborders, and geologic units (C). CX, Cambissolos Háplicos (Haplustepts); CY, Cambissolos Flúvicos (Fluventic Haplustepts); GM, Gleissolos Melânicos (Argiaquolls); GX, Gleissolos Háplicos (Fluvaquents, Endoaqualfs); LA, Latossolos Amarelos (Haplustox); LV, Latossolos Vermelhos (Haplustox, Eutrustox); LVA, Latossolos Vermelho-Amarelos (Haplustox, Eutrustox); MX, Chernossolos Háplicos (Haplustolls, Argiustolls); RY, Neossolos Flúvicos (Ustifluvents, Ustorthents); and VX, Vertissolos Háplicos (Haplusterts) (Soil Survey Staff, 2014). Soil suborder map compiled from Coelho et al. (2013). Figure 1. Location of study area, that is, of Parque Estadual da Mata Seca, a dry forest state park in Brazil (A), as well as digital elevation model (B), and sampling design, soil suborders, and geologic units (C). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 , , , , p , DOI: 10.1590/S0100-204X2016000900036 Materials and Methods CX, Cambissolos Háplicos (Haplustepts); CY, Cambissolos Flúvicos (Fluventic Haplustepts); GM, Gleissolos Melânicos (Argiaquolls); GX, Gleissolos Háplicos (Fluvaquents, Endoaqualfs); LA, Latossolos Amarelos (Haplustox); LV, Latossolos Vermelhos (Haplustox, Eutrustox); LVA, Latossolos Vermelho-Amarelos (Haplustox, Eutrustox); MX, Chernossolos Háplicos (Haplustolls, Argiustolls); RY, Neossolos Flúvicos (Ustifluvents, Ustorthents); and VX, Vertissolos Háplicos (Haplusterts) (Soil Survey Staff, 2014). Soil suborder map compiled from Coelho et al. (2013). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 , , , , p , DOI: 10.1590/S0100-204X2016000900036 1374 G.M. Vasques et al. G.M. Vasques et al. Environmental data layers representing soil-forming factors were compiled in a geographic information system and extracted from the soil sampling sites to be used as model covariates (Table 1). A digital elevation model (DEM) was derived from an Ikonos (DigitalGlobe Inc., Westminster, CO, USA) stereo pair from December 2012 with 1-m spatial resolution, and then resampled to 10 m using bilinear interpolation (Figure 1 B). Relief covariates were calculated from the resampled DEM. Vegetation covariates were represented by two sets of RapidEye imagery (Planet Labs Inc., San Francisco, CA, USA) with 5-m spatial resolution, one from the MPa) and at permanent wilting point (PWP, -1.5 MPa). Soil organic carbon stock (OCS, kg m-2) was derived by multiplying OC, BD, and layer thickness, corrected for the percent of coarse fragments (>2 mm), using the following equation: OCS = 0.01OC × BD × D × (1 - CF/1000), in which OCS is the organic carbon stock (kg m-2), OC is the organic carbon content (g kg-1), BD is bulk density (kg dm-3), D is layer thickness (cm), and CF is the coarse fragment (>2 mm) content (g kg- 1). Available water (AW, volumetric percentage) was calculated by subtracting WFC from PWP. Table 1. Environmental data layers used as model covariates(1). Materials and Methods Abbreviation Description Unit ELEV Elevation m ASP, SLO Slope aspect and gradient Radian SHT, SLG, LSF Slope height, length, and LS factor m MSP, RSP Mid-slope position and relative slope position Unitless CNBL, VDCN Channel network base level and vertical distance to channel network m VDP Valley depth m PFCV, PLCV Profile and plan curvatures m-1 MRRTF Multiresolution index of ridge top flatness Unitless MRVBF Multiresolution index of valley bottom flatness Unitless CVI, MBI, SPI, TPI, TRI, TWI Convergence, mass balance, stream power, topographic position, terrain ruggedness, and topo- graphic wetness indices Unitless TSC, TST Terrain surface convexity and terrain surface texture Unitless VTR Vector terrain ruggedness Unitless DFIN, DRIN, TOIN Diffuse, direct, and total insolations kWh m-2 CAR, CHT, CSL Catchment area, height, and slope m2, m, radian REdryB1...B5, REwetB1...B5 Surface reflectance of bands 1 to 5 (blue, green, red, red edge, nir) of RE in the dry or wet season % (x102) REdryEVI, REwetEVI Enhanced vegetation index from RE in the dry or wet season [2.5 x (nir - red) / (nir + 6 x red - 7.5 x blue + 1)] Unitless REdryNDVI, REwetNDVI “Red” normalized difference vegetation index from RE in the dry or wet season [(nir - red) / (nir + red)] Unitless REdryNDVIg, REwetNDVIg “Green” normalized difference vegetation index from RE in the dry or wet season [(nir - green) / (nir + green)] Unitless REdryNDVIgr, REwetNDVIgr “Green-red” normalized difference vegetation index from RE in the dry or wet season [(green - red) / (green + red)] Unitless REdryRVI, REwetRVI Ratio vegetation index from RE in the dry or wet season [red / nir] Unitless REdrySAVI, REwetSAVI Soil-adjusted vegetation index from RE in the dry or wet season [(nir - red) / (nir + red + 0.5) x (1 + 0.5)] Unitless GMCTexp Exposure rate of the total count of gamma radiation µR h-1 GMK, GMTh, GMU K, equivalent Th, and equivalent U contents %, ppm, ppm GMUK, GMThK, GMUTh Ratios of U to K, Th to K, and U to Th Unitless MAG Intensity of the magnetic field nT MAGSig, MAG1der Analytical signal and first vertical derivative of the magnetic field nT m-1 (1)nir, near infrared; RE, RapidEye sensor; K, potassium; Th, thorium; U, uranium; kWh, kilowatt-hour; µR, microroentgen; ppm, parts per million; and nT, nanotesla. Table 1. Environmental data layers used as model covariates(1). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion Most soil properties had a close to normal distribution, as indicated by their mean, median, skewness, and kurtosis values (Table 2). On average, soils had more sand, followed by clay, then silt. However, particle-size distribution varied according to soil class. Latossolos Amarelos (Haplustox), Latossolos Vermelho-Amarelos (Haplustox, Eutrustox), and Neossolos Flúvicos (Ustifluvents, Usthorthents) had the largest sand content, which decreased with depth. Latossolos Amarelos and Latossolos Vermelho- Amarelos are formed on sand-rich parent material, mainly sandstone, whereas Neossolos Flúvicos are made up of sandy materials deposited on the floodplain of the São Francisco River. Clay content, however, increased with depth, with the largest values found in Vertissolos Háplicos (Haplusterts), Gleissolos Háplicos (Fluvaquents, Endoaqualfs), Cambissolos Háplicos (Haplustepts), and Latossolos Vermelhos (Haplustox, Eutrustox). These soils are formed from clay-richer rocks of the “Bambuí” Group. The 11 soil properties evaluated were mapped at the 0.0–0.10, 0.10–0.20, and 0.20–0.40-m depths, respectively. Horizon-based data from the 44 deep pits were recalculated for the three layers by depth- weighted averaging. Model training data was composed of all samples from the survey (222) plus cLHS (41) sets. External model validation data were composed of all 64 random transect samples from a separate field campaign. Descriptive statistics of the soil properties were made, and Pearson’s linear correlations among them were derived. Two geostatistical methods were compared to map soil properties, namely OK and RK. In RK, the soil properties were first predicted across the study area as a function of the 62 environmental covariates, using stepwise multiple linear regression (SMLR) as a global trend model. Second, the SMLR residuals were interpolated across the area by OK. Then, the SMLR predictions were added to the interpolated residuals to derive the final output maps. Semivariogram models – spherical, exponential, or Gaussian – were chosen visually and were fitted by ordinary least squares and/ or manually. Soil OC, OCS, and CEC were strongly correlated (Table  3), and decreased with depth; for OCS calculation, the 0.20–0.40-m layer has double the thickness of the first two. The highest values were found in Chernossolos Háplicos (Haplustolls, Argiustolls) and Vertissolos Háplicos, which also had high pH. Both soils are formed from nutrient-richer parent material; however, Chernossolos Háplicos occur on footslopes and Vertissolos Háplicos on depressions. It should be highlighted that water retention properties were stable across depths and were highly correlated with particle- size fractions and CEC. Materials and Methods 2016 DOI: 10.1590/S0100-204X2016000900036 Mapping soil carbon, particle-size fractions 1375 dry and the other from the wet season, that is, from August 2012 and May 2013, respectively. The original five bands were radiometrically and atmospherically corrected to surface reflectance, resampled to 10 m by bilinear interpolation, and then used to derive vegetation indices. Parent material covariates included gamma‑radiometric and magnetometric layers obtained from an aerial survey (Levantamento..., 2009) with 125‑m spatial resolution, which were resampled to 10 m by nearest neighbor resampling. The covariates were processed in the Saga geographic information system (Saga Development Team, University of Hamburg, Hamburg, Germany). prediction uncertainty was determined by mean error (ME) for biasedness and by root mean square error (RMSE) for accuracy. The RMSE of the validation was used to select the best geostatistical method for each soil property at each depth. Data analyses were conducted in the R software (R Core Team, 2015). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion Water retention was greater in Cambissolos Flúvicos (Fluventic Haplustepts), Vertissolos Háplicos, and Cambissolos Háplicos; the two latter with high clay contents. Latossolos Amarelos and Latossolos Vermelho-Amarelos had the lowest OC, OCS, pH, CEC, water retention, clay, and silt content values, since they were formed from poorer and sandier parent materials. These soil property values Stepwise multiple linear regression was implemented using Akaike’s information criterion (AIC) with a p-value of 0.05 for including or excluding variables. All linear regression assumptions were checked. Influential outliers were identified and removed by an outlier test consisting of comparing the Bonferroni corrected p-value from a t-test of the Studentized residuals against a threshold of 0.05. Multicollinearity was minimized by removing variables with variance inflation factors >10.i The goodness-of-fit of the models was assessed by the coefficient of determination (R2), whereas Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 1376 G.M. Vasques et al. at 0.20–0.40 m (Table  4), with all linear regression assumptions met. Models included from 2 to 14 covariates at 5% probability. Relief (elevation and terrain derivatives) and parent material (gamma radiometric and magnetometric) covariates were selected in 31 of the 33 models (11 properties at three are consistent with those of a previous study in the same region (Oliveira et al., 1998), in which relatively high OC, CEC, and pH were also found in Vertissolos (Vertisols). The SMLR models achieved R2 varying from 0.23, for OC at 0–0.10 m, to 0.84, for sand content Table 2. Descriptive statistics of soil properties(1). Table 2. Descriptive statistics of soil properties(1). (1)N, number of observations; SD, standard deviation; OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion Mapping soil carbon, particle-size fractions 1377 field (27 models) was by far the most selected variable, followed by diffuse insolation (15 models), ratio of thorium to potassium (13 models), and surface reflectance from RapidEye in the red-edge region (band depths). Vegetation covariates were selected in 29 models, with general preference for data from the dry (26 models) over the wet (20 models) season. Among the available covariates, the intensity of the magnetic Table 3. Pearson’s linear correlations among soil properties(1). Results and Discussion Property OC BD OCS pH CEC Clay Silt Sand WFC PWP AW 0.0–0.10 m OC 1 -0.44** 0.96** 0.50** 0.84** 0.49** 0.59** -0.61** 0.55** 0.54** 0.20* BD -0.44** 1 -0.25** 0.06ns -0.25** -0.26** -0.10ns 0.22** -0.06ns -0.13ns 0.09ns OCS 0.96** -0.25** 1 0.62** 0.83** 0.44** 0.57** -0.57** 0.64** 0.59** 0.30** pH 0.50** 0.06ns 0.62** 1 0.53** 0.24** 0.44** -0.38** 0.51** 0.42** 0.33** CEC 0.84** -0.25** 0.83** 0.53** 1 0.61** 0.70** -0.74** 0.78** 0.76** 0.28** Clay 0.49** -0.26** 0.44** 0.24** 0.61** 1 0.53** -0.90** 0.71** 0.76** 0.14ns Silt 0.59** -0.10ns 0.57** 0.44** 0.70** 0.53** 1 -0.85** 0.76** 0.72** 0.32** Sand -0.61** 0.22** -0.57** -0.38** -0.74** -0.90** -0.85** 1 -0.85** -0.86** -0.26** WFC 0.55** -0.06ns 0.64** 0.51** 0.78** 0.71** 0.76** -0.85** 1 0.87** 0.55** PWP 0.54** -0.13ns 0.59** 0.42** 0.76** 0.76** 0.72** -0.86** 0.87** 1 0.07ns AW 0.20* 0.09ns 0.30** 0.33** 0.28** 0.14ns 0.32** -0.26** 0.55** 0.07ns 1 0.10–0.20 m OC 1 0.04** 0.97** 0.53** 0.78** 0.42** 0.56** -0.55** 0.65** 0.65** 0.16* BD 0.04** 1 0.16** 0.33ns 0.07** -0.34** 0.10ns 0.18** -0.06ns -0.01ns -0.10ns OCS 0.97** 0.16** 1 0.72** 0.83** 0.43** 0.54** -0.55** 0.66** 0.66** 0.18** pH 0.53** 0.33ns 0.72** 1 0.57** 0.21** 0.47** -0.37** 0.54** 0.47** 0.27** CEC 0.78** 0.07** 0.83** 0.57** 1 0.61** 0.69** -0.73** 0.79** 0.77** 0.25** Clay 0.42** -0.34** 0.43** 0.21** 0.61** 1 0.52** -0.91** 0.72** 0.80** 0.03ns Silt 0.56** 0.10ns 0.54** 0.47** 0.69** 0.52** 1 -0.83** 0.78** 0.73** 0.29** Sand -0.55** 0.18** -0.55** -0.37** -0.73** -0.91** -0.83** 1 -0.86** -0.89** -0.16** WFC 0.65** -0.06ns 0.66** 0.54** 0.79** 0.72** 0.78** -0.86** 1 0.89** 0.46** PWP 0.65** -0.01ns 0.66** 0.47** 0.77** 0.80** 0.73** -0.89** 0.89** 1 0.00ns AW 0.16* -0.10ns 0.18** 0.27** 0.25** 0.03ns 0.29** -0.16** 0.46** 0.00ns 1 0.20–0.40 m OC 1 0.00** 0.97** 0.45** 0.65** 0.37** 0.47** -0.46** 0.54** 0.54** 0.07* BD 0.00** 1 0.21** 0.32ns 0.19** -0.28** 0.07ns 0.17** 0.04ns 0.10ns -0.12ns OCS 0.97** 0.21** 1 0.65** 0.76** 0.34** 0.53** -0.46** 0.56** 0.57** 0.05** pH 0.45** 0.32ns 0.65** 1 0.58** 0.19** 0.45** -0.33** 0.46** 0.44** 0.10** CEC 0.65** 0.19** 0.76** 0.58** 1 0.56** 0.66** -0.68** 0.78** 0.78** 0.10** Clay 0.37** -0.28** 0.34** 0.19** 0.56** 1 0.51** -0.93** 0.75** 0.78** 0.03ns Silt 0.47** 0.07ns 0.53** 0.45** 0.66** 0.51** 1 -0.79** 0.76** 0.74** 0.14** Sand -0.46** 0.17** -0.46** -0.33** -0.68** -0.93** -0.79** 1 -0.86** -0.87** -0.08** WFC 0.54** 0.04ns 0.56** 0.46** 0.78** 0.75** 0.76** -0.86** 1 0.89** 0.36** PWP 0.54** 0.10ns 0.57** 0.44** 0.78** 0.78** 0.74** -0.87** 0.89** 1 -0.11ns AW 0.07* -0.12ns 0.05** 0.10** 0.10** 0.03ns 0.14** -0.08** 0.36** -0.11ns 1 (1)OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Results and Discussion Property Depth (m) N Mean SD Minimum Median Maximum Skewness Kurtosis OC (g kg-1) 0.00–0.10 327 16.0 7.0 3.6 15.1 47.5 1.32 2.87 0.10–0.20 327 11.3 5.4 1.9 10.4 34.3 1.21 2.00 0.20–0.40 201 6.4 2.6 2.2 6.0 20.3 1.53 4.48 BD (kg dm-3) 0.00–0.10 146 1.36 0.13 1.07 1.37 1.59 -0.21 -0.77 0.10–0.20 146 1.41 0.13 1.16 1.40 1.77 0.31 -0.27 0.20–0.40 146 1.41 0.13 1.07 1.42 1.75 0.09 -0.23 OCS (kg m-2) 0.00–0.10 134 2.1 0.7 0.8 2.0 4.6 0.90 1.34 0.10–0.20 134 1.3 0.5 0.4 1.2 2.8 0.72 -0.01 0.20–0.40 134 1.9 0.8 0.6 1.7 5.5 1.54 3.52 pH 0.00–0.10 327 6.2 0.8 4.1 6.4 8.1 -0.37 0.07 0.10–0.20 327 6.1 0.8 3.9 6.2 8.2 -0.21 -0.05 0.20–0.40 201 6.1 0.9 4.1 6.1 8.3 0.10 -0.51 CEC (cmolc kg-1) 0.00–0.10 327 11.8 5.6 2.8 10.9 36.9 1.24 2.17 0.10–0.20 327 10.6 5.5 2.3 9.5 36.8 1.38 2.64 0.20–0.40 201 10.1 5.5 1.9 8.9 36.7 1.57 3.71 Clay (g kg-1) 0.00–0.10 327 317 121 61 340 659 -0.08 -0.71 0.10–0.20 327 342 130 44 360 659 -0.21 -0.84 0.20–0.40 201 398 133 60 418 660 -0.43 -0.67 Silt (g kg-1) 0.00–0.10 327 221 101 28 219 566 0.42 0.04 0.10–0.20 327 207 96 12 203 566 0.44 0.24 0.20–0.40 201 192 80 32 186 557 0.76 1.92 Sand (g kg-1) 0.00–0.10 327 462 195 23 442 869 0.19 -0.69 0.10–0.20 327 451 198 23 424 892 0.31 -0.68 0.20–0.40 201 410 186 12 382 847 0.50 -0.46 WFC 0.00–0.10 146 28.0 6.0 8.9 28.8 42.8 -0.78 1.14 0.10–0.20 146 26.9 5.5 11.0 27.3 43.4 -0.47 1.11 0.20–0.40 146 26.8 5.6 11.9 27.0 42.9 -0.12 0.85 PWP 0.00–0.10 146 14.6 5.0 3.0 14.8 29.7 0.18 0.71 0.10–0.20 146 14.5 4.9 3.5 14.7 31.4 0.13 0.52 0.20–0.40 146 14.5 5.2 4.1 14.5 31.3 0.43 0.53 AW 0.00–0.10 146 13.4 3.0 5.2 13.5 21.3 -0.34 0.16 0.10–0.20 146 12.4 2.5 4.8 12.5 24.0 0.67 4.00 0.20–0.40 146 12.3 2.6 4.3 12.4 24.2 0.79 3.77 (1)N, number of observations; SD, standard deviation; OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion ** and *Significant at 1 and 5% probability, respectively. nsNonsignificant at 5% probability. Table 3. Pearson’s linear correlations among soil properties(1). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 , , , , p , DOI: 10.1590/S0100-204X2016000900036 G.M. Vasques et al. 1378 (Filella & Peñuelas, 1994). The global trend SMLR models of all soil properties are presented in Table 5.i 4, 690–730 nm) from the wet season (13 models). Red edge is the region where the reflectance of live plants increases from the red (chlorophyll absorption) to the near-infrared region, which has been strongly linked to chlorophyll content, leaf area index, and plant moisture These findings show the strong parent material control of the soil property patterns in the area, but also the effect of sun heat and vegetation. This These findings show the strong parent material control of the soil property patterns in the area, but also the effect of sun heat and vegetation. This Table 4. Stepwise multiple linear regression model fit and external validation results, with the best geostatistical method in bold for each property(1). (1)N, number of observations (training/validation); R2, training coefficient of determination; ME, validation mean error; RMSE, validation root mean square error; OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion The “Urucuia” Group, “Bambuí” Group, and “Quaternary sediments” geologic units hold 6, 69, and 24% of the OCS at 0.40 m, respectively, with the largest mean (5.79 kg m-2) in the “Bambuí” Group. makes sense in this region where plant patterns are controlled both by climatic conditions, which regulate evapotranspiration, leaf fall, and phenology (Pezzini et al., 2014), and by soil conditions, which regulate water and nutrient availability (Souza et al., 2007). Oliveira‑Filho et al. (1998) identified available light, that is, canopy gaps, as a critical factor controlling the plant species’ abundance distribution in a tropical dry forest in Central Brazil. Considering external validation, OK outperformed RK for 21 out of 33 property x depth combinations, though, for most cases, OK and RK had similar prediction quality (Table  4). The preference for OK over RK did not follow a clear pattern. In fact, in some cases, the best method varied among depths for the same soil property. Nonetheless, this preference still indicates that the local variation of soil properties was, in general, a stronger predictor than environmental covariates. This is regardless of the quality of the SMLR models with RK, which, in some cases, had R2 >0.60 but still performed worse than OK, as observed for clay and sand contents, and PWP. As a rule, the quality of soil predictions depends on soil property, number and distribution of samples, environmental covariates, and soil-landscape correlations, among other factors. It should be noted that neither the number of samples nor the pool of environmental covariates (soil-forming factor groups) was tested in the present study. The spatial patterns of the soil properties were related, either positively or negatively (Table  3 and Figures 2 and 3). In the long range, they responded to the variation of geology, which, to some extent, also controls the variation of vegetation. In the short range, they reflected the spatial distribution of soil classes and their properties. The sampling density of about 3.2 samples per square kilometer was enough to derive better OK than RK maps using many (and some costly) environmental covariates for most cases; however, the effect of sampling density on prediction quality was not tested. Since soil properties were correlated, all OK maps look similar. Results and Discussion Property Depth (m) N R2 Ordinary kriging Regression kriging ME RMSE ME RMSE OC 0.00–0.10 260/64 0.41 -1.3 5.8 -1.8 5.7 0.10–0.20 262/64 0.23 -4.1 4.9 -4.5 5.1 0.20–0.40 137/64 0.27 -0.3 2.7 -0.3 2.9 BD 0.00–0.10 83/63 0.29 0.1 0.1 0.1 0.1 0.10–0.20 83/63 0.60 0.1 0.1 0.1 0.1 0.20–0.40 83/63 0.48 0.0 0.1 0.1 0.1 OCS 0.00–0.10 71/63 0.65 0.0 0.6 -0.1 0.6 0.10–0.20 71/63 0.47 -0.2 0.4 -0.2 0.5 0.20–0.40 71/63 0.47 -0.2 0.8 -0.2 0.8 pH 0.00–0.10 263/64 0.48 0.0 0.7 -0.1 0.5 0.10–0.20 262/64 0.44 -0.2 0.8 -0.3 0.7 0.20–0.40 137/64 0.50 -0.3 0.7 -0.2 0.7 CEC 0.00–0.10 259/64 0.45 0.0 3.8 0.0 3.0 0.10–0.20 254/64 0.47 -0.2 3.8 0.0 3.6 0.20–0.40 136/64 0.56 -0.1 4.4 -0.4 4.4 Clay 0.00–0.10 262/64 0.64 24 61 11 61 0.10–0.20 263/64 0.64 37 64 23 65 0.20–0.40 136/64 0.69 14 71 9 79 Silt 0.00–0.10 263/64 0.61 -6 65 -9 70 0.10–0.20 262/64 0.61 -11 64 -12 62 0.20–0.40 136/64 0.69 -15 57 -20 59 Sand 0.00–0.10 263/64 0.69 -16 63 -3 79 0.10–0.20 262/64 0.70 -23 63 -15 81 0.20–0.40 133/64 0.84 0 76 14 86 WFC 0.00–0.10 83/63 0.62 -0.1 4.2 0.0 3.5 0.10–0.20 83/63 0.74 -0.4 3.6 -0.1 2.8 0.20–0.40 83/63 0.68 -0.1 3.6 0.0 3.3 PWP 0.00–0.10 83/63 0.70 -2.6 3.9 -2.7 4.2 0.10–0.20 83/63 0.72 -1.5 2.9 -1.4 3.6 0.20–0.40 83/63 0.68 -1.3 3.2 -1.5 4.1 AW 0.00–0.10 83/63 0.25 2.7 3.7 2.7 3.6 0.10–0.20 81/63 0.68 1.2 2.4 0.9 2.2 0.20–0.40 81/63 0.63 1.3 2.7 1.1 2.7 (1)N, number of observations (training/validation); R2, training coefficient of determination; ME, validation mean error; RMSE, validation root mean square error; OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW available water (volumetric percentage) linear regression model fit and external validation results, with the best geostatistical method in Mapping soil carbon, particle-size fractions 1379 tons of soil OC in the first 40 cm, of which 34% are found in Cambissolos Háplicos and 30% in Latossolos Vermelhos, due to their large coverage. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion Despite this, the RK maps show differences related to the covariates used in the global trend models, even though they globally resemble the other maps; in addition, the short-range variation of these maps is more detailed according to the variation of the environmental covariates. Semivariogram parameters varied among soil properties (Table 6). Sand content and PWP had the highest ranges, whereas pH and BD had the lowest ones. Across depths, the same semivariogram model was chosen and similar ranges were found for the same property, indicating similar spatial dependence structures. The strength of spatial continuity, as evidenced by the nugget:sill ratio, was as follows: higher for the particle-size fractions and water retention properties; intermediate for the chemical properties, such as pH and CEC; and lower for OC, OCS, and BD. After global trend modeling by SMLR, partial sill and total sill variances decreased in all semivariograms, when compared to the original properties, whereas nugget variance and range changed inconsistently. These results are expected since the SMLR models explained part of the total variance of soil properties, but not necessarily their short- (nugget) or long- (range) distance variations. In any case, changing the semivariogram parameters affected kriging weights and derived spatial patterns, as well as kriging variances. The western and northwestern portions of the study area are on sand-rich parent material. These portions have: the sandiest and poorest soils in terms of fertility, that is, low carbon and CEC; low water holding capacity, that is, low WFC, PWP, and AW; and the poorest vegetation of the park, the “carrasco”. The eastern portion constitutes the São Francisco River floodplain, with soils with fluvic characteristics, good water holding capacity, and medium fertility. The central portion corresponds to the area of the “Bambuí” Group, with variable lithology and the presence of calcareous materials, occasionally as rock outcrops. The most common soils in the area are Latossolos Vermelhos and Cambissolos Háplicos, each occupying 31% of the area. Soils with largest carbon stocks, that is, Vertissolos Háplicos and Chernossolos Háplicos, appear in this portion and together occupy less than 3% of the study area. Total soil OCS at 0.40 m was determined by adding the values obtained at the three depths derived using OK. The study area stores approximately 5.65x105 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 1380 G.M. (1)R2, training coefficient of determination; OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Covariates are described in Table 1. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion Vasques et al. Table 5. Stepwise multiple linear regression models for soil properties(1). Table 5. Stepwise multiple linear regression models for soil properties(1). Results and Discussion Stepwise multiple linear regression models for soil properties(1). Table 5. Stepwise multiple linear regression models for soil properties(1). Results and Discussion Property Depth (m) Equation R2 OC 0.00–0.10 -49.44 + 0.05 x CNBL - 0.01 x SLG - 0.01 x REdryB2 - 35.64 x REdryNDVIg + 0.03 x REwetB2 + 70.02 x REwetNDVIg + 1.49 x GMCTexp - 0.07 x GMThK - 0.03 x MAG 0.41 0.10–0.20 -550.29 + 683.51 x DFIN - 0.005 x SLG + 2.61 x TRI - 0.22 x VDCN - 0.26 x GMUK - 0.03 x MAG 0.23 0.20–0.40 -1.19 - 2.90 x MBI + 1.60 x TRI - 0.01 x REwetB3 + 0.01 x REwetB4 - 0.02 x MAG + 2.88 x GMK 0.27 BD 0.00–0.10 1.48 - 1.32 x CSL + 0.005 x CVI - 0.02 x MRRTF - 0.13 x RSP 0.29 0.10–0.20 2.76 - 0.003 x CNBL + 0.004 x CVI + 0.0002 x SLG + 0.01 x VDP - 0.0000004 x CAR - 0.22 x REdryEVI + 0.0007 x MAG 0.60 0.20–0.40 3.35 - 0.004 x CNBL + 0.01 x VDP - 0.84 x REdryNDVIg + 0.01 x REwetRVI 0.48 OCS 0.00–0.10 -1.18 - 0.02 x CVI + 0.10 x MRVBF - 0.12 x TWI + 0.01 x REdryB1 - 0.004 x REdryB4 - 2.03 x REdryNDVIgr + 0.003 x REwetB4 + 3.54 x REwetNDVI - 0.03 x GMUK - 0.01 x MAG 0.65 0.10–0.20 -1.94 - 0.03 x VDCN + 0.01 x REdryB1 - 0.002 x REdryB3 - 0.01 x REwetB2 + 0.004 x REwetB4 - 0.01 x MAG 0.47 0.20–0.40 63.41 - 77.83 x DFIN + 0.01 x REdryB1 - 0.002 x REdryB4 - 0.01 x REwetB2 + 0.005 x REwetB4 - 0.03 x GMUK - 0.004 x MAG 0.47 pH 0.00–0.10 2.39 + 0.85 x MBI - 0.14 x TPI + 1.02 x TST - 4.85 x REdryNDVIg + 0.002 x REwetB4 + 5.17 x REwetNDVIg - 0.06 x GMTh - 0.01 x GMThK - 0.01 x MAG + 1.75 x MAG1der 0.48 0.10–0.20 2.34 - 3.23 x REdryNDVIg + 0.002 x REwetB4 + 4.47 x REwetNDVIg - 0.01 x GMThK - 0.01 x MAG 0.44 0.20–0.40 5.30 + 0.30 x LSF - 0.59 x RSP - 3.19 x REdryNDVIg + 0.002 x REwetB4 + 1.6 x GMK - 0.004 x MAG 0.50 CEC 0.00–0.10 7.93 + 3.33 x TRI - 0.15 x VDCN - 951.32 x VTR + 10.97 x REdryNDVIgr - 0.01 x REwetB3 + 0.01 x REwetB4 + 1.05 x GMCTexp - 0.06 x GMThK - 0.02 x MAG 0.45 0.10–0.20 -734.23 - 0.06 x CVI + 893.47 x DFIN + 3.53 x MSP + 6.75 x TRI - 0.26 x VDCN - 671.22 x VTR + 6.79 x REdryEVI + 0.004 x RE- wetB4 + 3.74 x GMK - 0.05 x GMThK - 0.02 x MAG 0.47 0.20–0.40 -415.06 + 530.77 x DFIN - 15.36 x MBI + 1615.38 x PFCV - 0.005 x SLG + 4.85 x TRI - 0.24 x VDCN - 11.74 x REdryEVI + 46.67 x REdryNDVIgr - 0.07 x GMThK - 0.02 x MAG 0.56 Clay 0.00–0.10 -23352.67 - 0.11 x ASP - 8.09 x CHT + 26944.57 x DFIN + 190.55 x DRIN + 136.11 x TRI - 109.84 x TSC - 79.57 x TST + 4.9 x VDP - 0.08 x REdryB3 + 15.92 x GMTh - 1.25 x GMThK + 40.07 x GMU - 0.59 x MAG + 183.51 x MAGSig 0.64 0.10–0.20 -24889.92 - 0.12 x ASP - 6.00 x CHT + 29032.08 x DFIN + 163.26 x DRIN + 136.8 x TRI - 99.55 x TSC + 3.63 x VDP - 0.06 x REdryB3 + 9.55 x GMTh + 87.57 x GMU - 6.87 x GMUK - 0.75 x MAG 0.64 0.20–0.40 -18521.85 + 20634.83 x DFIN + 217.54 x DRIN + 18.23 x MRVBF - 25704.15 x PFCV + 141.18 x TRI - 12.10 x TWI - 4.40 x VDCN + 75.95 x GMCTexp - 0.97 x MAG 0.69 Silt 0.00–0.10 -549.62 + 2.10 x ELEV + 17678.05 x PFCV - 31.45 x TPI - 2.04 x VDCN - 0.25 x REdryB1 - 556.71 x REdryNDVIg + 31.93 x REdryR- VI + 192.89 x GMK - 0.63 x GMThK - 0.70 x MAG - 162.96 x MAGSig 0.61 0.10–0.20 -67.81 + 17101.05 x PFCV - 27.10 x TPI - 0.24 x REdryB1 - 812.53 x REdryNDVIg + 41.92 x REdryRVI + 0.11 x REwetB4 + 664.67 x REwetNDVIg - 5.33 x REwetRVI + 33.27 x GMCTexp - 1.09 x GMThK - 0.45 x MAG - 226.31 x MAGSig 0.61 0.20–0.40 -590.52 + 766.95 x CSL + 1.59 x ELEV + 19926.25 x PFCV - 3.55 x VDCN + 0.63 x REdryB2 - 0.63 x REdryB4 + 0.16 x REdryB5 + 35.00 x GMCTexp - 95.57 x GMU - 0.65 x MAG 0.69 Sand 0.00–0.10 26683.60 - 29920.03 x DFIN - 203.24 x DRIN + 24.21 x TPI - 94.91 x TRI + 99.66 x TSC + 652.32 x REdryNDVIg - 75.55 x REdryRVI + 0.54 x REwetB3 - 0.23 x REwetB4 + 15.88 x REwetRVI - 91.49 x GMCTexp + 2 x GMThK + 1 x MAG 0.69 0.10–0.20 2993.77 - 4.60 x CNBL + 25.13 x TPI - 50.9 x REdryRVI + 0.44 x REwetB3 - 0.24 x REwetB4 + 16.76 x REwetRVI - 86.5 x GMCTexp + 2.20 x GMThK + 1.12 x MAG 0.70 0.20–0.40 36670.32 + 9.57 x CHT - 41016.27 x DFIN - 354.69 x DRIN - 0.26 x SPI - 267.19 x TRI + 137.58 x TSC - 7.35 x VDP + 0.07 x REdryB5 - 891.81 x REdryNDVIgr + 12.64 x REwetRVI - 57.61 x GMCTexp + 10.79 x GMUK + 0.72 x MAG 0.84 WFC 0.00–0.10 2.48 + 3.42 x TRI - 0.24 x VDCN + 23.55 x REwetNDVIg + 1.96 x GMCTexp - 0.07 x GMThK - 0.04 x MAG 0.62 0.10–0.20 -624.27 - 0.01 x ASP + 678.61 x DFIN + 11.77 x DRIN + 4.34 x LSF + 2.87 x MSP - 6.19 x TSC + 13.88 x GMK - 0.15 x GMUK - 0.04 0.74 Table 5. 0.20–0.40 44.51 - 0.08 x ELEV + 0.14 x SHT + 0.50 x SLO + 5.32 x TST + 0.25 x TWI - 0.18 x VDP - 1365.85 x VTR - 0.01 x REdryB2 + 0.02 x REwetB1 + 4.98 x REwetEVI + 1.27 x GMCTexp - 4.66 x GMK - 0.02 x GMThK + 9.31 x MAG1der 0.63 Results and Discussion Property Depth (m) Equation R2 OC 0.00–0.10 -49.44 + 0.05 x CNBL - 0.01 x SLG - 0.01 x REdryB2 - 35.64 x REdryNDVIg + 0.03 x REwetB2 + 70.02 x REwetNDVIg + 1.49 x GMCTexp - 0.07 x GMThK - 0.03 x MAG 0.41 0.10–0.20 -550.29 + 683.51 x DFIN - 0.005 x SLG + 2.61 x TRI - 0.22 x VDCN - 0.26 x GMUK - 0.03 x MAG 0.23 0.20–0.40 -1.19 - 2.90 x MBI + 1.60 x TRI - 0.01 x REwetB3 + 0.01 x REwetB4 - 0.02 x MAG + 2.88 x GMK 0.27 BD 0.00–0.10 1.48 - 1.32 x CSL + 0.005 x CVI - 0.02 x MRRTF - 0.13 x RSP 0.29 0.10–0.20 2.76 - 0.003 x CNBL + 0.004 x CVI + 0.0002 x SLG + 0.01 x VDP - 0.0000004 x CAR - 0.22 x REdryEVI + 0.0007 x MAG 0.60 0.20–0.40 3.35 - 0.004 x CNBL + 0.01 x VDP - 0.84 x REdryNDVIg + 0.01 x REwetRVI 0.48 OCS 0.00–0.10 -1.18 - 0.02 x CVI + 0.10 x MRVBF - 0.12 x TWI + 0.01 x REdryB1 - 0.004 x REdryB4 - 2.03 x REdryNDVIgr + 0.003 x REwetB4 + 3.54 x REwetNDVI - 0.03 x GMUK - 0.01 x MAG 0.65 0.10–0.20 -1.94 - 0.03 x VDCN + 0.01 x REdryB1 - 0.002 x REdryB3 - 0.01 x REwetB2 + 0.004 x REwetB4 - 0.01 x MAG 0.47 0.20–0.40 63.41 - 77.83 x DFIN + 0.01 x REdryB1 - 0.002 x REdryB4 - 0.01 x REwetB2 + 0.005 x REwetB4 - 0.03 x GMUK - 0.004 x MAG 0.47 pH 0.00–0.10 2.39 + 0.85 x MBI - 0.14 x TPI + 1.02 x TST - 4.85 x REdryNDVIg + 0.002 x REwetB4 + 5.17 x REwetNDVIg - 0.06 x GMTh - 0.01 x GMThK - 0.01 x MAG + 1.75 x MAG1der 0.48 0.10–0.20 2.34 - 3.23 x REdryNDVIg + 0.002 x REwetB4 + 4.47 x REwetNDVIg - 0.01 x GMThK - 0.01 x MAG 0.44 0.20–0.40 5.30 + 0.30 x LSF - 0.59 x RSP - 3.19 x REdryNDVIg + 0.002 x REwetB4 + 1.6 x GMK - 0.004 x MAG 0.50 CEC 0.00–0.10 7.93 + 3.33 x TRI - 0.15 x VDCN - 951.32 x VTR + 10.97 x REdryNDVIgr - 0.01 x REwetB3 + 0.01 x REwetB4 + 1.05 x GMCTexp - 0.06 x GMThK - 0.02 x MAG 0.45 0.10–0.20 -734.23 - 0.06 x CVI + 893.47 x DFIN + 3.53 x MSP + 6.75 x TRI - 0.26 x VDCN - 671.22 x VTR + 6.79 x REdryEVI + 0.004 x RE- wetB4 + 3.74 x GMK - 0.05 x GMThK - 0.02 x MAG 0.47 0.20–0.40 -415.06 + 530.77 x DFIN - 15.36 x MBI + 1615.38 x PFCV - 0.005 x SLG + 4.85 x TRI - 0.24 x VDCN - 11.74 x REdryEVI + 46.67 x REdryNDVIgr - 0.07 x GMThK - 0.02 x MAG 0.56 0.00–0.10 -23352.67 - 0.11 x ASP - 8.09 x CHT + 26944.57 x DFIN + 190.55 x DRIN + 136.11 x TRI - 109.84 x TSC - 79.57 x TST + 4.9 x VDP - 0.64 Equation 0.10–0.20 -550.29 + 683.51 x DFIN - 0.005 x SLG + 2.61 x TRI - 0.22 x VDCN - 0.26 x GMUK - 0.03 x MAG .10–0.20 2.76 - 0.003 x CNBL + 0.004 x CVI + 0.0002 x SLG + 0.01 x VDP - 0.0000004 x CAR - 0.22 x REdryEVI + 0.0007 x MAG .20–0.40 3.35 - 0.004 x CNBL + 0.01 x VDP - 0.84 x REdryNDVIg + 0.01 x REwetRVI .00–0.10 -1.18 - 0.02 x CVI + 0.10 x MRVBF - 0.12 x TWI + 0.01 x REdryB1 - 0.004 x REdryB4 - 2.03 x REdryNDVIgr + 0.003 x REwe 3.54 x REwetNDVI - 0.03 x GMUK - 0.01 x MAG .10–0.20 -1.94 - 0.03 x VDCN + 0.01 x REdryB1 - 0.002 x REdryB3 - 0.01 x REwetB2 + 0.004 x REwetB4 - 0.01 x MAG 20–0.40 63.41 - 77.83 x DFIN + 0.01 x REdryB1 - 0.002 x REdryB4 - 0.01 x REwetB2 + 0.005 x REwetB4 - 0.03 x GMUK - 0.004 x MA 0.20–0.40 63.41 - 77.83 x DFIN + 0.01 x REdryB1 - 0.002 x REdryB4 - 0.01 x REwetB2 + 0.005 x REwetB4 - 0.03 x GMUK - 0.004 x MAG 0.47 .00–0.10 2.39 + 0.85 x MBI - 0.14 x TPI + 1.02 x TST - 4.85 x REdryNDVIg + 0.002 x REwetB4 + 5.17 x REwetNDVIg - 0.06 x GMTh - GMThK - 0.01 x MAG + 1.75 x MAG1der .10–0.20 2.34 - 3.23 x REdryNDVIg + 0.002 x REwetB4 + 4.47 x REwetNDVIg - 0.01 x GMThK - 0.01 x MAG .20–0.40 5.30 + 0.30 x LSF - 0.59 x RSP - 3.19 x REdryNDVIg + 0.002 x REwetB4 + 1.6 x GMK - 0.004 x MAG 0.00–0.10 7.93 + 3.33 x TRI - 0.15 x VDCN - 951.32 x VTR + 10.97 x REdryNDVIgr - 0.01 x REwetB3 + 0.01 x REwetB4 + 1.05 x GMCTexp - 0.06 x GMThK - 0.02 x MAG 0.45 0.00–0.10 7.93 + 3.33 x TRI - 0.15 x VDCN - 951.32 x VTR + 10.97 x REdryNDVIgr - 0.01 x REwetB3 + 0.01 x REwetB4 + 1.05 x GMCTexp - 0.06 x GMThK - 0.02 x MAG 0.45 CEC 0.10–0.20 -734.23 - 0.06 x CVI + 893.47 x DFIN + 3.53 x MSP + 6.75 x TRI - 0.26 x VDCN - 671.22 x VTR + 6.79 x REdryEVI + 0.004 x RE- wetB4 + 3.74 x GMK - 0.05 x GMThK - 0.02 x MAG 0.47 CEC 0.10–0.20 -734.23 - 0.06 x CVI + 893.47 x DFIN + 3.53 x MSP + 6.75 x TRI - 0.26 x VDCN - 671.22 x VTR + 6.79 x REdryEVI + 0.004 x RE- wetB4 + 3.74 x GMK - 0.05 x GMThK - 0.02 x MAG 0.47 0.20–0.40 -415.06 + 530.77 x DFIN - 15.36 x MBI + 1615.38 x PFCV - 0.005 x SLG + 4.85 x TRI - 0.24 x VDCN - 11.74 x REdryEVI + 46.67 x REdryNDVIgr - 0.07 x GMThK - 0.02 x MAG 0.56 0.00–0.10 -23352.67 - 0.11 x ASP - 8.09 x CHT + 26944.57 x DFIN + 190.55 x DRIN + 136.11 x TRI - 109.84 x TSC - 79.57 x TST + 4.9 x VDP - 0.08 x REdryB3 + 15.92 x GMTh - 1.25 x GMThK + 40.07 x GMU - 0.59 x MAG + 183.51 x MAGSig 0.64 .10–0.20 -24889.92 - 0.12 x ASP - 6.00 x CHT + 29032.08 x DFIN + 163.26 x DRIN + 136.8 x TRI - 99.55 x TSC + 3.63 x VDP - 0.06 x R + 9.55 x GMTh + 87.57 x GMU - 6.87 x GMUK - 0.75 x MAG 0.20–0.40 -18521.85 + 20634.83 x DFIN + 217.54 x DRIN + 18.23 x MRVBF - 25704.15 x PFCV + 141.18 x TRI - 12.10 x TWI - 4.40 x VDCN + 75.95 x GMCTexp - 0.97 x MAG 0.69 0–0.40 -18521.85 + 20634.83 x DFIN + 217.54 x DRIN + 18.23 x MRVBF - 25704.15 x PFCV + 141.18 x TRI - 12.10 x TWI - 4.40 x VDCN + 75 95 x GMCTexp - 0 97 x MAG 0.69 0.00–0.10 -549.62 + 2.10 x ELEV + 17678.05 x PFCV - 31.45 x TPI - 2.04 x VDCN - 0.25 x REdryB1 - 556.71 x REdryNDVIg + 31.93 x REdryR- VI + 192.89 x GMK - 0.63 x GMThK - 0.70 x MAG - 162.96 x MAGSig 0.61 Silt 0.10–0.20 -67.81 + 17101.05 x PFCV - 27.10 x TPI - 0.24 x REdryB1 - 812.53 x REdryNDVIg + 41.92 x REdryRVI + 0.11 x REwetB4 + 664.67 x REwetNDVIg - 5.33 x REwetRVI + 33.27 x GMCTexp - 1.09 x GMThK - 0.45 x MAG - 226.31 x MAGSig 0.61 .20–0.40 -590.52 + 766.95 x CSL + 1.59 x ELEV + 19926.25 x PFCV - 3.55 x VDCN + 0.63 x REdryB2 - 0.63 x REdryB4 + 0.16 x REdry 35.00 x GMCTexp - 95.57 x GMU - 0.65 x MAG 0.00–0.10 26683.60 - 29920.03 x DFIN - 203.24 x DRIN + 24.21 x TPI - 94.91 x TRI + 99.66 x TSC + 652.32 x REdryNDVIg - 75.55 x REdryRVI + 0.54 x REwetB3 - 0.23 x REwetB4 + 15.88 x REwetRVI - 91.49 x GMCTexp + 2 x GMThK + 1 x MAG 0.69 Sand 0.10–0.20 2993.77 - 4.60 x CNBL + 25.13 x TPI - 50.9 x REdryRVI + 0.44 x REwetB3 - 0.24 x REwetB4 + 16.76 x REwetRVI - 86.5 x GMCTexp + 2.20 x GMThK + 1.12 x MAG 0.70 0.20–0.40 36670.32 + 9.57 x CHT - 41016.27 x DFIN - 354.69 x DRIN - 0.26 x SPI - 267.19 x TRI + 137.58 x TSC - 7.35 x VDP + 0.07 x REdryB5 - 891.81 x REdryNDVIgr + 12.64 x REwetRVI - 57.61 x GMCTexp + 10.79 x GMUK + 0.72 x MAG 0.84 0.20–0.40 36670.32 + 9.57 x CHT - 41016.27 x DFIN - 354.69 x DRIN - 0.26 x SPI - 267.19 x TRI + 137.58 x TSC - 7.35 x VDP + 0.07 x REdryB5 - 891.81 x REdryNDVIgr + 12.64 x REwetRVI - 57.61 x GMCTexp + 10.79 x GMUK + 0.72 x MAG 0.84 0 00–0 10 2 48 + 3 42 x TRI - 0 24 x VDCN + 23 55 x REwetNDVIg + 1 96 x GMCTexp - 0 07 x GMThK - 0 04 x MAG 0 62 WFC 0.10–0.20 -624.27 - 0.01 x ASP + 678.61 x DFIN + 11.77 x DRIN + 4.34 x LSF + 2.87 x MSP - 6.19 x TSC + 13.88 x GMK - 0.15 x GMUK - 0.04 x MAG 0.74 7 - 0.01 x ASP + 678.61 x DFIN + 11.77 x DRIN + 4.34 x LSF + 2.87 x MSP - 6.19 x TSC + 13.88 x GMK - 0.15 x GMUK - 0.04 G 0.74 WFC 0.10–0.20 -624.27 - 0.01 x ASP + 678.61 x DFIN + 11.77 x DRIN + 4.34 x LSF + 2.87 x MSP - 6.19 x TSC + 13.88 x GMK - 0.15 x GMUK - 0.04 x MAG 0.74 0.20–0.40 -941.16 - 0.01 x ASP + 1165.41 x DFIN + 4.45 x LSF - 0.30 x VDCN + 6.58 x REdryEVI + 14.91 x GMK - 0.05 x MAG 0.68 0 00 0 10 943 34 0 00001 CAR + 1157 62 DFIN + 6 07 LSF 8 53 MBI + 3 29 MSP + 1216 84 PFCV 0 41 VDCN 0 31 RE 0 70 .20–0.40 -941.16 - 0.01 x ASP + 1165.41 x DFIN + 4.45 x LSF - 0.30 x VDCN + 6.58 x REdryEVI + 14.91 x GMK - 0.05 x MAG .00–0.10 -943.34 - 0.00001 x CAR + 1157.62 x DFIN + 6.07 x LSF - 8.53 x MBI + 3.29 x MSP + 1216.84 x PFCV - 0.41 x VDCN - 0.31 wetRVI + 15.27 x GMK - 0.03 x MAG - 23.62 x MAG1der PWP 0.10–0.20 -1022.86 - 0.01 x ASP + 1245.28 x DFIN + 5.23 x LSF - 0.72 x MRVBF + 5.09 x RSP - 0.70 x VDCN + 0.19 x VDP - 0.01 x REdryB4 + 0.02 x REwetB4 + 12.77 x GMK - 24.94 x MAG1der 0.72 0.20–0.40 974.67 - 11.46 x DFIN - 0.44 x MBI + 8.06 x SHT + 1902 x TRI + 18.32 x VTR + 0.36 x CHT - 95.76 x CSL - 0.00001 x CAR + 0.01 x REdryB2 - 0.04 x GMK - 18.6 x MAG - 799.99 x MAG1der 0.68 AW 0.10–0.20 617.8 - 739.46 x DFIN + 5.9 x TST - 0.17 x VDP - 0.004 x REdryB4 + 0.39 x REwetRVI + 0.97 x GMCTexp 0.68 AW 0.20–0.40 44.51 - 0.08 x ELEV + 0.14 x SHT + 0.50 x SLO + 5.32 x TST + 0.25 x TWI - 0.18 x VDP - 1365.85 x VTR - REwetB1 + 4.98 x REwetEVI + 1.27 x GMCTexp - 4.66 x GMK - 0.02 x GMThK + 9.31 x MAG1der 1381 Mapping soil carbon, particle-size fractions of soil properties based on their best geostatistical methods: A, organic carbon content (OC) at 0.0–0.10-m depth b 20 and 0.20–0.40 m by ordinary kriging (OK); B, bulk density (BD) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by OK .10, 0.10–0.20, and 0.20–0.40 m by OK; D, pH at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by RK; and E, cation excha –0.20 m by RK, and at 0.10–0.20 m by OK. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Results and Discussion 2016 DOI: 10.1590/S0100-204X2016000900036 Mapping soil carbon, particle-size fractions Mapping soil carbon, particle-size fractions 1383 Previous studies have also shown little or no improvement of RK over OK, at plot (Kravchenko & Robertson, 2007), farm/catchment (Zhu & Lin, 2010), or watershed scale (Vasques et al., 2010). Kravchenko & Robertson (2007) found that RK produced only a modest improvement in accuracy compared to OK and performed poorly in data sets with strong spatial correlation in the target variable, even when the Table 6. Semivariogram parameters for original soil properties and residuals from stepwise multiple linear regression (SMLR)(1). Results and Discussion Figure 2. Final maps of soil properties based on their best geostatistical methods: A, organic carbon content (OC) at 0.0–0.10-m depth by regression kriging (RK), and at 0.10–0.20 and 0.20–0.40 m by ordinary kriging (OK); B, bulk density (BD) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by OK; C, organic carbon stock (OCS) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by OK; D, pH at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by RK; and E, cation exchange capacity (CEC) at 0.0–0.10 and 0.10–0.20 m by RK, and at 0.10–0.20 m by OK. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 , , , , p , DOI: 10.1590/S0100-204X2016000900036 1382 G.M. Vasques et al. operties based on their best geostatistical methods: A, clay content at 0.0–0.10, 0.10–0.20, and 0.2 0 m by OK, at 0.10–0.20 m by regression kriging (RK), and at 0.10–0.20 m by OK; C, sand co ter retention at field capacity (WFC) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by RK; E, water r –0.20, and 0.20–0.40 m by OK; and F, available water (AW) at 0.0–0.10 and 0.10–0.20 m by RK li 51 9 1371 1385 2016 operties based on their best geostatistical methods: A, clay content at 0.0–0.10, 0.10–0.20, and 0.20 10 m by OK, at 0.10–0.20 m by regression kriging (RK), and at 0.10–0.20 m by OK; C, sand co ater retention at field capacity (WFC) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by RK; E, water re –0.20, and 0.20–0.40 m by OK; and F, available water (AW) at 0.0–0.10 and 0.10–0.20 m by RK Figure 3. Final maps of soil properties based on their best geostatistical methods: A, clay content at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by ordinary kriging (OK); B, silt content at 0.0–0.10 m by OK, at 0.10–0.20 m by regression kriging (RK), and at 0.10–0.20 m by OK; C, sand content at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by OK; D, water retention at field capacity (WFC) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by RK; E, water retention at permanent wilting point (PWP) at 0.0–0.10, 0.10–0.20, and 0.20–0.40 m by OK; and F, available water (AW) at 0.0–0.10 and 0.10–0.20 m by RK, and at 0.10–0.20 m by OK. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 , , , , p , DOI: 10.1590/S0100-204X2016000900036 (1)OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW, available water (volumetric percentage). Conclusions GRUNWALD, S. Multi-criteria characterization of recent digital soil mapping and modeling approaches. Geoderma, v.152, p.195- 207, 2009. DOI: 10.1016/j.geoderma.2009.06.003. 1. Soil property patterns are linked to each other, as well as to topographic, geologic, and vegetation patterns in a tropical dry forest area in Brazil. GRUNWALD, S.; VASQUES, G.M.; RIVERO, R.G. Fusion of soil and remote sensing data to model soil properties. Advances in Agronomy, v.131, p.1-109, 2015. DOI: 10.1016/ bs.agron.2014.12.004. 2. Ordinary kriging is a relatively simple and efficient method to create soil property maps, but it is conditioned to a good sampling design, since, contrary to regression kriging, it only relies on soil samples and not on environmental covariates. HENGL, T.; JESUS, J.M. de; MACMILLAN, R.A.; BATJES, N.H.; HEUVELINK, G.B.M.; RIBEIRO, E.; SAMUEL-ROSA, A.; KEMPEN, B.; LEENAARS, J.G.B.; WALSH, M.G.; GONZALEZ, M.R. SoilGrids1km – global soil information based on automated mapping. PLoS ONE, v.9, p.e105992, 2014. DOI: 10.1371/journal.pone.0105992. HENGL, T.; JESUS, J.M. de; MACMILLAN, R.A.; BATJES, N.H.; HEUVELINK, G.B.M.; RIBEIRO, E.; SAMUEL-ROSA, A.; KEMPEN, B.; LEENAARS, J.G.B.; WALSH, M.G.; GONZALEZ, M.R. SoilGrids1km – global soil information based on automated mapping. PLoS ONE, v.9, p.e105992, 2014. DOI: 10.1371/journal.pone.0105992. 3. There is no straightforward decision on whether to use ordinary kriging or regression kriging for mapping soil properties, because the quality of predictions depends on soil property, number and distribution of samples, environmental covariates, and soil-landscape correlations, among other factors, which indicates that, for similar studies, the employed geostatistical methods should be compared on a case-by-case basis. KNOTTERS, M.; BRUS, D.J.; OUDE VOSHAAR, J.H. A comparison of kriging, co-kriging and kriging combined with regression for spatial interpolation of horizon depth with censored observations. Geoderma, v.67, p.227-246, 1995. DOI: 10.1016/0016-7061(95)00011-C. KRAVCHENKO, A.N.; ROBERTSON, G.P. Can topographical and yield data substantially improve total soil carbon mapping by regression kriging? Agronomy Journal, v.99, p.12-17, 2007. DOI: 10.2134/agronj2005.0251. References regression model was relatively strong. The same behavior was observed in the present study. In two contrasting landscapes (agricultural versus forested) in the United States, RK was superior to OK only when the spatial structure could not be well captured by point-based observations or when a strong relationship existed between the target soil property and the covariates (Zhu & Lin, 2010). In the same country, in Florida, Vasques et al. (2010) observed that the preference for RK over OK depended on the depth of the measurement and on the regression method used to estimate soil total carbon in a 3,585-km2 watershed. Studies in tropical dry forest with similar extents or sampling densities were not found to compare with the results obtained in the present study. BOUCNEAU, G.; VAN MEIRVENNE, M.; THAS, O.; HOFMAN, G. Integrating properties of soil map delineations into ordinary kriging. European Journal of Soil Science, v.49, p.213-229, 1998. DOI: 10.1046/j.1365-2389.1998.00157.x. COELHO, M.R.; DART, R. de O.; VASQUES, G. de M.; TEIXEIRA, W.G.; OLIVEIRA, R.P. de; BREFIN, M. de L.M.; BERBARA, R.L.L. Levantamento pedológico semi-detalhado (1:30.000) do Parque Estadual da Mata Seca, município de Manga-MG. Rio de Janeiro: Embrapa Solos, 2013. 264 p. (Embrapa Solos. Boletim de pesquisa e desenvolvimento, 217). BERBARA, R.L.L. Levantamento pedológico semi-detalhado (1:30.000) do Parque Estadual da Mata Seca, município de Manga-MG. Rio de Janeiro: Embrapa Solos, 2013. 264 p. (Embrapa Solos. Boletim de pesquisa e desenvolvimento, 217). DONAGEMA, G.K.; CAMPOS, D.V.B. de; CALDERANO, S.B.; TEIXEIRA, W.G.; VIANA, J.H.M. (Org.). Manual de métodos de análise de solos. 2.ed. rev. Rio de Janeiro: Embrapa Solos, 2011. 230p. (Embrapa Solos. Documentos, 132). FILELLA, I.; PEÑUELAS, J. The red edge position and shape as indicators of plant chlorophyll content, biomass and hydric status. International Journal of Remote Sensing, v.15, p.1459-1470, 1994. DOI: 10.1080/01431169408954177. FILELLA, I.; PEÑUELAS, J. The red edge position and shape as indicators of plant chlorophyll content, biomass and hydric status. International Journal of Remote Sensing, v.15, p.1459-1470, 1994. DOI: 10.1080/01431169408954177. Results and Discussion Vasques et al. 1384 Results and Discussion Property Depth (m) Model Nugget Sill Range (m) Model Nugget Sill Range (m) Original property SMLR residuals OC 0.00–0.10 Gaussian 26.45 72.49 6,116 Gaussian 18.83 29.26 4,345 0.10–0.20 Spherical 16.09 39.22 10,152 Spherical 15.98 26.55 9,722 0.20–0.40 Spherical 3.24 6.80 8,284 Spherical 3.27 4.60 7,038 BD 0.00–0.10 Spherical 0.01 0.02 3,017 Spherical 0.01 0.02 3,707 0.10–0.20 Spherical 0.004 0.03 3,337 Spherical 0.005 0.01 1,884 0.20–0.40 Spherical 0.01 0.03 3,225 Spherical 0.01 0.01 1,473 OCS 0.00–0.10 Spherical 0.27 0.57 7,881 Gaussian 0.15 0.20 4,118 0.10–0.20 Spherical 0.19 0.35 7,785 Spherical 0.11 0.19 6,122 0.20–0.40 Spherical 0.28 0.70 8,709 Spherical 0.23 0.34 7,380 pH 0.00–0.10 Exponential 0.18 0.63 1,092 Spherical 0.25 0.32 3,000 0.10–0.20 Exponential 0.16 0.69 881 Spherical 0.20 0.39 1,122 0.20–0.40 Exponential 0.23 0.80 1,099 Spherical 0.32 0.42 4,048 CEC 0.00–0.10 Spherical 9.49 46.83 11,045 Spherical 7.65 18.40 8,079 0.10–0.20 Spherical 10.16 40.17 9,201 Spherical 7.19 12.33 8,059 0.20–0.40 Spherical 10.13 38.18 7,857 Spherical 7.71 13.15 8,270 Clay 0.00–0.10 Exponential 2,152 19,609 3,776 Spherical 3635 5382 6,068 0.10–0.20 Exponential 1,128 21,955 3,218 Spherical 3755 6385 6,181 0.20–0.40 Exponential 2,689 29,258 5,967 Gaussian 3678 7079 6,197 Silt 0.00–0.10 Gaussian 3,697 20,910 8,156 Spherical 2720 4814 10,423 0.10–0.20 Gaussian 3,599 19,458 8,572 Spherical 2196 4274 8,501 0.20–0.40 Gaussian 2,404 7,504 4,237 Spherical 1230 2322 5,046 Sand 0.00–0.10 Spherical 7,289 62,729 13,402 Gaussian 8644 13901 4,371 0.10–0.20 Spherical 7,833 62,357 12,733 Spherical 7633 14209 9,840 0.20–0.40 Spherical 6,432 52,580 11,538 Gaussian 3779 6203 5,073 WFC 0.00–0.10 Gaussian 12.98 53.93 5,433 Gaussian 8.69 19.53 4,478 0.10–0.20 Gaussian 11.71 55.26 6,701 Spherical 6.36 10.46 7,367 0.20–0.40 Gaussian 13.14 49.49 5,551 Spherical 4.00 11.00 1,500 PWP 0.00–0.10 Spherical 4.65 41.63 10,734 Gaussian 5.17 11.68 4,368 0.10–0.20 Spherical 4.10 44.48 12,691 Spherical 3.00 8.15 3,000 0.20–0.40 Spherical 5.04 44.89 9,616 Spherical 6.63 11.91 8,068 AW 0.00–0.10 Gaussian 3.66 9.66 2,432 Gaussian 3.70 7.49 2,502 0.10–0.20 Spherical 1.99 6.32 3,274 Spherical 2.01 2.66 6,562 0.20–0.40 Spherical 1.95 7.16 5,239 Spherical 1.71 2.70 6,947 (1)OC, organic carbon content (g kg-1); BD, bulk density (kg dm-3); OCS, organic carbon stock (kg m-2); CEC, cation exchange capacity (cmolc kg-1); clay, silt, and sand contents (g kg-1); WFC, water retention at field capacity (volumetric percentage); PWP, water retention at permanent wilting point (volumetric percentage); and AW available water (volumetric percentage) Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 , , , , p , DOI: 10.1590/S0100-204X2016000900036 G.M. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Acknowledgements LEVANTAMENTO aerogeofísico do Estado de Minas Gerais: área 11A (Jaíba – Montes Claros – Bocaiúva). Rio de Janeiro: Serviço Geológico do Brasil, 2009. To Dr. Mário Marcos do Espírito Santo, Dr. Ricardo Luís Louro Berbara, Dr. Maria de Lourdes Mendonça Santos Brefin, to Instituto Estadual de Florestas (IEF) of the state of Minas Gerais, Brazil, to the staff of Parque Estadual da Mata Seca, and to Serviço Geológico do Brasil (CPRM), for support; and to Embrapa (grant No. 03.10.06.013.00.00) and Inter-American Institute for Global Change Research-United States National Science Foundation (grant No. GEO-04523250), for financial support. MILES, L.; NEWTON, A.C.; DEFRIES, R.S.; RAVILIOUS, C.; MAY, I.; BLYTH, S.; KAPOS, V.; GORDON, J.E. A global overview of the conservation status of tropical dry forests. Journal of Biogeography, v.33, p.491-505, 2006. DOI: 10.1111/j.1365- 2699.2005.01424.x. MINASNY, B.; MCBRATNEY, A.B. A conditioned Latin hypercube method for sampling in the presence of ancillary information. Computers and Geosciences, v.32, p.1378-1388, 2006. DOI: 10.1016/j.cageo.2005.12.009. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Mapping soil carbon, particle-size fractions 1385 R CORE TEAM. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2015. MURPHY, P.G.; LUGO, A.E. Ecology of tropical dry forest. Annual Review of Ecology and Systematics, v.17, p.67-88, 1986. DOI: 10.1146/annurev.es.17.110186.000435. SOIL SURVEY STAFF. Keys to soil taxonomy. 12nd ed. Washington: United States Department of Agriculture, 2014. OLIVEIRA, C.V.; KER, J.C.; FONTES, L.E.F.; CURI, N.; PINHEIRO, J.C. Química e mineralogia de solos derivados de rochas do Grupo Bambuí no norte de Minas Gerais. Revista Brasileira de Ciência do Solo, v.22, p.583-593, 1998. DOI: 10.1590/S0100-06831998000400003. SOUZA, J.P. de; ARAÚJO, G.M.; HARIDASAN, M. Influence of soil fertility on the distribution of tree species in a deciduous forest in the Triângulo Mineiro region of Brazil. Plant Ecology, v.191, p.253-263, 2007. DOI: 10.1007/s11258-006-9240-2. OLIVEIRA-FILHO, A.T.; CURI, N.; VILELA, E.A.; CARVALHO, D.A. Effects of canopy gaps, topography, and soils on the distribution of woody species in a central Brazilian deciduous dry forest. Biotropica, v.30, p.362-375, 1998. DOI: 10.1111/j.1744- 7429.1998.tb00071.x. VASQUES, G.M.; GRUNWALD, S.; COMERFORD, N.B.; SICKMAN, J.O. Regional modelling of soil carbon at multiple depths within a subtropical watershed. Geoderma, v.156, p.326- 336, 2010. DOI: 10.1016/j.geoderma.2010.03.002. PEZZINI, F.F.; RANIERI, B.D.; BRANDÃO, D.O.; FERNANDES, G.W.; QUESADA, M.; ESPÍRITO-SANTO, M.M.; JACOBI, C.M. Changes in tree phenology along natural regeneration in a seasonally dry tropical forest. Plant Biosystems, v.148, p.965-974, 2014. DOI: 10.1080/11263504.2013.877530. ZHU, Q.; LIN, H.S. Pesq. agropec. bras., Brasília, v.51, n.9, p.1371-1385, set. 2016 DOI: 10.1590/S0100-204X2016000900036 Acknowledgements Comparing ordinary kriging and regression kriging for soil properties in contrasting landscapes. Pedosphere, v.20, p.594-606, 2010. DOI: 10.1016/S1002-0160(10)60049-5. Received on August 27, 2015 and accepted on January 27, 2016
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Book review: Savannas of Our Birth: People, Wildlife, and Change in East Africa
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Book details Reid, RS Savannas of Our Birth: People, Wildlife, and Change in East Africa. Berkeley, CA: University of California Press; 2012. Hardcover, 416 pages; ISBN: 9780520273559 Keywords: Savanna ecology; Human-environment interaction; Environmental change The influence of human activity on ecological systems has long been a polarizing topic within the scientific community and beyond. Savanna ecosystems in particu- lar highlight much that is at stake in such debates: often romanticized pristine untouched nature on the one hand or as the site of human beginnings on the other. Varying interpretations often diverge regarding the influence and importance of humans in African savanna systems. This most often occurs along disciplinary lines, with ecolo- gists focusing on ecological processes while either ignor- ing the role of humans or demonizing their behaviors, pastoralists in particular. Social scientists, in contrast, often focus on the ways pastoralists work and survive in such areas, with only cursory treatment of the complex ecologies they continue to co-create. It is therefore re- freshing - perhaps even inspiring - to find an analysis of East African savannas where various camps of thought are placed in conversation and a broad spectrum of posi- tions are embraced as Robin Reid so eloquently does in Savannas of Our Birth: People, Wildlife and Change in East Africa. In this path-breaking book, Reid pursues a middle ground that blends dialogues from conservation science, pedology, and disturbance ecology with human ecology, anthropology, and human geography. Reid also blurs the boundaries between popular and scientific literature and knowledge by writing in an accessible manner (endnotes are used to keep references from cluttering the text) and by weaving the words of pasto- ralists into the text itself. The book addresses the over-arching question of how humans can be seen as part of the savanna ecosystem. In doing so, Reid refers to global discourses on pastoralism, wildlife conservation, desertification, wildlife poaching, climate change, human-wildlife conflict, and population growth, amongst other topics. Reid combines her own empirical work as an ecologist in Kenya and Tanzania with the work of other scholars, as well as the views of pasto- ralists with whom she has worked. Savannas of Our Birth is a powerful book for teaching and research on human- environment interactions and conservation, while also contributing to policymaking as well as conservation and development practice. © 2014 Lovell and Goldman; licensee Springer. 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. Book review: Savannas of Our Birth: People, Wildlife, and Change in East Africa Eric J Lovell1* and Mara J Goldman1,2 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 * Correspondence: eric.lovell@colorado.edu 1Department of Geography, University of Colorado, Boulder, CO, USA Full list of author information is available at the end of the article Book details While this analysis provokes greater research into the spatially and temporally dynamic ecologies of human- wildlife interactions, it demonstrates that the existence of wildlife cannot be separated from land tenure re- gimes, land-use management, and culture. Reid uses Chapters 6 and 7 to expand her analysis of human-wildlife interactions, by asking when, why, and how pastoralists, livestock, and wildlife co-exist. Reid ad- vances her argument towards a middle ground by pre- senting positive and negative perspectives of grazing pressures on the foundation of savanna ecologies. Indi- genous management regimes are presented that have been used by herders to sustain grazing pressures on the rangelands and maintain a compatible relationship with grazing wildlife. She also suggests that this balance has been challenged by shifting livelihood practices, from herding towards crop cultivation. The diversification of livelihood practices has altered the how, when, and where humans, livestock, and wildlife can co-exist. Reid uses the subsequent chapters to present African savannas as multifaceted and complex ecosystems, dri- ven by variegated rainfall regimes and/or disturbances (i.e. infectious diseases, fires). She provides an in-depth analysis of the physical geography and biophysical pro- cesses that constitute African savannas and situates them as part of the essential building blocks of the cre- ation of life. By starting at the Pre-Cambrian period and moving into what is now considered the Anthropocene, a period dominated by humans as ‘ecosystem engineers’, Reid balances the role of nonhumans and humans in the engineering of African savannas. The East African sa- vanna and the Great Rift Valley, in particular, are noted as the site of the oldest footprints of humankind, a relic of society's foundation. Reid uses this long telescope on our history to connect the reader to the African savanna as the nucleus of human society. The most encapsulating process that has shifted human- wildlife relations across African rangelands has been the emergence of management strategies that align with Western ideals of protecting ‘nature’. In the four chapters (Chapters 8 to 11) that lead up to the concluding chapter, Reid examines specific case studies of parks and protected areas that occupy approximately 10% of the East African savanna and that happen to be where humans, livestock, and wildlife congregate. Her analysis illustrates how land- scapes that have been set aside under the veil of biological preservation and the protection of ‘natural’ ecological systems have been driven by Western ideals of nature. Book details It is well suited for teaching in both undergraduate and graduate classes and the chapters can be used independently. We used Chapter 2 in an upper- level undergraduate course on Africa with great success. Inevitably, taking on a project with such a large scope means that not all areas of inquiry will be covered in ne- cessary depth. We suggest that an important area that could have received more attention in the book includes a comprehensive analysis on the global political eco- nomic aspects of savanna ecosystems. Our review and critique comes from the perspective of nature-society geographers with a political ecology focus. This means that we are both delighted to see such an interdisciplin- ary endeavor and yet yearning for more recognition of politics and power. Chapter 1 is perhaps Reid's most notable contribution to studies of human-environment interactions. In this * Correspondence: eric.lovell@colorado.edu 1Department of Geography, University of Colorado, Boulder, CO, USA Full list of author information is available at the end of the article Page 2 of 3 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 chapter, Reid introduces the project's core concept of understanding savannas from the middle ground, which unfolds in greater detail throughout the remaining 11 chapters. The middle ground is presented as an inter- mediary perspective that rests between those who value pastoral livelihood practices (human-centric) and those who value the protection of wildlife (eco-centric), the latter of which coincides with the Western philosophical notions that humans should be separate from nature. The middle ground perspective is, in many ways, an at- tempt to demystify the distinction between nature and culture by reimagining a savanna where human activities play a fundamental role in the construction of the savanna ecosystems that we understand today. This position moves scientific interpretations of savanna ecology away from early equilibrium-based thinking that has predominately shaped enduring global discourses, such as desertification, (neo)Malthusianism, and Hardin's tragedy of the com- mons, to instead engage with place-based and empirically driven connections among international social, political, and economic processes and landscape change. Tanzanian rangelands come through in these sections as her own methodologies and research projects have led to fascinating discoveries of human-wildlife dynamics. For instance, by conducting nighttime population counts, Reid and colleagues have revealed astonishing patterns of coexistence between wildlife grazing and human settle- ment. Book details Reid uses the iconic landscapes of Kenya and Tanzania (Serengeti-Mara, Amboseli, and Ngorongoro) to demon- strate how the engineering of these landscapes for conser- vation has actually failed to capture ‘wild Africa’ through the very act of excluding the essential ‘ecosystem engineer’, humankind. She thus adds to a growing literature that challenges the Western conservation model by illustra- ting how pastoralism is essential to a healthy savanna ecosystem. In Chapters 3 to 5, Reid provides a detailed analysis of the livelihood practices common in savanna ecosystems. In some instances, Reid steps out of Africa entirely to demonstrate the similarities between African nomadic systems and nomadic movements found in central Asia during large-scale climatic perturbations. While much anthropological work has focused on nomadism and stra- tegies of mobility, Reid's constant engagement with the cultural and political ecological contexts more closely mat- ches the work of Galaty and Johnson (1990) and Turner (2004), respectively. For Reid, the middle ground perspective - of maintain- ing savannas for people and wildlife - can be accom- plished through moderation, a skill that Reid so strongly conveys in her book. Moderation means moving away from blaming pastoralists for environmental degradation towards understanding the ways that pastoral practices may in fact facilitate a greater net primary productivity for savanna systems. However, moderation also means Central to the analysis in these chapters is the close relationships among livestock herders, livestock, and mi- gratory wildlife, particularly grazing and ungulate popu- lations. Reid's long-term experience and commitment to socially engaged ecological research in the Kenyan and Page 3 of 3 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 Lovell and Goldman Pastoralism: Research, Policy and Practice 2014, 4:14 http://www.pastoralismjournal.com/content/4/1/14 acknowledging that there are limits to the magnitude and extent of grazing pressures that savanna systems can tolerate. A tolerance - in Reid's words - between pastoralists and ecosystem health (including wildlife) has always existed in African savannas. When there is recognizable degradation in ecosystem health, wildlife dynamics, or livelihood security, this is a sign that the tolerance is in decline and needs to be strengthened. Reid suggests strengthening savanna tolerance by build- ing the capacity of local and international pastoral insti- tutions. Author details 1 Author details 1Department of Geography, University of Colorado, Boulder, CO, USA. 2Institute for Behavioral Studies, University of Colorado, Boulder, CO, USA. 1Department of Geography, University of Colorado, Boulder, CO, USA. 2Institute for Behavioral Studies, University of Colorado, Boulder, CO, USA. Received: 12 August 2014 Accepted: 16 August 2014 Received: 12 August 2014 Accepted: 16 August 2014 Book details This includes relying more on the voices and knowledges of pastoralist leaders, lawyers, and activists and viewing pastoralists as dynamic, highly educated (formally and informally), and innovative women and men that strive for opportunities within and beyond the savanna ecosystem. interactions in African savanna ecosystems. The book is long and dense, making it sometimes hard to get through. However, Reid's willingness to engage with different epis- temologies, lifelong dedication to the study of savanna ecology, and her commitment to a socially and ecologic- ally balanced and just perspective on human-environment relations results in a job well done. Savannas of Our Birth: People, Wildlife, and Change in East Africa is an excep- tional project that warrants multiple readings by students and researchers alike. The scattered empirical findings and in-depth descriptions of indigenous land-use manage- ment position the project to be a necessary text for conser- vation biology, anthropology, and geography curriculums. Robin Reid is to be thanked for securing the space for pastoralists within ecology and strengthening the ground for understanding human-environment systems that con- stitute the African savanna. If social science has contributed anything to studies of nature-society, it is that the shifting biophysical dynam- ics of the savanna ecosystem are intricately linked to multi-scalar social and political systems - systems that are also in constant flux. For Reid, an ecologist by train- ing, presenting the middle ground means engaging with the complex nature of such systems that are often ex- cluded from ecological studies. With this in mind, Reid has beautifully enfolded the political and the social dimen- sions into the ecological - a task that is tremendously daunting for many scientists and researchers. Even from the perspective of social scientists, Reid gives equity to the ecological and the socio-political, in a balance that many political ecologists struggle to maintain. Competing interests The authors declare that there is no competing interests. Competing interests The authors declare that there is no competing interests. References Galaty, John G, and Douglas L Johnson. 1990. The world of pastoralism: Herding systems in comparative perspective. New York: The Guilford Press. Turner, Matthew D. 2004. The political ecology and the moral dimensions of “resource conflicts”: The case of farmer-herder conflicts in the Sahel. Political Geography 23:863–889. One area where Reid's analysis could have taken a step further is wrangling with the global political economic processes that influence savanna systems. The most ob- vious route to connect savannas to global processes is through conservation. Conservation is certainly front and centre to Reid's analysis. However, one may find that while she does provide a detailed analysis of how the Western gaze has shaped African savannas as pristine nature in need of saving, she does not emphasize how contemporary institutions continue to perpetuate such visions. These institutions include not only international conservation organizations but also media outlets, tourist pressures through social media movements, and growing concerns over international security (i.e. ‘Africa's War on Poaching’). The ever-growing connections between the African savanna and global political economy may unravel endless connections extending beyond Reid's original focus for this book. Yet, more direct connections to contemporary networks that shape discourses of ecol- ogy, conservation, and human-wildlife interaction may place her work in better conversation with domestic and foreign policies. doi:10.1186/s13570-014-0014-5 Cite this article as: Lovell and Goldman: Book review: Savannas of Our Birth: People, Wildlife, and Change in East Africa. Pastoralism: Research, Policy and Practice 2014 4:14. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com Reid's inclusive analysis is one of the most comprehensive and well-balanced examinations of human-environment
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https://hal.archives-ouvertes.fr/hal-03188179/file/539073-Design%20as%20an%20interactive%20boundary%20object_2020-Journal_of_Organization_Design.pdf
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Design as an interactive boundary object
Journal of organization design
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To cite this version: Thinley Tharchen, Raghu Garud, Rebecca L. Henn. Design as an interactive boundary object. Journal of Organization Design, 2020, 9, 34 p. ￿hal-03188179￿ Thinley Tharchen, Raghu Garud, Rebecca L. Henn. Design as an interactive boundary object. Journal of Organization Design, 2020, 9, 34 p. ￿hal-03188179￿ Abstract What is it about the term ‘design’ that facilitates the emergence of interdisciplinary interactions even though the term may hold different meanings for those involved? To address this question, we analyzed the vocabularies, practices and orders of worth  proposed by the members of an interdisciplinary Center for Design. Our analysis revealed similarities and differences in the meanings accorded by these individuals to the term design. The analysis also revealed an awareness on their part that their notions of design were incomplete, and that they had to rely on the inputs of others. Such reflexivity was an important factor in fostering meaningful interactions between these individuals. Based on these findings, we argue that design is an interactive bound- ary object, which enables different meaning structures to co-exist and co-inform actors from multiple disciplines and domains. Within such a view, the emergence of interac- tions occurs not despite but because of the diversity of views about the notion of design itself. Keywords:  Design, Vocabularies, Practices, Orders of worth, Design games, Reflexivity, Interactive boundary object Keywords:  Design, Vocabularies, Practices, Orders of worth, Design games, Reflexivity, Interactive boundary object The term ‘design’ is of considerable interest to academics and practitioners alike. For instance, well-known organizations such as Apple, Samsung and PepsiCo have been implementing design thinking and practices (Ignatius 2015; Kolko 2015; Yoo and Kim 2015). In management, a robust body of literature has now emerged around design prac- tices (e.g., Boland and Collopy 2004; Burton et al. 2006; Liedtka and Ogilvie 2011; Mar- tin 2009). A review of these developments highlights how design thinking and practices are key to the emergence of organizational cultures enabling innovations (Elsbach and Stigliani 2018; Garud et al. 2006). Riding on this wave of promise around design, we investigate how design and emer- gence are interrelated. Of particular interest is the ability of actors from different dis- ciplines to continue interacting with one another even when individuals might accord different meanings to the notion of design including technical interoperability, effi- ciency, aesthetics, customer satisfaction, and societal sustainability. Indeed, understand- ing how and why such interactions occur is all the more intriguing given that some of these meanings may be at odds with one another. What is it about design that makes it possible for interdisciplinary interactions to emerge given that the term itself has multiple meanings? © The Author(s) 2020. 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://creat​iveco​ mmons​.org/licen​ses/by/4.0/. Design as an interactive boundary object Thinley Tharchen1*  , Raghu Garud2 and Rebecca L. Henn3 *Correspondence: tharchen@em‑lyon.com 1 Emlyon Business School, 23 Avenue Guy de Collongue, 69130 Écully, France Full list of author information is available at the end of the article *Correspondence: tharchen@em‑lyon.com 1 Emlyon Business School, 23 Avenue Guy de Collongue, 69130 Écully, France Full list of author information is available at the end of the article RESEARCH Open Access HAL Id: hal-03188179 https://hal.science/hal-03188179v1 Submitted on 1 Apr 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. Tharchen et al. J Org Design (2020) 9:21 https://doi.org/10.1186/s41469-020-00085-w Abstract To address this question, we build on the “linguistic turn” in social science research (Alvesson and Kärreman 2000) Tharchen et al. J Org Design (2020) 9:21 Page 2 of 34 wherein language and vocabularies are constitutive of meaning (e.g., Douglas 1986; Knorr-Cetina 1999; Wittgenstein 2009). Not only are practices implicated in the lan- guage we use (Loewenstein et al. 2012; Pickering 1993, 1995), but, in addition, language and vocabularies constitute the orders of worth (Boltanski and Thévenot 2006; Stark 2009) by which a community values what it does and establishes its identity (Garud and Rappa 1994; Jones and Livne-Tarandach 2008). Based on this understanding, we explored the vocabularies, practices and orders of worth employed by individuals affiliated with a Center for Design at a large public uni- versity. The Center attracted the participation of individuals from different disciplines with self-declared interests in exploring questions around design as it relates to educa- tion and research. Consequently, this setting served as an exemplary case (Yin 2003), one that allowed us to investigate how design could provide both interpretive flexibility, and yet still hold value for interdisciplinary interactions to emerge. The findings from this study confirmed our assumption that individuals across dis- ciplines differed in the meanings they accorded to design. Building on Wittgenstein’s notion of language games, we label the combination of vocabularies, practices, and orders of worth around which differences in meanings surfaced as constituting design games. Yet, we also found “common ground” (Puranam et  al. 2009) and “interlacing” (Tuertscher et al. 2014) across design games. Besides this glue holding them together, we found individuals to be “reflexive” (Cunliffe 2003; Cunliffe and Jun 2005) in their accounts of what it meant to design. Specifically, our informants highlighted an incom- pleteness in their own understandings of design. Such reflexivity allowed them to appre- ciate the value of others’ expertise, which, in turn generated interactions. By highlighting how design can induce such a culture of interdisciplinary interactions, our findings speak to the link between design and emergence (e.g., Elsbach and Stigli- ani 2018; Garud et al. 2008; Hunter et al. 2020; Koçak and Puranam 2018). Specifically, polycentricity implicated in the notion of design enabled a culture of reflexive interac- tions wherein design served as an interactive boundary object allowing different mean- ing structures to co-exist. Background and inquiry framework In The Sciences of the Artificial (Simon 1996), Simon offered a theory of design as a sci- entific enterprise for the creation of artifacts that are adapted to human goals and pur- poses. Given bounded rationality, Simon (1996) formulated “near decomposability” as the principle underlying the partitioning of complex systems into sub-problems (Alex- ander 1964; Parnas 1972) held together by an architecture (Baldwin and Clark 2000; Garud et al. 2008). Though such an approach to design finds its early roots in the man- agement writings on the division of labor and administrative hierarchy (e.g., Simon 1947; Taylor 1911), the literature on organization design (Burton and Obel 2018; Miles and Snow 1978; Nadler and Tushman 1997) and product design (Baldwin and Clark 2000; Sanchez and Mahoney 1996; Ulrich 1995) highlight the concept of modularity as a dis- tinguishing facet of this approach. Modularity, which draws on the mirroring hypothesis (Colfer and Baldwin 2010), advocates “information hiding” such that “each module is informationally self-sufficient, [and] hence can be designed independently of the rest of the system” (Colfer and Baldwin 2010: 4). Once interface specifications between mod- ules have been specified, modularity will lead to reduced dependencies or need for com- munication across design teams. Though design using the principles of modularity has received empirical support (Colfer and Baldwin 2010), studies show that self-organized groups also make contribu- tions across the entire system (Garud and Kotha 1994; Tuertscher et al. 2014). Moreover, certain technological problems may not be decomposable (Colfer and Baldwin 2010), and hence require not “information hiding”, but “richly connected contributions” (Colfer and Baldwin 2010: 19) across participating actors. Indeed, modularization, if taken too far, may also make the design process predictable, and so reduce the likelihood of breakthrough innovations (Fleming and Sorenson 2001). Furthermore, robust designs (Hargadon and Douglas 2001) do not emerge in splendid isolation, but in and through interactions with other participating actors. These observations suggest that design, rather than being a stable architecture built around pre-specified "design rules" (Baldwin and Clark 2000), is instead an evolving system (Simon 1996) where new goals constantly emerge through interactions between human and material artifacts. Such a focus is also implicit in an approach to design that seeks to harness the benefits of multiple interacting perspectives (e.g., Boland and Col- lopy 2004; Martin 2009), with “social interaction [seen as]…a key resource of design pro- cesses” (Hatchuel 2001: 261). Abstract Most importantly, the actors were able to continue interacting with one another not despite but because of the overlapping structure of similarities and differences in the vocabularies, practices, and the orders of worth used by them across the different disciplines. f These findings hold practical implications. Design’s capacity to foster interactions among individuals across diverse disciplines can be used to approach complex problems such as sustainability, which requires the inputs and participation of various stakehold- ers. Such design-induced collaboration can enable joint problem definition as well as the co-creation of solutions that balance the competing demands of the different interest groups involved (Garud and Karnøe 2003; Garud et al. 2015; Tuertscher et al. 2014). To develop these points, we begin by considering different notions of design, start- ing with a definition that emerges from the work by Simon (1996), a scholar who has played a pivotal role in shaping management thinking in general, and design in particu- lar. After reviewing subsequent management thinking on design, we present our inquiry framework comprising vocabularies, practices, and orders of worth. We then detail the research site and methods guiding this inquiry before outlining the findings. Finally, we theorize that design is an interactive boundary object that allows different meaning Tharchen et al. J Org Design (2020) 9:21 Page 3 of 34 structures to co-exist and co-inform across disciplinary boundaries generating meaning- ful interactions. Background and inquiry framework Background and inquiry framework Individuals are “not drawn together because they share a common definition of design, a common methodology, a common philosophy, or even a common set of objects to which everyone agrees that the term "design" should be applied” (Buchanan 1992: 14), but because they are motivated to learn from and mean- ingfully contribute to each other’s perspectives. Organizational forms such as TopCoder and Wikipedia wherein multiple parties from “experts” to “hobbyists” work collectively embody such a design approach (Garud et al. 2008; Kolbjørnsrud 2018). Design seen from such  an interactional perspective is not just a set of principles, structures and tools for problem solving, but equally importantly implicates the cultural Tharchen et al. J Org Design (2020) 9:21 Tharchen et al. J Org Design (2020) 9:21 Page 4 of 34 components enabling generative interactions across different stakeholder groups and material artifacts leading to the emergence of innovative outcomes (Elsbach and Stigli- ani 2018; Garud et al. 2006). For instance, Tuertscher et al. (2014) showed how a culture of collaborative participation across groups of scientists and engineers distributed all over the world led to the design of an innovative particle detector capable of identify- ing the elusive Higgs boson particle. Relatedly, Garud and Karunakaran (2018) detailed how product design (Gmail and AdSense) and an organizational culture of participative experimentation co-emerged at Google. However, such an interactional approach to design also surfaces a paradox. Specifi- cally, in interdisciplinary interactions, the term design takes on a global meaning, which has interpretive flexibility (Pinch and Bijker 1987). At the same time, though, design is also inherently local, tied to the practices within specific disciplines. While we expect a global meaning of design to render it a “boundary object” (Star and Griesemer 1989) that enhances co-ordination across disciplinary boundaries, the local meanings that become salient during interdisciplinary interactions could on the other hand be a source of frag- menting and conflict. Indeed, conflict arising from differences in meanings across disci- plines have been the subject of books such as Architect and Engineer: A Study in Sibling Rivalry (Saint 2007) and Bridging the Gap: Rethinking the Relationship of Architect and Engineer (Building Arts Forum 1991). What is it then about design that enables interdisciplinary interactions to emerge even when the term itself implicates different meanings across multiple disciplines? Background and inquiry framework To gain an understanding of these dynamics, we used Wittgenstein’s notion of language games, i.e., “language and the activities into which it is woven” (Wittgenstein 2009: 8) to inves- tigate into the vocabularies, practices and orders of worth of individuals from different disciplinary backgrounds affiliated with an interdisciplinary Center for Design. fi Vocabularies not only constitute language, but also provide the cultural toolkits (Swidler 1986) with which social collectives constitute their identities within professions such as architecture and medical care (Dunn and Jones 2010; Jones and Livne-Tarandach 2008). They do so by functioning as “terministic screens” (Burke 1966), guiding individu- als to consider what is important within their profession (Jones and Livne-Tarandach 2008). Professional architects, for instance, use vocabularies from certain cultural reg- isters to appeal to their clients and audiences (Jones and Livne-Tarandach 2008). Thus, examining vocabularies provides a window into the values and practices of collectives shaping their thoughts and actions (Loewenstein et  al. 2012). Indeed, translating the importance of studying vocabularies in the context of design, Boland and Collopy (2004: 14) noted: “engaging in good design is choosing a vocabulary or language to use in defin- ing the design task, generating alternatives, and making judgments of balance, fit, and scale.” Boland and Collopy’s (2004) observation regarding defining the design task and gen- erating alternatives speaks to the practices implicated in the vocabularies used in design. A consideration of practices goes beyond a representational view of language as a mir- ror of reality by taking a performative turn wherein words ‘do things’ (Austin 1975), and where such sayings and doings involve interactions with material tools (Barad 2003; Pickering 1995) generating “different domains of possible action” (Nicolini 2011: 616). In the context of design, these material tools may involve the use of persuasive material Tharchen et al. J Org Design (2020) 9:21 Page 5 of 34 artifacts that “carry conviction for the design of a particular solution, invite others into a dialogue, stimulate their imagination, and facilitate and accommodate their contribu- tions” (Wagner 2004: 159). Consequently, by examining the vocabularies used by those engaged in design, it is possible to gain an appreciation of interwoven practices [i.e., gain “interactional expertise” (Collins and Evans 2002)]. In addition, underlying vocabularies are deeper axiological considerations (Hart 1971) that are manifest in the values that individuals across different disciplines associate with design. Background and inquiry framework This relates to the second observation of Boland and Collopy (2004) regarding making judgments on design. The judgments that individuals make while evaluating or justifying the outcomes of any activity invoke “orders of worth” (Boltanski and Thévenot 2006) employing different “evaluation criteria” (Garud and Rappa 1994) (such as effi- ciency, market success, innovation, public recognition, family tradition, and collective interests, respectively). When it comes to design, an interdisciplinary concept, orders of worth are all the more salient, as individuals must be accountable to others during their interactions, i.e., justify their “beliefs, feelings, and actions to others” (Ferraro et al. 2005: 17). Moreover, as Wittgenstein (2009: 94) noted: "It is not only agreement in definitions, but also (odd as it may sound) agreement in judgments that is required for communica- tion by means of language." Finally, it is in these values that a “design attitude” is thought to manifest and distinguish itself from more mainstream modes of engagement such as those guided by a “decision attitude” (Boland and Collopy 2004; Michlewski 2008). In sum, vocabularies, practices and orders of worth form the three elements of the framework that we employ in this study to inquire into the meaning of design. By exam- ining these dimensions at an interdisciplinary Center for Design, we explore the different meanings accorded to design by the different actors involved, as well as inquire into how interdisciplinary interactions emerged despite the differences that existed between the individuals. Contributing to the growing interest in design and the emergence of col- laborative organizational cultures (Elsbach and Stigliani 2018; Garud et al. 2008; Koçak and Puranam 2018), this study showcases the possibility of reflexive individuals engag- ing in interdisciplinary interactions not despite but because of the diversity in meanings associated with the term design, providing them with the opportunities to learn from one another. Research site and methods Using a case study design (Yin 2003), a Center for Design at a large public university served as the research site for this study. The Center was founded in 2008 through an NSF grant investigating “interdisciplinary design as instructional discipline” with four national workshop locations. Both the grant and the Center aimed to promote interdis- ciplinary education and collaboration across individuals from different disciplines with expertise on different areas of design such as innovation, decision-making, organiza- tions, products, systems, visualization, etc. The Center’s vision was articulated in one of its inception documents: Design is an intellectual fulcrum that integrates concepts and skills across dis- ciplines and professions to shape and reshape the world. We live in an age of design. Most disciplines practice some form of it, but in order to create truly effec- tive solutions we need people skilled in its practice who can master the breadth Tharchen et al. J Org Design (2020) 9:21 Page 6 of 34 and depth of technical knowledge and skills in the context of diverse and subtle human and societal issues. The Center will bring together diverse faculty and lev- erage, integrate, and expand a wide range of on-going interdisciplinary Design research. (emphasis added). Over the years, the collaborations among individuals affiliated with the Center have resulted in numerous publications on topics such as the logic of design, bridg- ing design cultures, product design, additive manufacturing, in addition to initiatives to launch a university-wide interdisciplinary graduate degree program in Design. Besides, the Center has also been organizing workshops on design thinking, featuring speakers from the academia and the industry. Because of the Center’s commitment to design as an interdisciplinary activity, we took advantage of this unique context— a particular exemplary case (Tsoukas 2009) to generate “theoretical refinement” on how individuals from different disciplines can come together to interact on a hybrid forum (Callon et al. 2009). Data collection We started our data collection in March 2012 in the backdrop of some familiarity with the research setting. Two among the three of us were members of the Center and had already attended some of their meetings and interdisciplinary workshops. Indeed, it was at these events that we were struck by the multiple and sometimes competing meanings of design that arose during the discussions. This piqued our interest into understand- ing how despite the differences in meaning across disciplines ‘design’ enabled individu- als from multiple disciplines to come together to interact with each other. In order to investigate this, we began approaching individual members from the Center and started to interview them. For our interviews, we theoretically and purposively sampled our informants (Glaser and Strauss 1967; Lincoln and Guba 1985) ensuring that the indi- viduals we interviewed were from different academic disciplines. We continued inter- viewing individuals at the Center until further interviews yielded no further insights, reaching theoretical saturation (Glaser and Strauss 1967; Strauss and Corbin 1998). To supplement our analysis, we analyzed the resumes of the people that we interviewed; details of our informants are in Appendix A. In all, we conducted in-depth interviews with 14 members at the Center between March and November 2012. The interviews were semi-structured, consisting of open-ended questions such as: “What do you do as a designer?”, “What words come to mind when you think about ‘design’?” (Appendix B). The interviews that we conducted lasted approximately 45 min on average, and were all audio recorded and subsequently transcribed. Data analysis We content analyzed vocabularies (Krippendorff 2004) and generated first- and second- order codes (Gioia et al. 2012) from the data. Examining vocabularies enables analysis of data at the level of individual words to surface a semantic network of design vocabular- ies. Thematic analysis by coding makes it possible for us to understand the larger theo- retical categories that the vocabularies were constitutive of. Tharchen et al. J Org Design (2020) 9:21 Page 7 of 34 Content analysis We generated a list of individual words from the interview transcripts using the Word- Stat text analysis module of QDA Miner (Péladeau 2004), a process that resulted in a large number of words. In line with prior studies that have analyzed vocabularies, we then identified words that were frequent enough (Jones and Livne-Tarandach 2008; Nag et al. 2007) in the interview transcripts to meaningfully constitute the distinctive lexi- con of a topic—design in our case. Following Merleau-Ponty’s (2012) “Phenomenology of Perception”, we found that a cutoff frequency of five occurrences of a word (in total across the 14 informants) offered a level of granularity and parsimony that generated a gestalt understating of the meaning of design as accorded by the informants. Using this cutoff, we generated an initial list of words for each informant. f We then looked at these words in context, excluding those occurrences that were unrelated to our informant’s meaning of design, including words such as common prep- ositions, articles, common descriptors, and proper nouns. We consolidated the remain- ing words by their commonly occurring variant or stem (Nag et al. 2007), for example ‘create’, ‘creative’ and ‘creativity’ collapsed into their stem ‘create’. Using this process, we came up with a distinctive list of 116 words that our informants used while talking about design, which we also verified as appropriate by reading the interview transcripts. Following this, we organized this list of words as vocabularies related to practices, and orders of worth. Separately, we also classified words that were more general to the notion of design. Table 1 shows the complete list of words across all informants that surfaced from our analysis of the interview transcripts. Additionally, we sorted these words alphabetically and transferred them into a two- mode matrix wherein the rows correspond with the individual words and the columns with informants. In other words, the cells contain the frequency of use of individual words across informants. We used the two-mode matrix as input to UCINET NetDraw (Borgatti et al. 2002) to generate a semantic network that mapped individual words to each informant as shown in Fig. 1. Coding g Besides generating this network as a tool to visualize the different meanings associated with design, we also analyzed the data to generate first-order and second-order codes (Gioia et al. 2012) following the conventions of grounded theory (Glaser and Strauss 1967). While the first-order codes were based on informants’ statements, the second- order codes distilled and assembled the first-order codes into higher order themes. The first-order coding process involved reviewing the interview transcripts to identify ini- tial concepts and ideas of the informants that were significant, using labels in the terms actually used by the informants. This process of coding continued via the constant comparative method (Glaser and Strauss 1967) whereby new data units over time and across informants were either categorized under existing codes, or with new codes when themes analytically different from existing codes surfaced. f In all, our analysis yielded 14 first-order codes. We collapsed these 14 codes into five second-order themes, which in turn were parsed across the two aggregate theo- retical categories of practices and orders of worth. Tables 2 and 3 present a summary Page 8 of 34 Tharchen et al. J Org Design (2020) 9:21 Table 1  Vocabularies of practices, orders of worth, and general design terms Table 1  Vocabularies of practices, orders of worth, and general design terms Table 1  Vocabularies of practices, orders of worth, and general design terms Practices ALGORITHM APP ARCHITECT BIM BRAINSTORM CAD CIRCUIT CIVIL CODE COGNITIVE COMPLEX COMPUTER CONSTRUCTION CONTRACTOR COPYRIGHT DEVICE DIAGRAM DISSECTION DRAW​ ELECTRICAL ENGINEER EXPERIMENT GRAPH HUMAN INDUSTRY​ ITERATE JAVA LAB LAYOUT LICENSE MAP MATH MECHANICAL MOCKUP MODEL MODULE OBJECTIVE OPTIMIZE ORGANIZATION ORGANIZE PATENT PREDICT PROBLEM PROCESSOR PROGRAM PROTOTYPE PSYCHOLOGY QUALITATIVE QUANTIFY REDESIGN REPRESENTATION REVIEW ROBOT SATELLITE SCHEDULE SIMULATE SKETCH SOFTWARE STATISTIC STRATEGY STUDIO SYSTEM TECHNIQUE TECHNOLOGY VIRTUAL VISUALIZE WELD Orders of worth AESTHETICS BEAUTY​ BUDGET BUSINESS COMFORT COST CUSTOMER EFFICIENCY ELEGANT ETHICS FUNCTION INNOVATE LEGAL LOGIC MARKET MONEY NEW NOVEL PERFORM PROFIT QUALITY RATIONAL RELIABILITY RISK TRADEOFF USE UTILITY General design terms ART​ ARTIFACT​ BUILD CLIENT CREATE CRITIQUE FEEDBACK FORM IDEA INTERACT​ INTERFACE MAKE OWNER PATTERN PLATFORM PROCESS PRODUCT​ PROJECT SPACE STAKEHOLDER THEORY TIME of this data analysis, highlighting the supporting illustrative quotes (column 1), the informant-based first-order codes (column 2), and the induced second-order themes (column 3), under each of the two aggregate theoretical categories of practices and orders of worth, respectively. Findings In this section, we provide details of the vocabularies, practices, and orders of worth used by the members of the design Center. The findings show that different disciplines have their own distinctive set of vocabularies, practices, and orders of worth associ- ated with their understanding of what it means to design. They also show a structure Tharchen et al. J Org Design (2020) 9:21 Page 9 of 34 Legend Disciplinary background of Informants Orders of Worth Practices General Design terms Cluster 1 Cluster 2 Fig. 1  Semantic network showing interlacing of vocabularies, practices and orders of worth across informants Fig. 1  Semantic network showing interlacing of vocabularies, practices and orders of worth across informants of overlapping similarities and differences across the vocabularies, practices, and orders of worth in use. Design as open-ended engagement and constantly ‘evolving’, ‘changing’, having a ‘life’, etc. Design using qualitative tools such as ‘sketches’, ‘studio’, ‘brainstorming’, ‘dia- grams’, ‘maps’, ‘layouts’, etc. Design as problem solving including meet- ing ‘objectives’ and ‘requirements’, and establishing functionality Design approaches Table 2  Data structure underlying theoretical category of practices Second-order themes Illustrative quotes Design using quantitative tools such as ‘equations’, ‘simulations’, statistical ‘mod- els’, ‘algorithms’, ‘optimization’, etc. When we teach design as a faculty group one of the funniest things is we hate projects that don’t have spreadsheets, graphs and equations. We don’t think its design unless it has spreadsheets, graphs and equations and analysis and stuff like that. (engineering designer 4) [What are the words that come to your mind when you think about design?] Innovation, time to innovation, evaluation, simulation, fidelity of representation, understanding, risk—so all these things. (civil engineer) When we teach design as a faculty group one of the funniest things is we hate projects that don’t have spreadsheets, graphs and equations. We don’t think its design unless it has spreadsheets, graphs and equations and analysis and stuff like that (engineering designer 4) Innovation, time to innovation, evaluation, simulation, fidelity of representation, understanding, risk—so all these things. (civil engineer) [H]ow do you drive that model to explore the different options and tradeoff between this set of features versus that set of features, the costs, reliability. And so from a—from a design perspective— it’s optimization, it’s visualization, those sort of things (mechanical engineer) My training was hand drawing entirely, and doing everything through sketch- ing. (architect) [S]o you have to design a site layout, that would allow for you to deliver the project per your schedule but also make sure you don’t violate anything that the owner may have as a requirement or a need (architectural engineer) I try to create an environment where when we brainstorm ideas the weirdest and goofiest ones are welcomed. (industrial psychologist) We’re trying to make students understand that before you can actually get to a physical design you have to understand the problem. (engineering designer 1) Design is just making an attempt to meet some objective (management expert 2) [D]esign is part of the process of both understanding the user’s needs and requirements (industrial engineer) [D]esign is part of the process of both understanding the user’s needs and requirements (industrial engineer) I think it’s a constantly evolving process and no—even legally it doesn’t end. Vocabularies For illustrative purposes, we zoom into the vocabularies of two of our informants, com- puter scientist and architect as shown in Fig. 2a, b, respectively. As anticipated, data anal- ysis revealed that the vocabularies used by these informants were consistent with their disciplinary training. For example, the vocabulary of computer scientist (Fig. 2a) includes words such as ‘code’, ‘platform’, ‘program’, ‘app’, ‘circuit’, ‘efficiency’, etc., while that of architect (Fig. 2b) includes words such as ‘sketch’, ‘art’, ‘representation’, ‘studio’, etc. Moreover, as noted earlier, the vocabularies of both informants contained the prac- tices (tools and artifacts such as ‘code’, ‘sketch’, etc.) and orders of worth (such as ‘effi- ciency’, ‘beauty’, etc.) of their discipline, and are hence constitutive of their design games in the discipline of computer science and architecture, respectively. Yet, as Fig. 2c shows, words such as ‘idea’, ‘make’, ‘build’, ‘model’, ‘process’, ‘theory’, ‘project’, etc., were common to both the computer scientist and the architect. This structure of overlapping similarities and differences in vocabularies was common across other informant pairs as well. To make sense of this structure of similarities and Page 10 of 34 Tharchen et al. J Org Design (2020) 9:21 Interaction with designers outside of one’s own discipline Practicesi We first identified and elaborated on the categories of practices that emerged from our grounded analyses of the data. This step then served as the basis for examining the simi- larities and differences in the practices across members of the design Center. Illustrative quotes Interaction with material artifacts such as ‘prototypes’, and ‘mockups’ Interactions And, how do we convert those [customer needs] into requirements to drive the design? By having some processes to develop various concepts, selecting those concepts, prototyping them and eventually to the final design. And, it is highly iterative. I mean at any point you may need to go back. (engineering designer 3)i [I]nnovation is defined as the implementa- tion of creative ideas, [you] come up with a new and different idea, sketch it out, prototype it, [and then] actually see it made, implemented and tested. (industrial psychologist) [I]nnovation is defined as the implementa- tion of creative ideas, [you] come up with a new and different idea, sketch it out, prototype it, [and then] actually see it made, implemented and tested. (industrial psychologist) So I interact with a lot of discipline specific designers, so circuit designers, mechani- cal hardware designers, spacecraft mechanism designers [and] with other systems engineers. (engineering designer 3) We led—the series of workshops a couple of years ago on interdisciplinary design [which] was really good exposure to—how does architecture come at the problem versus industrial design versus engineering design versus IST or some- body else? (mechanical engineer) I study designers …And so my interaction with them is really learning what they do and what their process is like and how it differs from the traditional engineer- ing model and how it can kind of incorporate some of that back together. (engineering designer 2) differences, we examined how these words were tied to the practices and orders of worth of design across disciplines. differences, we examined how these words were tied to the practices and orders of worth of design across disciplines. Table 2  (continued) Table 2  (continued) Illustrative quotes I mean there are ways the legal process keeps moving not just with a single idea that might get litigated but ideas can be pursued as follow-on inventions and creativities that iterate a process (patent lawyer) It’s really emphasizing how important design is, to get it right, because it’s something you’re going to live with—for any of the long-lived software (computer scientist) You can evaluate a design after three months, after six months and after a year and after ten years and it can succeed in a lot of different ways and fail in a lot of different ways. So you cannot anticipate all the ways in which the design will get used. (engineering designer 4) Tharchen et al. J Org Design (2020) 9:21 Page 11 of 34 Page 11 of 34 Categories of practices (industrial psychologist) Novelty There is a philosophy that is aligned with the research that I do that says at least some aspects of design can be driven by what customers want. (management expert 2) Then we go look [to] capturing customer needs—how do we convert those into requirements to drive the design (engineering designer 3) I am trained to help people conceptualize what their inventions—and in this case we might even extend that to designs—help capture them as property rights and potentially exploit them. I also think about how that is likely to impact business (patent lawyer) Market success We’ll look at the interaction of the product and the person. So it can be anything from comfort—to the interaction in terms of fun factor. Engagement is a really important fac- tor. (engineering designer 2) Another aspect of design that I’ve gotten into more [is] user interface design, you’ve got to think about “well, how is the user going to interact with this? How is that going to hap- pen?” (computer scientist) Interactivity First-order codes Categories of practices Table 2 highlights illustrative quotes supporting three major categories of practice that emerged—design tools (quantitative and qualitative), design approaches (problem-solv- ing and open-ended engagement), and interactions (with material artifacts and individu- als outside of one’s own discipline). We describe these in greater detail below. Design tools  It is not surprising that design tools emerged prominently as one of the cat- egories of practice. Consistent with the performative turn (Pickering 1993, 1995), design is to be understood in its implementation, and from this vantage point, the “tools of the trade” (Beunza and Stark 2004) are central. We found two kinds of design tools being Page 12 of 34 Page 12 of 34 Tharchen et al. J Org Design (2020) 9:21 Table 3  Data structure underlying theoretical category of orders of worth Illustrative quotes First-order codes Second-order themes A number of aspects [define a good code]. One [is] efficiency in terms of the amount of memory or processor time—space–time are [the] two aspects of efficiency. (computer scientist) We’ve got too many products already out there. There’s not enough commonality. How do we get better? How do we standardize that? How do we consolidate it?” …to try and get cost savings that are efficiency-reduced complexity? (mechanical engineer) There’s no reason you couldn’t do that to say “is this an efficient courtroom design layout” from some automated calculation. (architec- tural engineer) Product efficiency Product oriented orders of worth But I like the idea that we concentrate more on designing fewer things and more beautiful things (engineering designer 4) If you look at what goes into a really polished app, it would include things like good art- work (computer scientist) So design typically involves coming up with something that has maybe an aesthetic appeal (industrial psychologist) Visual and symbolic beauty So a design is a new description, so there is a concept of newness. (management expert 2) Maybe the redemption of the computer is that we need to find ways to—to tweak it and use it in ways that it was never intended to be used. (architect) But what I really like studying is the people and places that come up with new and different things and to me that’s always going to be interesting. Table 3  (continued) Illustrative quotes First-order codes Second-order themes I do engineering design research. And to me, that means what methods and tools, [and] processes make designers more efficient or effective. (mechanical engineer) [I have] done a lot of research in building information modeling providing practical guidance to project teams to design efficient BIM implementation strategies [and] written and developed guides that allow people to design a process for modeling for a project. (architectural engineer) Process efficiency Process oriented orders of worth We did the creative campus project—it was engineering, architecture, landscape architecture and dance …very interesting project of trying to get all those groups work- ing together [which] was fun. (mechanical engineer) Well I think what [interactions have] done is it’s helped me understand design as done in dif- ferent disciplines (engineering designer 3) Interdisciplinary interactions Engineers are notoriously bad [laughs] at understanding how important design is, because most of our curriculum here never addresses that. (computer scientist) Design specifically, I think I didn’t understand as much. And so that’s kind of what motivated me towards thinking about design and things like that more formally. (civil engineer) I think if it [design] ultimately lies somewhere it’s at the overlap of disciplines. I don’t think any discipline owns it nor should they, with it being as broad and applicable as it is to everyone. (industrial psychologist) Reflexivity Illustrative quotes Product efficiency A number of aspects [define a good code]. One [is] efficiency in terms of the amount of memory or processor time—space–time are [the] two aspects of efficiency. (computer scientist) A number of aspects [define a good code]. One [is] efficiency in terms of the amount of memory or processor time—space–time are [the] two aspects of efficiency. (computer scientist) We’ve got too many products already out there. There’s not enough commonality. How do we get better? How do we standardize that? How do we consolidate it?” …to try and get cost savings that are efficiency-reduced complexity? (mechanical engineer) We’ve got too many products already out there. There’s not enough commonality. How do we get better? How do we standardize that? How do we consolidate it?” …to try and get cost savings that are efficiency-reduced complexity? (mechanical engineer) p y g There’s no reason you couldn’t do that to say “is this an efficient courtroom design layout” from some automated calculation. (architec- tural engineer) There is a philosophy that is aligned with the research that I do that says at least some aspects of design can be driven by what customers want. (management expert 2) Market success Then we go look [to] capturing customer needs—how do we convert those into requirements to drive the design (engineering designer 3) I am trained to help people conceptualize what their inventions—and in this case we might even extend that to designs—help capture them as property rights and potentially exploit them. I also think about how that is likely to impact business (patent lawyer) We’ll look at the interaction of the product and the person. So it can be anything from comfort—to the interaction in terms of fun factor. Engagement is a really important fac- tor. (engineering designer 2) Another aspect of design that I’ve gotten into more [is] user interface design, you’ve got to think about “well, how is the user going to interact with this? How is that going to hap- pen?” (computer scientist) Page 13 of 34 Tharchen et al. J Org Design (2020) 9:21 Table 3  (continued) Interdisciplinary interactions Illustrative quotes I do engineering design research. And to me, that means what methods and tools, [and] processes make designers more efficient or effective. (mechanical engineer) I do engineering design research. And to me, that means what methods and tools, [and] processes make designers more efficient or effective. (mechanical engineer) [I have] done a lot of research in building information modeling providing practical guidance to project teams to design efficient BIM implementation strategies [and] written and developed guides that allow people to design a process for modeling for a project. (architectural engineer) We did the creative campus project—it was engineering, architecture, landscape architecture and dance …very interesting project of trying to get all those groups work- ing together [which] was fun. (mechanical engineer) g Well I think what [interactions have] done is it’s helped me understand design as done in dif- ferent disciplines (engineering designer 3) alluded to—what we label as quantitative and qualitative. Quantitative tools include mathematical and statistical representations such as ‘equations’ (engineering designer 4), ‘simulations’ (management expert 2, civil engineer), statistical ‘models’ (management expert 2, engineering designer 1), ‘algorithms’ (computer scientist), ‘optimization’ (mechan- ical engineer, civil engineer), etc. For example, an industrial engineer described the use of statistical tools in the design process: alluded to—what we label as quantitative and qualitative. Quantitative tools include mathematical and statistical representations such as ‘equations’ (engineering designer 4), ‘simulations’ (management expert 2, civil engineer), statistical ‘models’ (management expert 2, engineering designer 1), ‘algorithms’ (computer scientist), ‘optimization’ (mechan- ical engineer, civil engineer), etc. For example, an industrial engineer described the use of statistical tools in the design process: [Our] design problem basically was grounded in a lot of statistical comparisons. So we look at risk perceptions between different stakeholders and boil them down in terms of statistical comparisons—whether it be inferential statistics like t-tests, ANOVAs or clustering methods. (industrial engineer). Quantitative tools also assumed a wide variety of functions such as: predicting the per- formance of a design (management expert 2), converting customer needs into require- ments (engineering designer 3), generating tradeoffs in designs (civil engineer), etc. For some, quantitative tools were central to their practice of design. As an engineering designer mentioned: The engineering design process can be quantified and replicated, and there is evi- dence that it actually influences solutions. So, when people do not try to move Page 14 of 34 Tharchen et al. Illustrative quotes J Org Design (2020) 9:21 Vocabulary of Computer Scientist a b c Vocabulary of Architect Similarities and Differences in Vocabularies of Computer Scientist and Architect Fig. 2. a Vocabulary of computer scientist. b Vocabulary of architect. c Similarities and differences in vocabularies of computer scientist and architect Vocabulary of Computer Scientist a b c Vocabulary of Architect Similarities and Differences in Vocabularies of Computer Scientist and Architect b Vocabulary of Architect Vocabulary of Computer Scientist a c Similarities and Differences in Vocabularies of Computer Scientist and Architect Similarities and Differences in Vocabularies of Computer Scientist and Architect Fig. 2. a Vocabulary of computer scientist. b Vocabulary of architect. c Similarities and differences in vocabularies of computer scientist and architect Fig. 2. a Vocabulary of computer scientist. b Vocabulary of architect. c Similarities and differences in vocabularies of computer scientist and architect towards quantification gives me cause for concern. (engineering designer 1). In addition to quantitative tools, the data also showcased the use of multiple quali- tative tools such as ‘sketches’ (architect), ‘diagrams’ (computer scientist, engineering designer 3), ‘maps’ (architectural engineer, management expert 2), ‘layouts’ (architectural engineer), ‘word cards’ (engineering designer 2), ‘brainstorming’ (architect, industrial psy- chologist, engineering designer 1, engineering designer 3, mechanical engineer), ‘studios’ (architect, architectural engineer, mechanical engineer), and ‘interviews’ (engineering designer 2, architectural engineer) in the design practices of informants. As an example, an architect described the use of sketches in aiding improvisation: I intentionally only had a very rough sketch [of a tree house] and tried to build it like a kid would build it, which was mostly improvisational. (architect). I intentionally only had a very rough sketch [of a tree house] and tried to build it like a kid would build it, which was mostly improvisational. (architect). Other qualitative tools such as diagrams, maps, and layouts aided visualization of the design process by “map[ping] out modeling tasks that are going to be performed, Tharchen et al. J Org Design (2020) 9:21 Page 15 of 34 Page 15 of 34 and identifying information exchanges that go between those tasks” (architectural engineer). These tools also enabled effective translation of ideas to other individuals. To illustrate: So one of the tools that we use is what’s called a concept of operations diagram. It’s essentially like an executive summary of how the system is working…it’s almost like a pictorial of some complex thing and it’s got ‘Here’s how this whole thing works.’ It takes a while to develop that, and when you have that tool, it’s used to communicate between the system acquirer or the person that wants the system and the people that are developing the system to make sure that they’re on the same page. (engineering designer 3). Informants also used ‘studios’ in their design practices. An industrial psychologist dis- tinguished it from quantitative engineering tools: Studio approaches always fascinated me–just so much hands on and so much con- stant feedback and it’s just so different from what we do and what the engineers do. (industrial psychologist). Engineering designer 2 mentioned the use of ‘word cards’, and ‘interviews’ to enable the designer to get better feedback on user engagement with the design: We draw from industrial design too where people pull in words [from word cards] that they think represented their experiences with it [the design]…Or you can inter- view them afterwards and ask them “How did you feel about your engagement with this device? (engineering designer 2). In summary, the analysis of the data showcased both quantitative and qualitative tools. While the former set of tools was based on measurable facts, the latter set was based on intuitive approaches that enabled visualization, facilitated improvisation, and generated feedback during the design process. It is in the combination of these two types of tools that design appeared to unfold. Design approaches  A second category of practice had to do with the approaches par- ticipants used to design, which were manifest as problem-solving and open-ended engage- ment. I intentionally only had a very rough sketch [of a tree house] and tried to build it like a kid would build it, which was mostly improvisational. (architect). The problem-solving approach to design was ‘rational’ (management expert 1), ‘log- ical’ (engineering designer 1) and focused on arriving at a desirable ‘solution’ to the ‘design problem’ at hand (e.g., computer scientist, architectural engineer, engineering designer 1, management expert 1, etc.). An engineering designer best described the problem-solving approach in the following terms: The engineering design process—what we do is we try to define the problem. So first we’ve got to figure out what the problem is…[then] you need to solve it…So scoping that problem is a very critical part. What is it that you are working towards? (engi- neering designer 3). For the engineering designer, the practice of design begins with problem definition and then moves onwards to find a solution to the problem. Another engineering designer we spoke to contrasted a problem-solving approach with the practice of design in fashion design: Tharchen et al. J Org Design (2020) 9:21 Page 16 of 34 I view design more as working towards a problem that I can understand. When I look at a catwalk, I don’t understand it, and I know that [fashion] designers design these things. Of course, there is value in there, but I think it’s just a different use of the term [design] than how I use it. (engineering designer 2) (emphasis added). As evidenced, for engineering designer 2, what fashion designers do is not a facet of design he understood, as it lacked an emphasis on problem solving. Yet, he saw some value in fashion design, even though he did not fully understand it. We return to this notion of reflexivity, an attribute that we found in all the people we interviewed, later in the document. The focus on problem solving also extended to disciplines outside of engineering. To illustrate: I clearly think of my concept of design as a pretty rational kind of process. What are you trying to achieve and how should you go about it? …There is the beast on the table. What’s the best design that we can come up with so that it has a good chance of being successful in its environment? (management expert 1). Management expert 1 likewise associated design with first identifying and defining the problem, and then generating the ‘best’ solution to address the problem. Reflect- ing his background, he offered that an organization must be designed to increase the likelihood of its success in competitive environments. I intentionally only had a very rough sketch [of a tree house] and tried to build it like a kid would build it, which was mostly improvisational. (architect). However, there were informants who were critical towards the ‘problem solving’ approach. For instance, an engineering designer considered this approach as limiting the scope of what could be achieved through design as an activity: [Design] is not just problem solving, which is engineering culture. So engineers in the design process—they view it as a problem and solve it in 30 min… Engineers have no clue what a vision is. Their vision—is a neat little technical problem they have to solve. (engineering designer 4). This observation draws attention to a second approach to design, one characterized by greater open-endedness. To illustrate: [Design] is a projectile thrown into the future. I don’t like the problem-solving definition that often is used…to me it’s always somewhat provisional…You make design proposals, but I never necessarily assume they’re right or perfect—so they’re always open to modification, to rethinking. (architect). The above quote highlights that design is always provisional and open to modifica- tion, in contrast to being a final solution. Such an open-ended approach to design was also emphasized by a computer scientist who emphasized the constantly changing nature of software designs: One of the aspects of software is that it’s not a static object. It’s something you build once but it will have a life of its own. It’s going to go into production, it might be used for 10, 20 years. Someone else is going to look at it and modify it, extend it. So you can’t just have something that works, it’s also got to be able to live, in terms of other people looking at it, modifying it, extending it, changing it. (computer scientist). Tharchen et al. J Org Design (2020) 9:21 Page 17 of 34 Other informants attributed to design a ‘living quality’ in the following terms: I believe that design is something that maybe starts with some initial ideas but gets developed and changed and grows and sometimes retracts over time. (patent lawyer). I think it’s a lot like life—a building has a life. It’s not a static thing. (architect). J Org Design (2020) 9:21 Page 18 of 34 I am collaborating with computer scientists, neurologists, artists… It’s one of those things that we’re synthesizing knowledge and creating new ideas. (engineering designer 1). An architect reflected that collaboration with engineers made him fluent in their design practices and the terms to use that they would value: In the last four or five years I’ve been doing a lot of collaborative research with engi- neering faculty, so I’m getting to the point where I speak engineer … but obviously I still root it in architecture (architect). Such interactions arose because design projects such as buildings “[are] too complex for any one person to understand all aspects of it” (architect), and also because the out- comes of interdisciplinary interactions in collaborative projects were likely to be new to all. A civil engineer shared with us the novel outcome of an interdisciplinary collabora- tion with aerospace engineers: I talked to some aerospace people [to design] the software, a primary design tool for US satellite assets … it wouldn’t happen without collaboration, openness, going across disciplinary boundaries. (civil engineer). I think it’s a lot like life—a building has a life. It’s not a static thing. (architect). In sum, we found two approaches to design: problem solving, which prescribes com- pleteness in problem definition and aims at arriving at the ‘optimum’ or ‘best’ solu- tion, and open-ended engagement, which anticipates designs to be always incomplete and “perpetually in the making” (Garud et al. 2008: 356). Interactions  Besides the tools of the design trade and the approaches to design, the data revealed another important facet of the design process: interactions with material artifacts and with designers outside of one’s own discipline. Interaction with material artifacts such as ‘prototypes’, (engineering designer 1, engineering designer 3, engineer- ing designer 4, mechanical engineer, industrial psychologist), and ‘mockups’ (architec- tural engineer) enabled iteration and feedback in the design process. An engineering designer explained the use of prototypes in the design process: You brainstorm and think of things that you hadn’t thought about. You build a couple prototypes. You test. You see the things about the final solution that weren’t in the problem statement and then you continue to iterate until you actu- ally end up with…[the solution] (engineering designer 1). Another informant, an architectural engineer highlighted the use of ‘mockups’ to improve the quality of feedback received from prospective users of the design. We’ve done a lot of virtual mockups that allow a user group to live navigate those models inside of an immersive display on a one-on-one scale. They can walk around, look at the space and get a better sense of scale and the environment and can provide better feedback. (architectural engineer). Interaction with material artifacts such as ‘prototypes’ and ‘mockups’ helped designers concretize design concepts. They also enabled meaningful iterations by “expressing, developing, detailing, communicating, and presenting an evolving design concept” (Wagner 2000: 379) for testing and feedback in the design process, and thereby enabling different stakeholders to easily understand the design: I think we’ve found that having the models can be a very productive communica- tion tool…I think it … levels the playing field for everyone’s understanding of what the design actually is. (architectural engineer). Interactions were not just limited to material artifacts, but also extended to inter- actions with designers from other disciplines. This was manifest in the references informants made to design projects involving collaboration with individuals outside of their own discipline. Engineering designer 1 described in the following terms one of his collaborative projects: Tharchen et al. Similarities and differences in practices across disciplines So far, we examined the design practices used by the members of the Center. Our inves- tigation revealed that these practices varied across members we investigated. Although we already alluded to some similarities and differences, it is useful to explore them in greater detail. For instance, how did different disciplinary groups use the practices they mentioned? Did practices neatly separate out across the individuals, or were there some commonalities? The results of our analysis are summarized in Table 4. Specifically, the table highlights the similarities and differences in the practices across all informants. As an illustration, consider the practices of an architectural engineer (Table 4) and those of an industrial engineer (Table 4). Both share commonalities in practices such as in their problem-solv- ing approaches to design and in their use of quantitative tools. However, architectural engineer used qualitative tools and interactive material artifacts, which were absent in the practices of industrial engineer. This pattern of similarities and differences in prac- tices exists across all informants as illustrated in Table 4. Whereas the differences in practices are a result of differences in disciplinary training and orientation of informants, the similarities point to overlaps in some of the practices across different disciplines. For example, informants from the mainstream engineering disciplines placed emphasis on quantitative tools and a problem-solving approach (e.g., engineering designer 1, engineering designer 3, civil engineer, mechanical engineer). By contrast, other informants emphasized qualitative tools (e.g., architect, industrial psy- chologist, architectural engineer) and open-ended engagement (e.g., architect, patent lawyer). These differences in orientations were persuasively articulated by the architect who challenged the assumption that every aspect of the design could be quantified. To illustrate: Page 19 of 34 Tharchen et al. Similarities and differences in practices across disciplines J Org Design (2020) 9:21 Table 4  Similarities and differences in practices across informants Biographical sketch of each informant is summarized in Appendix A Practices Informants Computer scientist Management expert 1 Management expert 2 Architect Architectural engineer Engineering designer 1 Engineering designer 2 Engineering designer 3 Engineering designer 4 Industrial psychologist Patent lawyer Civil engineer Industrial engineer Mechanical engineer Design tools  Quantita- tive tools X + X + X X + X + + X X X  Qualitative tools + + X X + X + X X + Design approaches  Problem solving X X X − X X X X − X X X X X  Open- ended engage- ment X + X + X X Interactions  Interaction with material artifacts X X X X X X  Interaction with design- ers outside of one’s discipline + + X X X X X X X X X X + X Tharchen et al. J Org Design (2020) 9:21 Page 20 of 34 [The assumption] that everything can be measured and everything can be put in some kind of numerical form… I don’t think that’s actually true. (architect). The same architect also critiqued the problem-solving approach to design advo- cated by others: I know a lot of people like to talk about ‘we solve problems.’ But, I don’t think everything that we do, intends to be a solution—certainly not a final solution. To me, [the notion of a] problem always has some difficult connotations. It’s a very technological world view. I design and build a tree house for my son, and at no point in that process did I ever consider it a problem. I considered it an opportu- nity, I considered it a challenge, I considered it a chance to express. (architect). Notwithstanding these differences, an emphasis on interactions with designers outside of their own discipline was common across informants. Indeed it was such an emphasis that led these individuals to interact with each other. Informants across disciplines emphasized such interactions in their design practices in the following ways: Interdisciplinary work is very important. Just working with a bunch of engineers doesn’t excite me as much anymore (mechanical engineer). Similarities and differences in practices across disciplines We don’t embrace technology in the way that architects and engineers do and so spending time with folks outside of my discipline makes me realize that there is a rapidly changing world and we need to pay better attention to it to understand really what’s truly happening now—right now—in the creativity world (indus- trial psychologist). I was looking at collaboration between architects and engineers. What were the issues? What were the barriers? What were the impediments to more effective collaboration between architects and engineers? (architect). In sum, although we found less convergence on the practice dimensions of design tools (quantitative and qualitative), and design approaches (problem-solving and open-ended engagement), we found convergence on the degree to which informants across disci- plines emphasized interdisciplinary interactions in their design practices. These findings suggest that the desire to interact is important for these individuals despite and even because of differences in design practices across disciplines. Together these practices, i.e., design tools, design practices and interactions inscribe the horizon of possibilities (Nicolini 2012) of design outcomes across disciplines such as buildings (architect, archi- tectural engineer), surgical tools (engineering designer 2), organization designs (manage- ment expert 1), apps (computer scientist) and others. Categories of orders of worth Our data analysis revealed two major categories of orders of worth—product oriented and process oriented (Table 3). Product oriented orders of worth covered facets of the designed product such as ‘product efficiency’, ‘visual and symbolic beauty’, ‘novelty’, ‘market utility’, and ‘interactivity’. Process oriented orders of worth had to do with ‘pro- cess efficiency’, ‘interdisciplinary interactions’ and ‘reflexivity’ in the design process. Product oriented orders of worth  Informants invoked ‘product efficiency’ as an order of worth in the design of various artifacts such as: programming codes that efficiently utilized memory space and processor time (computer scientist), risk management tools that optimized search and visualization of tradeoffs (civil engineer), product designs that enabled cost savings (mechanical engineer), and courtrooms and work environments designed for efficiency (architectural engineer, industrial engineer). Or, they alluded to designs that effectively performed some function (management expert 2, engineering designer 1, engineering designer 3). A management expert alluded to the effective design of organizations in the following way: [We] are designing, for example, organizations that have desirable properties that we believe will make them effective in their environments and can accomplish their purposes…What are the desirable properties that you want to try to build in to the artifact called …an organization (management expert 1). Informants also invoked ‘visual and symbolic beauty’ as an order of worth, mentioning designs such as ‘[user] interfaces’ that looked beautiful (computer scientist), or generally advanced the value of beauty and aesthetics (engineering designer 4, industrial psycholo- gist). Symbolic beauty as an order of worth was strongly invoked by an architect who championed its intrinsic importance, challenging the premise that designs should be based only on functional utility. To illustrate: Drawings have symbolic importance as well. They are beautiful in their own right. But, if a drawing is just an instrumental representation of something that can be made, then it eliminates our ability to think of things that can’t be made. It con- strains your imagination, I think, in not good ways. (architect). Novelty also emerged as an important order of worth across informants. Orders of worth Just as with practices, we first identified from the interview data the orders of worth surfaced by our informants. This step served as the basis for examining the similari- ties and differences in these orders of worth across members of the design Center. Tharchen et al. J Org Design (2020) 9:21 Page 21 of 34 agement expert 2). agement expert 2). agement expert 2). Across informants, ‘market utility’ of the design emerged as an important order of worth. For instance, informants mentioned design as the creation of products acceptable to customer needs (management expert 2, engineering designer 3), or even exploiting a given market (civil engineer, patent lawyer). To some informants, market utility assumed utmost priority. For instance, a mechanical engineer noted: In product family [design] amazingly, the Best Buys and the Targets and the Walmarts of the world are dictating the entry-level product on the shelf. A company has to figure out what features need to be packed into the product given that the only way it is on the shelf is if it sells for $[x]. (mechanical engineer). Management expert 2 also called for the need to appreciate market-oriented design as a welcome correction to the conventional performance-oriented design. To illustrate: So, all the engineering models work in the wrong direction—from design to perfor- mance to market acceptability. Instead it should be from market acceptability to performance to design. (management expert 2). Closely related to the market, informants invoked ‘interactivity’ of the product as an important order of worth related to comfort (engineering designer 2) and ease of interac- tion (computer scientist) with the designed product. To illustrate this order of worth, an Engineering Designer pointed out the interactive features of the Apple iPhone: The first iPhone revolutionized the industry as a whole; it was a paradigm shift in how people interacted and how they perceived phones. It became more than just a phone. (engineering designer 1). Informants also mentioned “drawings that have changed the way we think about space” (architect), “technologies that have transformed what it means to be human” (engineer- ing designer 4), or “design of user experiences” as examples of interactive designs. Process oriented orders of worth  Informants valued efficiency in the design process in terms of ‘optimization’ to get the best product design (mechanical engineer), ‘minimizing’ the number of iterations to generate a solution (engineering designer 1), effective design processes founded on design principles (management expert 1, computer scientist), and the use of statistical models and algorithms (management expert 2, civil engineer, engineering designer 3). An architectural engineer highlighted making ‘efficient’ use of resources as a distinguishing feature of the construction design process: The construction process is probably much more specific and discrete. Categories of orders of worth Informants who valued ‘novelty’ emphasized its importance observing: “if you were just doing what has already been done, you’re not designing” (civil engineer), “so when I think about somebody designing something, or creating a design, or participating in the design pro- cess, it’s about creating something new that hasn’t existed before” (patent lawyer), “when you are doing design you are trying to build things that never existed before” (engineer- ing designer 4). Recalling his interactions with a circuit designer, management expert 2 also emphasized novelty as a virtue that distinguished design from other activities: The guy [circuit designer] pulls out a book, flips to the page where that circuit is and says, “There it is.” So I thought, I wanted to be a designer; I don’t want to be a cook. This guy has a cookbook and any time he needs a circuit he opens up the cookbook and there is the circuit. He just has to plug in the numbers for his particular use. … So a chef could create something, a chef is a designer, a cook is not a designer. (man- Tharchen et al. J Org Design (2020) 9:21 Tharchen et al. J Org Design (2020) 9:21 Page 22 of 34 agement expert 2). Certainly, effi- cient use of resources is probably one of the keys. From a construction standpoint, how effectively are they using equipment, crews, materials? How effectively are they meeting the budget for that project? (architectural engineer). Informants also valued their ‘interdisciplinary interactions’ in the design process. An engineering designer commented that working with interdisciplinary teams generated multiple ways of approaching the ‘design problem’. To illustrate: When I’m hiring…, I’ll have mechanical engineers, computer scientists, industrial Tharchen et al. J Org Design (2020) 9:21 Page 23 of 34 engineers all working together…I believe that any good design starts from viewing the problems in a different way. This is central to the design process; you have people in varying degrees of expertise that come together and show different outlooks on the problem. (engineering designer 2). A mechanical engineer mentioned how interactions with architects had enhanced his learning about the design process in architecture: It was very interesting working with a different group. I was surprised how much I learned and got out of interacting with the architectural design process in compari- son to the engineering one. (mechanical engineer). Interdisciplinary interactions were also the focus of various design workshops organ- ized by the Center. These workshops focused on topics such as designing an interdis- ciplinary graduate design curriculum and fostering better collaboration on design research across disciplines. For example, one workshop organized by the Center in 2010 titled: “When Engineering Design Meets Architecture” focused on overcoming the “lan- guage barriers” that impeded successful collaboration between architecture and engi- neering disciplines, and recommended the development of design curriculum that led to “T-shaped people”, who not only had in-depth knowledge of design within their own dis- cipline, but were also knowledgeable about design as practiced across other disciplines. Supporting these interdisciplinary interactions was the notion of reflexivity (Cunliffe 2003; Cunliffe and Jun 2005) in the design process, i.e., an awareness of the assumptions and biases in any design approach, and a realization that any notion of design is always incomplete. To illustrate: For me, design is like an elephant. Each blind man comes up to the elephant and gets a different perspective on what design is. There are many different characteris- tics of design. I have two or three blind men’s views of the elephant. So I can appre- ciate multiple perspectives, but they are still incomplete. Each one [perspective] is incomplete. (management expert 2). Similarities and differences in orders of worth across disciplines Table 5 provides a summary of the orders of worth across informants. For example, we see from Table 5 that management expert 2 (Table 5) places primary emphasis on ‘prod- uct efficiency’, ‘novelty’, ‘market utility’, and ‘process efficiency’ besides valuing ‘interdis- ciplinary interaction’ and ‘reflexivity’ in the design process. The other orders of worth namely ‘visual and symbolic beauty’ and ‘interactivity’ are subordinate in importance for management expert 2. Thus management expert 2 invokes multiple orders of worth simultaneously, and also has in place a hierarchy of values (Henn 2013). We see this pat- tern in the orders of worth of all other informants. Table 5 also highlights the structure of similarities and differences in these orders of worth across informants. As in the case of practices, we found these orders of worth to be closely tied to informants’ disciplinary training. For example, we found both ‘product efficiency’ and ‘process efficiency’ to be orders of worth of primary emphasis amongst informants from the engineering disciplines (e.g., engineering designer 1, engineering designer 3, mechanical engineer, civil engineer, etc.). In contrast, an architect questioned the very premise of efficiency as a driver of design, attributing negative value to it in his practice. If efficiency puts lots of people who formerly enjoyed their jobs out of work, and makes them do drudgery work, then have you actually improved the world? My feel- ing is, even if they can do it more efficiently, who cares? I want to have fun. The rea- son I’m an architect is because it’s fun, not because it’s efficient. (architect). However in contrast to practices, we found greater variability across orders of worth even among individuals from the same discipline. For example, engineering designer 2 also placed ‘product efficiency’ lower in her ordering vis-à-vis ‘interactivity’ with the design. To illustrate: In software design [computer engineers are] just thinking about processing speed and nothing to do with the human behavior [and interaction]. (engineering designer 2) Further while informants from the engineering disciplines placed ’visual and symbolic beauty’ lower down in their hierarchy of values referring to these values as “those kind of things that don’t let you make a phone call” (engineering designer 3), and “that’s not so important” (mechanical engineer), other informants, such as computer scientist and engi- neering designer 4, placed primary emphasis on these attributes within their hierarchy of values. agement expert 2). By invoking the parable of The Blind Men and the Elephant, the management expert was acknowledging incompleteness of his own viewpoints on design. The fact that any perspective on design is partial was also echoed by the architect and an engineering designer: Design is too slippery a human activity for anyone to claim that they own or control it (architect). I’ve always known that different disciplines sort of viewed it [design] in different ways. ((engineering designer 3). We found such reflexivity in all members of the design Center we interviewed. Because of such reflexivity, informants respected others’ perspectives on design despite discipli- nary differences. We illustrate this with a set of remarks offered by a mechanical engineer to open a design workshop organized by the Center that we attended: We come here to become aware. We don’t have agreement [on design], but we respect each other’s positions. (mechanical engineer). Tharchen et al. J Org Design (2020) 9:21 Page 24 of 34 Page 24 of 34 Table 5  Similarities and differences in orders of worth across informants X P i h i b di d h i i h i Orders of worth Informants Computer scientist Management expert 1 Management expert 2 Architect Architectural engineer Engineering designer 1 Engineering designer 2 Engineering designer 3 Engineering designer 4 Industrial psychologist Patent lawyer Civil engineer Industrial engineer Mechanical engineer Product oriented  Product effi- ciency X X X − X X + X + + X X X  Visual and sym- bolic beauty X + + X + + − X X + + + +  Novelty + + X X X + X X X X X + +  Market utility + X X + + + + X + − X X X  Interac- tivity X − + X X X X + X X X Process oriented  Process effi- ciency X X X − X X + X + + X X X  Interdisci- plinary interac- tion + + X X X X X X X X X X + X  Reflexiv- ity X X X X X X X X X X X X X X X: Primary emphasis; +: subordinated emphasis; −: negative emphasis Tharchen et al. J Org Design (2020) 9:21 Tharchen et al. J Org Design (2020) 9:21 Page 25 of 34 Similarities and differences in orders of worth across disciplines We found such pattern of similarities and differences in the orders of worth cutting across disciplinary boundaries. For example while novelty as an order of worth was regarded highly by informants across disciplines (e.g., civil engineer, patent lawyer, engi- neering designer 4, management expert 2), industrial engineer did not consider ‘novelty’ as a necessary hallmark of design. To illustrate: I actually think that those things that emerge—so even self-replicating patterns that emerge to be functional [to be] design. So, it doesn’t necessarily require that element of either novelty or innovation, in terms of something that’s completely new. (indus- trial engineer). Tharchen et al. J Org Design (2020) 9:21 Page 26 of 34 Likewise while ‘market utility’ was valued by individuals from different disciplinary backgrounds (e.g., management expert 2, civil engineer, management expert 1, mechani- cal engineer), industrial psychologist attributed negative value to it as illustrated below: And so where a lot of organizations make their mistakes is on the idea evaluation side… they apply financial metrics to new and different things, and the fact is when something is new and different it’s not clear that it’s going to make money, and if those are the only metrics you apply they typically throw out the best ideas. (indus- trial psychologist). Across such patterns of similarities and differences, a striking commonality across informants was their appreciation of the value of interdisciplinary interactions. Such appreciation reflected reflexivity on the part of the participants—that their notion of design was incomplete. We will explore further the implications of such reflexivity in the discussion section. Before doing so, we will provide an overall summary of what we found. Design games We summarize our findings by returning to the semantic network of vocabularies that include practices and orders of worth across the fourteen informants (Fig. 1). The figure shows the presence of two clusters labeled Cluster 1 and Cluster 2. While informants in Cluster 1 are engineers representing disciplines such as engineering design, computer science and industrial engineering, informants comprising Cluster 2 represent a more heterogeneous mix of disciplines such as law, architecture, psychology, management, and engineering. Not surprisingly, common to informants in Cluster 1 are design prac- tices such as ‘problem solving’, the use of ‘quantitative tools’, and the orders of worth of ‘product efficiency’ and ‘process efficiency’. In contrast, design practices across inform- ants in Cluster 2 emphasize ‘open-ended engagement’ and the use of ‘qualitative tools’, in addition to also valuing ‘visual and symbolic beauty’. However, despite these differences we also seen interlacing (Tuertscher et al. 2014) in the vocabularies, practices, and orders of worth of informants both across and within the two clusters. For example, the semantic network reveals common ground (Puranam et al. 2009) in the vocabularies related to practices and orders of worth, as well as design vocabularies such as ‘idea’, ‘create’, ‘build’, ‘product’, ‘process’, ‘project’, etc., across inform- ants. Overall this pattern of similarities and differences corresponds to what Wittgen- stein observed in the context of language games as “a complicated network of similarities overlapping and criss-crossing” (Wittgenstein 2009: 36). Design is similarly an intercon- nected network of design games with connections constituted by an overlapping struc- ture of similarities and differences in the vocabularies, practices, and orders of worth of design across the different disciplines. Discussion We began the article by asking the question: What is it about design that makes it pos- sible for interdisciplinary interactions to emerge given that the term itself has multiple meanings? Confirming the polysemy of this term our investigation revealed that the meaning accorded to design depends on the context of its use—i.e., the vocabularies, Tharchen et al. J Org Design (2020) 9:21 Tharchen et al. J Org Design (2020) 9:21 Page 27 of 34 practices, and orders of worth that constitute the various design games at play. By itself, such polysemy ought to generate isolated pockets that only interface with one another, with design serving as a boundary object enabling co-ordination without the need for consensus and intense interactions among individuals across different disciplines (Star and Griesemer 1989). The conceptual underpinning for such an approach is the decom- position of complex problems into parts (Simon 1996), with each part addressed by a specific group that only interfaces with others across standardized boundaries. Such a view of design continues to function as an important organizing principle in manage- ment, ranging from the design of assembly lines using principles of scientific manage- ment (Taylor 1911) to the development of products and services following modularity principles (Baldwin and Clark 2000; Sanchez and Mahoney 1996; Ulrich 1995). However, our analysis revealed a different notion of design, one in which individuals do not just interface but instead interact with one another, and in the process, open up the black boxed design module. In this design approach, individuals come together not despite but because of differences in their vocabularies, practices, and orders of worth. So, what motivates these individuals with different notions of design to come together and interact? Our analysis suggests that individuals do so because design evokes reflex- ivity (Cunliffe 2003; Cunliffe and Jun 2005), i.e., an awareness of the assumptions and biases in one’s own design approach leads to a realization that any notion of design is always incomplete, and design initiatives are always ongoing and full of future potentiali- ties (Garud et al. 2008). This interactional view of design resonates with the second notion of design in Simon’s work, i.e., the emergence of design options in and through interactions, a notion also pursued by scholars in management studying design (e.g., Boland and Collopy 2004; Dunbar and Starbuck 2006; Gruber et al. 2015). Discussion Not surprisingly, Simon called his work on design as the Sciences of the Artificial focused on “devis[ing] courses of action aimed at changing existing situations into preferred ones” (Simon 1996: 111). More recently, scholars building on Simon’s second view of design have also explored how such interac- tions function as a social resource that holds the capacity to expand rather than bound rationality (Hatchuel 2001). Such interactional views of design have also been advocated in practice. For instance, Takeuchi and Nonaka (1986) contrasted the linear interfacing approach that emerges from classical notions of design against a more complex interactional approach. Using sports metaphors, they likened the former to a relay race and the latter to a rugby game. Similarly, Romme and Endenburg (2006) offered the notion of organizational decisions occurring through a “circular design” process, wherein the inputs of individuals from one circle links with other circles to generate informed consent. Fostering continued interactions among parties (with often competing interests) is also central to the design and formation of collaborative communities for business development in non-preferen- tial economic zones such as Greenland (Kadenic 2017). The interactional view of design surfaces its own complexities though. For instance, the rugby approach to design is messy and inherently unstable, as the authors themselves point out (Takeuchi and Nonaka 1986). So, what then allows such an interactional pro- cess to cohere? Our analysis of the data from the Center suggests several related mech- anisms. One of them is the presence of common vocabularies across individuals from Tharchen et al. J Org Design (2020) 9:21 Page 28 of 34 different disciplines. The other is the presence of overlaps in the practices and the orders of worth of design. Together, these attributes result in the emergence of robust designs (Hargadon and Douglas 2001), i.e., designs that are both a participative process and an interactional outcome, one that bear the imprints of designers from multiple disciplines and social groups. What are the implications of these findings? The world increasingly confronts “wicked problems” (Buchanan 1992; Rittel and Webber 1974), i.e., problems that have no definite formulation and are characterized by complex interdependencies, such as the challenges posed by sustainability (Reinders et al. 2012), innovation and new product development (Brown 2009; Takeuchi and Nonaka 1986). Discussion Such problems demand a pragmatic focus on generating actionable knowledge (Romme 2003) that takes into consideration world- views shaped by different practices and orders of worth. It is to address issues of this kind that design has surfaced in popularity, allowing individuals from different social groups to meaningfully interact and generate discussion over “matters of concerns” (Latour 2004) as much as over “matters of facts”. Design is a concept that sets the stage for reflexive individuals to come together because of their differences to jointly formu- late and explore problems and solutions. Contributionshi The finding of this study on how design manifests itself as a complex network of similari- ties and differences in the vocabularies, practices, and orders of worth across disciplines offers several contributions to the literature on design, which we highlight below. Design scholars emphasize the need to pay attention to vocabularies that designers use (Boland and Collopy 2004). We extend this conversation through a more systematic examination of vocabularies used by designers across different disciplines to highlight the similarities and differences in how they understand design. An important contribution of this study is to introduce the concept of design games, which alludes to not just vocabularies, but also the practices and orders of worth that they constitute. Further, inquiring into how it is that designers from different disciplines cohere in interdisciplinary forums despite their disciplinary differences, we found designers to be reflexive (Cunliffe 2003; Cun- liffe and Jun 2005), an attribute that complements the presence of common ground and the interlacing of practices and orders of worth of design across disciplines. Reflexivity consists of the recognition by individuals that any notion of design is incomplete, and therefore the importance of inputs from people with other disciplinary backgrounds. By incorporating reflexivity into design games, we advance one compelling reason why interdisciplinary forums and projects on design cohere and do not fragment.i Our findings integrate the growing literature on the design approach focusing on inter- disciplinary interactions with the literature on boundary objects (Star and Griesemer 1989). However, in contrast to boundary objects, we found that design is an interactive boundary object that not only coordinates the activities of individuals across disciplinary boundaries but also induces interaction. Whereas boundary objects were conceptual- ized to enable interfacing of individuals from different social worlds or communities of practice (Brown and Duguid 1991; Lave and Wenger 1991; Wenger 1998) without the need for intense interactions, interactive boundary objects such as design view intense engagements not to be problematic, but instead generative of novel outcomes. Such Tharchen et al. J Org Design (2020) 9:21 Page 29 of 34 interactions do not just transfer knowledge from different perspectives, but also enable knowledge transformation whereby the meaning of design is always emerging in and through interactions as designers co-orient and co-inform each other across disciplinary boundaries. Overall, these findings shed light on the link between design and emergence (Garud et  al. 2008; Hunter et  al. Contributionshi 2020), and how particular cultures can be created through design (Koçak and Puranam 2018). Through our study of an interdisciplinary Center for Design, we found that design can foster a culture of interdisciplinary interactions by invoking reflexivity. This is because design functions as an interactive boundary object, wherein individuals are motivated to interact with one another not despite but because of their differences in meaning. As there is no one meaning of design, individuals are attentive to the design practices and the orders of worth of others that they are interact- ing with. Such interactions provide the foundations of a culture centered on collabora- tion and learning (Elsbach and Stigliani 2018). Indeed, such reflexive interactions extend beyond the collaborating individuals to also include the perspectives of others (such as users) in ways that the future possibilities that are emerging and being co-created bear the inputs of all stakeholders: Dealing with emergence requires designers and managers to understand their designs in relation to those who will enact them in practice. It requires a commit- ment to co-create with these others whose lives will be shaped and changed by their engagement with the designed world. It requires an inquiry into what and whose desired futures are to be enabled and a willingness to be open to and be changed by that understanding. It suggests engaging the respectful interaction among people that can lead to transformed meanings, identities, and intersubjectivity. (Orlikowski 2004 : 94). Indeed, the increasing use of the term ‘design’ by academics and practitioners alike when engaging with issues such as environmental sustainability and public health con- cerns speaks to this promise of design to generate meaningful solutions to these world’s complex problems (e.g., Centers of Disease Control and Prevention 2018; TUDelft 2020; United Nations Environment Programme 2009). In this regard, Garud and Karnøe (2003) showed how a design approach facilitating interactions between actors with dif- ferent perspectives was critical for the emergence of wind turbines in Denmark. Boundary conditions, limitations and future researchh The empirical site for our study was a Center for Design, which was established to pro- mote interdisciplinary collaboration. Hence, the individuals at the Center that we inter- viewed were self-selected individuals interested in exploring interdisciplinary work. While an openness and commitment to interdisciplinary collaboration is an important boundary condition for the set of findings we have presented, our study offers a model of interdisciplinary collaboration to serve as a model for other settings, especially ones where such interdisciplinary collaboration is missing. A key ingredient for success, as the findings highlight, is the cultivation of reflexivity to the perspective of others by the individuals involved, which emerges through frequent interactions. Tharchen et al. J Org Design (2020) 9:21 Tharchen et al. J Org Design (2020) 9:21 Page 30 of 34 Our study has other features that present opportunities for future research on design. First, we have shown how interlacing (involving similarities and differences) in vocabularies, practices and orders of worth, served as a glue which held members of an interdisciplinary design Center together to foster generative collaborative inter- actions. It might be the case that such interlacing could also lead to fragmenting and conflict. While we did not observe this outcome, future studies could examine how similarities and differences in the vocabularies, practices and orders of worth might lead to generative or problematic interactions. Second, our findings related to design games presents an opportunity to study the evolution of the interlaced structure involving the vocabularies, practices and orders of worth such as those used by the informants we studied. This is an important question, which future research can also explore. Third, our findings around reflexivity suggest that the desire to collaborate is not wholly determined by a logic of consequences based on success or failure alone. However, future studies could explore whether and how collaboration in the context of interdisciplinary design is contingent on the outcomes realized during the design process. Fourth, given our focus on analyzing individual designers, we have empha- sized reflexivity as the generative mechanism inducing interdisciplinary interactions while underplaying the role of organizational processes, protocols and infrastructures that generate and sustain such interactions (Snow et al. 2017). Future research can investigate how these organizational structures enable or constrain such mechanisms of self-reflexivity. Funding Funding We acknowledge funding from the CRDI, The Pennsylvania State University. CRDI did not play any role in the design of the study as well as interpreting the results and writing the manuscript. We also thank the H. Campbell and Eleanor R. Stuckeman Collaborative Design Research Fund for funding several of the interviews that we conducted. Competing interests h h d l h The authors declare that they have no competing interests. Authors’ contribution All h ib d All authors contributed equally to the project. All authors read and approved the final manuscript. Acknowledgements g We thank Robyn Engel and Gina Rossi for conducting several interviews. Conclusion We close by returning to the question that motivated this study—What is it about design that makes it possible for interdisciplinary interactions to emerge given that the term itself has multiple meanings? The findings from this study confirmed the pres- ence of differences in meanings across disciplines. At the same time, it also show- cased agreement amongst participants that design is an activity that benefits from the interactions between actors who approach the activity from different vantage points. Driving such interactions is reflexivity on the part of actors—they all realize that their expertise is but one part of a larger puzzle, which itself emerges in and through inter- actions. Future studies can further examine the nature of such boundary interactions and how they can generate novel outcomes to complex problems. 1 Emlyon Business School, 23 Avenue Guy de Collongue, 69130 Écully, France. 2 Smeal College of Business, The Pennsyl- vania State University, 431 Business Building, University Park, PA 16802, France. 3 The Pennsylvania State University, 328 Stuckeman Family Building, University Park, PA 16802, France. Received: 15 February 2020 Accepted: 22 September 2020 Author details 1 1 Emlyon Business School, 23 Avenue Guy de Collongue, 69130 Écully, France. 2 Smeal College of Business, The Pennsyl- vania State University, 431 Business Building, University Park, PA 16802, France. 3 The Pennsylvania State University, 328 Stuckeman Family Building, University Park, PA 16802, France. Tharchen et al. J Org Design (2020) 9:21 Page 31 of 34 Appendices Appendix A Details of informants Appendices Appendix A Details of informants Informant Designation Area of research 1 Computer scientist Programing language design, mathematical logic 2 Management expert 1 Organization design, innovation management 3 Management expert 2 Customer driven design, simulation, new product development 4 Architect Architectural design, sustainable design, green design 5 Architectural engineer Construction visualization research 6 Engineering designer 1 Complex system design, product family design, design optimization 7 Engineering designer 2 Human factors, human computer interaction, innovative engineering design 8 Engineering designer 3 System design, innovation in engineering design 9 Engineering designer 4 Design theory, social ethics of design, open source design 10 Industrial psychologist Creativity, innovation management, organizational climate 11 Patent lawyer Intellectual property law, technology law, patent law 12 Civil engineer Water resource management, visualization of risks and tradeoffs within complex systems, decision support, multi-objective optimization 13 Industrial engineer Human factors, human machine interaction, display visualization, discrete events simulation, human in the loop 14 Mechanical engineer Product design, product family design, engineering design Appendix B Informant Designation Area of research Informant Designation Interview protocol We followed an open-ended semi-structured interview protocol that were guided by the following questions: 1 What do you do as a designer? 2 Can you describe what it means to ‘design’ in your field? 3 What words come to mind when you think about the word ‘design’? 4 How do you evaluate designs? 5 How is your role as a designer seen by others? Is it accurate? 6 What other kinds of designers do you interact with? What is the nature of that inter- action? 7 Is there a notion of design that is not consistent with yours? Antithetical to yours? 8 Who do you compete with? 9 What enables your work? 10 What constrains your work? 1 What do you do as a designer? 2 Can you describe what it means to ‘design’ in your field? 3 What words come to mind when you think about the word ‘design’? 4 How do you evaluate designs? 5 How is your role as a designer seen by others? Is it accurate? 6 What other kinds of designers do you interact with? What is the nature of that inter- action? 7 Is there a notion of design that is not consistent with yours? Antithetical to yours? 8 Who do you compete with? 9 What enables your work? 10 What constrains your work? 1 What do you do as a designer? 2 Can you describe what it means to ‘design’ in your field? 5 How is your role as a designer seen by others? Is it accurate? 6 What other kinds of designers do you interact with? What is the nature of that inter- action? 7 Is there a notion of design that is not consistent with yours? Antithetical to yours? 8 Who do you compete with? 9 What enables your work? 10 What constrains your work? In all interviews, there were two people present. All interviews were recorded and transcribed. Page 32 of 34 Tharchen et al. J Org Design (2020) 9:21 References Van Nostrand Rein- hold, New York Burke K (1966) Language as symbolic action: Essays on life, literature, and method. Univ of California Press, Berkeleyi Burton RM, Obel B (2018) The science of organizational design: fit between structure and coordination. J Org Des 7(1):1–13 Burton RM, Eriksen B, Håkonsson DD, Snow CC (2006) Organization design: the evolving state-of-the-art. Springer Sci- ence & Business Media, Berlin Callon M, Lascoumes P, Barthe Y (2009) Acting in an uncertain world: an essay on technical democracy (G. 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https://europepmc.org/articles/pmc4480334?pdf=render
English
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In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients
Applied microbiology and biotechnology
2,015
cc-by
9,104
Appl Microbiol Biotechnol (2015) 99:6021–6033 DOI 10.1007/s00253-015-6492-6 Appl Microbiol Biotechnol (2015) 99:6021–6033 DOI 10.1007/s00253-015-6492-6 APPLIED MICROBIAL AND CELL PHYSIOLOGY P. Drevinek P. Drevinek Department of Medical Microbiology, University Hospital Motol and 2nd Faculty of Medicine, Charles University, 150 06 Prague, Czech Republic In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients Drulis-Kawa (*) Institute of Genetics and Microbiology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland e-mail: zuzanna.drulis-kawa@microb.uni.wroc.pl Keywords Myoviridae bacteriophages . Phage treatment . Pseudomonas aeruginosa . Cystic fibrosis . Galleria mellonella model Keywords Myoviridae bacteriophages . Phage treatment . Pseudomonas aeruginosa . Cystic fibrosis . Galleria mellonella model P. Zarnowiec: W. Kaca The Jan Kochanowski University, Swietokrzyska 15, 25-406 Kielce, Poland K. Danis-Wlodarczyk Division of Gene Technology, Catholic University of Leuven, Kasteelpark Arenberg 21, 3001 Leuven, Belgium In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients Tomasz Olszak & Paulina Zarnowiec & Wieslaw Kaca & Katarzyna Danis-Wlodarczyk & Daria Augustyniak & Pavel Drevinek & Anthony de Soyza & Siobhán McClean & Zuzanna Drulis-Kawa Tomasz Olszak & Paulina Zarnowiec & Wieslaw Kaca & Katarzyna Danis-Wlodarczyk & Daria Augustyniak & Pavel Drevinek & Anthony de Soyza & Siobhán McClean & Zuzanna Drulis-Kawa Received: 10 November 2014 /Revised: 3 February 2015 /Accepted: 14 February 2015 /Published online: 12 March 201 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract The goal of the study was to determine the relation- ship between in vitro/in vivo efficacy of environmental Abstract The goal of the study was to determine the relation- ship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diver- sity between particular isolates and determined phage sensi- tivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PAwere tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insen- sitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55–68 % of PA strains (72–86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bac- terial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro as- says, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations. T. Olszak K. Danis-Wlodarczyk D. Augustyniak Z. Introduction Pseudomonas aeruginosa (PA) is the key pathogen associated with lower respiratory tract illness in patients with cystic fi- brosis (CF), infecting up to 80 % of adult patients. Once established, the pathogen is difficult to eradicate (Davies 2002; Parkins et al. 2012). Increased viscosity of mucus asso- ciated with the CF-lung causes favorable conditions for per- sistent infection by opportunistic bacteria. Chronic infection A. de Soyza Institute of Cellular Medicine, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK S. McClean Centre of Microbial Host Interactions, Institute of Technology Tallaght, Tallaght, Dublin 24, Ireland Appl Microbiol Biotechnol (2015) 99:6021–6033 6022 and associated inflammatory responses lead to progressive loss of lung function and respiratory failure (Callaghan and McClean 2012; Costello et al. 2011). Antibiotic therapy is usually effective only in the early stages of infection. During the development of a chronic infection, even long-term anti- biotic therapy with high doses does not result in eradication of the pathogen. This is multifactorial (Bradbury et al. 2008; Cheng et al. 1996; Fothergill et al. 2012). Bacterial adaptation is a characteristic of CF-related PA strains. Alterations in fea- tures such as antibiotic resistance, adhesion, alginate, and mu- cus production are common and can affect the success of clinical treatment (Silbert et al. 2001). Moreover, the strains of PA isolated in the late stages of infection are very hetero- geneous and demonstrate microevolution within an individual strain with a characterized genetic profile. This phenotypic variability of strains underlies the frequent failure of antibiotic therapy and also creates a problem for the development of alternative treatments (Bragonzi et al. 2009; Cramer et al. 2011). Multidrug resistance and biofilm formation are addi- tional problems associated with the treatment of P. aeruginosa infection, implying that alternative treatment methods are needed for eradication or suppression of PA. Lytic bacterio- phages offer a potentially exciting alternative approach in treatment for PA infections. Nevertheless, phage therapy, es- pecially in the eradication of PA clones from CF patients, requires extensive preclinical testing of phage biology and antibacterial activity. strains (isolated from patients without CF) from the collec- tion of the Institute of Genetics and Microbiology, University of Wroclaw, Poland. Phage isolation Six water samples collected from a natural wastewater treatment plant (irrigated fields) located in Wroclaw, Poland, as phage sources were centrifuged at 15,000g for 15 min, and the supernatants were filtered through a 0.22-μm Millex-GP filter (Merck Millipore, Darmstadt, Germany, SLGP033RS). One milliliter of filtered water sample and 0.5 ml of a bacterial broth culture, grown overnight in TSB, were added to 10 ml of TSB and incu- bated at 37 °C for 18 h. The suspension was then centri- fuged again, treated with chloroform, and filtered through a 0.22-μm Millex-GP filter (Merck KGaA, Darmstadt, Germany). This procedure was repeated three times to eliminate any bactericidal activity by contaminating chemicals. Bacteriophage presence and titer in the filtrate were assessed by the plaques test using the double-agar layer technique (Adams 1959). Phages were propagated from a single plaque. The phage lysate was then subjected to PEG 8000 (Acros Organics, Geel, Belgium) precipita- tion. The 28 environmental bacteriophages lytic on PA strains (27 named KT and one phage PA5oct) were depos- ited in the phage collection of the Institute of Genetics and Microbiology, University of Wroclaw, Poland. In this study, we were interested in the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of selected strains. Therefore, we first selected two phages with the broadest spectrum and analyzed them against six isolates that were examined for mucoid slime production, twitching motility, biofilm-forming ability and biochemical composition variabil- ity as analyzed by Fourier transform infrared spectroscopy (FTIR) techniques. Phage lytic activity was tested both in vitro and in vivo using the Galleria mellonella larvae mod- el. The most active phages were characterized by electron microscopy and genome size. Introduction A total of 123 PA isolates were used for phage lytic potency: namely PAO1; 121 clin- ical CF PA isolates from the collection of the Prague CF Centre; and non-CF0038 clinical PA strain from the collec- tion of the Institute of Genetics and Microbiology, University of Wroclaw, Poland. Bacteria were stored at −70 °C in tryptic soy broth (TSB; Becton Dickinson and Company, Cockeysville, MD) supple- mented with 20 % glycerol. Materials and methods Prior to phage sensitivity testing bacteria were subcultured in TSB. Unless stated otherwise, bacteria were grown for 4–6 h. Prior to phage sensitivity testing bacteria were subcultured in TSB. Unless stated otherwise, bacteria were grown for 4–6 h. To determine bacterial susceptibility to phage-mediated lysis, bacteria grown on liquid TSB medium at 37 °C were trans- ferred directly onto TSA plates. After drying, a drop of the phage suspension (108 PFU ml−1) was put on the bacterial layer and incubated at 37 °C. The plates were checked after 4–6 h and again 18 h later for the presence of bacterial lysis. The phage lytic potency assay was repeated at least three times. Spot testing is a rapid and efficient method for deter- mining the host range in a large collection of bacteria (Adams 1959; Kutter 2009). Subsurface twitching assay The subsurface twitching assay was performed as previously described by Semmler et al. (1999) with a slight modification. Ten P. aeruginosa colonies taken randomly were tested for twitching motility. Each colony was stab-inoculated through the agar to the underlying Petri dish and incubated at 37 °C for 48 h. The zone of motility at the agar Petri dish interface was measured after 0.01 % crystal violet (CV) staining. Pulsed field gel electrophoresis analysis Pulsed field gel electrophoresis analysis Pulsed field gel electrophoresis (PFGE) analysis was per- formed by previously described method (Drulis-Kawa et al. 2014). Prepared blocks were placed in lysis buffer (50 mM Tris, 50 mM EDTA, 1 % SDS) and digested for 2 h with 100-μg ml−1 proteinase K solution at 54 °C. After digestion, plugs were rinsed four times with TE buffer. DNA samples were placed on a 1 % agarose gel using the Bio-Rad (Hercules, CA, USA) CHEF-DR III system (16.5 h, 6 V cm−1, 12 °C, switch time 1–50, angle 120°). Low Range PFG Marker (New England Biolabs, Ipswich, MA, USA, N0345S) was used as a size marker. FTIR is a scientific technique based on measurement of vibra- tional energy changes of organic compounds excited by IR radiation. Due to the fact that vibrational energy is quantized, the frequencies of various functional groups can be detected only at fixed wavelengths, specific for every molecular com- pound. Vibrational spectra typical for biological samples in most cases correspond to mid-IR (4000–400 cm−1), and there are five spectral windows defined within that spectrum (Alvarez-Ordóñez et al. 2011). For minimal interference with the test material, in this study, the IR spectrum measurement was conducted by attenuated total reflectance (ATR)-FTIR. The IR spectral range was limited to 4000–900 cm−1, and four spectral windows were taken into account. Every window was specific for one group of organic compounds, i.e., carbohy- drates (1200–900 cm−1), carboxylic groups (1500– 1200 cm−1), proteins (1700–1500 cm−1), and lipid compounds (3000–2800 cm−1) (Alvarez-Ordóñez et al. 2011). Electron microscopy (nonadhered cells were removed from each peg, and lids were rinsed using 200 μl of physiological saline). The peg lids were submerged into 200 μl of 0.01 % CV solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) added to each well. The excess CV was removed, and bound CV was released by adding 200 μl of 96 % ethanol (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). The absorbance was measured at 595 nm using a multilabel microtiter plate reader (UVM 340, AsysHitech, Eugendorf, Austria). The positive control consisted of 100 μl of 0.01 % CV solution, while the negative control was 96 % ethanol. All assays were repeated 24 times per strain. The filtered high-titer phage lysate was centrifuged at 25,000g for 60 min, using a Beckman (Palo Alto, CA) J2-21 centrifuge and a JA19.1 fixed-angle rotor. The pellets were washed two times in ammonium acetate (0.1 M, pH 7.0) under the same conditions. Phages were deposited on copper grids with carbon-coated Formvar films (Agar Scientific, Elektron Technology UK Ltd, Stansted, UK) and stained for 10 s with uranyl acetate (2 %, pH 4.5) or phosphotungstate (2 %, pH 7). Excess liquid was blotted off, and phages were examined under a Philips EM 300 electron microscope. Magnification was controlled by means of T4 phage tails (Ackermann 2009). Bacterial strains Eighteen PA strains were used as hosts for phage propaga- tion: PAO1 (ATCC 15692) purchased from the American Type Culture Collection (ATCC); two clinical CF PA strains (isolated from a CF patient) from the collection of the Prague CF Centre, Czech Republic; one clinical CF PA strain (isolated from the lung infection of a CF patient) from the collection of Newcastle University Medical School, Newcastle upon Tyne, UK; and 14 clinical non-CF PA 6023 Appl Microbiol Biotechnol (2015) 99:6021–6033 Experimental procedure The biochemical profile of bacterial samples was measured by ATR-FTIR spectroscopy (Spotlight 400 FTIR Imaging System, Perkin Elmer, Waltham, MA, USA). Sample prepa- ration procedures for ATR include direct transfer of ten single bacterial colonies to the crystal, separately. The spectra were collected at room temperature over the wave space number range of 4000 to 900 cm−1 with a resolution of 4 cm−1, and 50 repeats were averaged to improve the signal to noise ratio. The spectra were displayed in terms of absorbance, which was calculated using Perkin Elmer software. A background mea- surement of the crystal was taken before each sample was applied. The spectrum obtained for each clone was analyzed in definite ranges, allowing to estimate the variability of spe- cific groups of biochemical components. The range 1200– 900 cm−1 is typical for carbohydrates, range 1500– 1200 cm−1 corresponds to the carboxyl groups, the range of Pretreatment of spectra The preprocessing was done as follows: baseline correction in the range of 2404 to 2275 cm−1 and smoothing of the spectral region from 2725 to 1556 cm−1 were done. The spectra were normalized such that the smallest recorded absorbance was set to 0 and the highest was set to 1 for each spectrum and then the first derivatives (Savitzky and Golay) with a window of 5 were used for chemometrics. The derivation of the spectra to the second order was used to increase the number of discrim- inant features present in the spectra. Chemometric analysis Reproducibility and discriminatory power were calculated for the working spectrum (range 4000–900 cm−1) and, indepen- dently, for the particular sections according to the important regions of infrared spectroscopy. To measure the degree of similarity between ten replicates (ten colonies), we used the differentiation index D expressed as D=(1−r)×1000, where r indicates the Pearson correlation coefficient (Mouwen et al. 2005). Smaller D values indicate more similar colonies (for identical strains, D=0). All statistical computations were car- ried out using the program R version 2.15.1 (R Core Team 2012) (http://www.r-project.org/). 1700–1500 cm−1 is characteristic of proteins and within the 3000–2800 cm−1 the lipid fraction can be analyzed. 1700–1500 cm−1 is characteristic of proteins and within the 3000–2800 cm−1 the lipid fraction can be analyzed. 10 μl of bacterial suspension and, within 1 h, 10 μl of phage lysate at the titration equal to multiplicity of infection (MOI) 100. Each experiment was carried out for 72 h at 37 °C, and results were read at 8, 24, 48, and 72 h. The results were expressed as the percentage survival rates. The experiments were performed at least three separate occasions. The experi- ments were controlled by observation of uninfected larvae, sham-infected larvae, larvae receiving phage lysate only, and infected but phage untreated larvae. Statistical analysis The analysis of survival curves was performer by log-rank Mantel-Cox test. p Values <0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, USA). Phage characteristics The lytic potency of the phage collection (28) specific for PA was performed against 121 CF isolates including 29 mucoid strains. All strains used had been previously characterized by genotyping methods such as multilocus sequence typing (MLST) and random amplification of polymorphic DNA (RAPD). Only unique clones were selected for this study (Maiden et al. 1998). In the phage lytic potency experiments (Fig. 1), several phages exhibited high antibacterial lytic po- tency, with 55 % (phage PA5oct) and 68 % isolates (phage KT28) in total and 72 and 86 % of mucoid isolates showing potency, respectively. Completely insensitive PA were also present (6.4 %). The CF PA strains showed strong heteroge- neity in phage susceptibility presented as confluent clear lysis, confluent opaque lysis, and single resistant colonies on plaque. Disparate results in three consecutive experiments may suggest the existence of multiple clones within each CF isolate. We have also analyzed isolates, classified to particular RAPD cluster, cultured form one patient at different time of infection. Seventy-one isolates from 31 patients (31 strains total) were typed to evaluate the changes in phage patterns. It turned out that during the colonizing process, 21 strains did not change their susceptibilities to the phage collection, 8 strains lost their ability to propagate phages, and 8 strains become susceptible to more bacteriophages from the collection. Biofilm quantification by crystal violet assay on peg lid plates Biofilm quantification by crystal violet assay on peg lid plates The biofilm formation by P. aeruginosa strains was measured by O’Toole and Kolter standard biomass assays using crystal violet (CV) with modifications (Aaron et al. 2002; O’Toole and Kolter 1998; Waszczuk et al. 2012). For the experiments, overnight cultures of bacterial strains were suspended in a physiological saline and diluted to an optical density of 0.5 McFarland units. The culture was then serially diluted in TSB medium to obtain a final density of 5×105 colony-forming units (CFU) ml−1. Two hundred microliters of prepared sus- pension was added into each well of a round-bottomed poly- styrene 96-well microtiter peg lid plate. The plates were then incubated for 24 h at 37 °C. After 24 h of incubation the peg lid was transferred to new titer plate and washed two times Appl Microbiol Biotechnol (2015) 99:6021–6033 6024 Fig. 2 Electron micrograph of PA5oct phage (a) and KT28 phage (b) negatively stained with uranyl acetate. The bar indicates 100 nm Galleria mellonella larvae model The in vivo assay was conducted on a wax moth larvae model (G. mellonella, Livefoods Direct, Sheffield, UK). Prior to each experiment the larvae were subjected to a 7-day acclimatiza- tion period in the dark at 15 °C. Experiments were performed in triplicate (ten larvae per trial). For survival control, we observed both untouched larvae and larvae injected with 20 μl of sterile saline buffer. Depending on the growth rate of the tested bacterial strains, experiments were conducted up to 96 h. Freshly plated bacterial cultures were inoculated into Luria Broth (LB, Sigma-Aldrich) tubes and incubated at 37 °C overnight under agitation. After 18 h, the bacterial suspension was diluted with physiological saline to OD600=0.1 and this was used as a starting point for serial dilutions. For the mea- surement of P. aeruginosa virulence, 10 μl of diluted bacterial suspension (serially diluted to 10−8 CFU) were used to infect larvae. Both bacteria and bacteriophages were administered to larvae by injection into the ventral side of the last pair of pseudopods. After injection, the larvae were incubated for 72 h at 37 °C. The effects of infection were checked at 8, 24, 48, 72, and 96 h after injection by assessment of survival and macroscopic appearance. For assessment of the antibac- terial activity of selected phages, larvae were injected with The two phages with the strongest lytic activity (PA5oct and KT28) were examined by transmission electron micros- copy and classified on the basis of their morphological fea- tures in the order Caudovirales and the family Myoviridae (Fig. 2). Phage KT28 is PB1-like viruses with an approximate head diameter of 70 nm between opposite apices and tails of Appl Microbiol Biotechnol (2015) 99:6021–6033 6025 0 10 20 30 40 50 60 70 80 90 100 % of sensitive strains Bacteriophage Mucoid Total Fig. 1 In vitro susceptibility of 123 P. aeruginosa strains to the collection of environmental Pseudomonas phages. Phage-mediated lysis was performed by spot technique on TSA plates at 37 °C by application of 108-PFU/ml phage suspension. The assay was repeated at least three times Fig. 1 In vitro susceptibility of 123 P. aeruginosa strains to the collection of environmental Pseudomonas phages. Phage-mediated lysis was performed by spot technique on TSA plates at 37 °C by application of 108-PFU/ml phage suspension. The assay was repeated at least three times Fig. 1 In vitro susceptibility of 123 P. PA strains diversity The high morphological diversity among PA strains influenc- ing variation in phage susceptibility was further tested by the following features: (i) mucoidy, (ii) production of specific type IV pili–twitching motility, (iii) biofilm-forming ability, and (iv) biochemical composition fluctuation measured by ATR-FTIR spectroscopy. Bacterial morphology diversity tests (i–iii) are presented in Table 1. Five strains were similarly immutable in their lack of mucoidy, with mucoid CF832 being the exception. There was no significant clonal diversity among particular isolates in type IV pilus-dependent motility. The non-CF0038 and CF217 PA isolates exhibited a high de- gree of twitching motility with zone diameters reaching 30.4± 2.5 and 17.2±1.6 mm, respectively, within 48 h. The lowest value of motility was presented by PA CF708, suggesting lack of type IV pili. The third examined feature was the ability of biofilm formation which ranged from an OD of 0.04 (CF832) up to OD 1.92 (CF217). Testing 24 randomly taken PA colo- nies revealed the strongest clone diversity in biofilm forma- tion of PA CF217, which showed a range of OD from 1.36 to 2.48. Fig. 3 Pulsed field gel electrophoresis analysis of PA5oct phage DNA and KT28 phage DNA. Low Range PFG Marker (LMR) was used as a size marker homogeneous sensitivity to phages whereas CF217, CF708, and CF832 isolates showed clonal diversity during four pas- sages of the phage treatment assay. The PAO1 and CF532 were fully sensitive to both phages, giving confluent clear lysis in almost all passages. The non-CF isolate (0038) was also susceptible to phage lysis, but in the case of PA5oct ap- plication, single resistant colonies were detected in the plaque zone. The CF708 and CF832 were generally susceptible to the chosen phages but exhibited various profiles during four pas- sages of treatment, which suggested strong clonal diversity. The CF217 isolate was almost totally resistant to the applied phages, although phage PA5oct and KT28 gave a slightly The total chemical profile of bacterial samples was mea- sured by ATR-FTIR spectroscopy over the wavenumber range of 4000 to 900 cm−1 (Fig. 5). Galleria mellonella larvae model aeruginosa strains to the collection of environmental Pseudomonas phages. Phage-m by spot technique on TSA plates at 37 °C by application of 108-PFU/ml phage suspension. The assay was repeated at lea 113×17 nm, a base plate, and fibers of 40 nm in length (Ceyssens et al. 2009). Phage PA5oct is a giant unclassified myovirus with an isometric head of about 131 nm in diameter and a contractile tail about 136 nm long (Drulis-Kawa et al. 2014). The isolates were named according to a recent nomen- clature system as vB_PaeM_PA5oct and vB_PaeM_KT28 (Kropinski et al. 2009). The phages’ genome size was deter- mined by PFGE (Fig. 3). KT28 DNAs were about 70 kbp in size, whereas that of PA5oct was approximately 375 kbp (Drulis-Kawa et al. 2014). To explore possible variation in phage susceptibility within a given strain, six PA isolates were chosen: PAO1 as a refer- ence strain; non-CF0038, a clinical strain isolated from a wound infection; CF217 (unique cluster, small colony, early infection); CF708 (cluster 1, small colony, slow growth, late infection), and two sequential isolates CF532 nonmucoid and CF832 mucoid from one CF patient (Table 1). The variability of phage lytic activity examined in four bacterial culture pas- sages is illustrated in Fig. 4. Reference PAO1, the non-CF strain, and the paired isolate CF532 exhibited almost Fig. 2 Electron micrograph of PA5oct phage (a) and KT28 phage (b) negatively stained with uranyl acetate. The bar indicates 100 nm 6026 Appl Microbiol Biotechnol (2015) 99:6021–6033 Fig. 3 Pulsed field gel electrophoresis analysis of PA5oct phage DNA and KT28 phage DNA. Low Range PFG Marker (LMR) was used as a size marker inhibitory effect on bacterial growth. The application of a phage cocktail composed of both tested viruses showed ho- mogeneous confluent clear lysis in all four passages (PAO1, non-CF0038, CF708, CF532, CF832), suggesting an additive effect of the phage mixture. No activity of the cocktail was observed for PA CF217. PA strains diversity The vibrations assigned to lipids were around 3000–2800 cm−1 (window 1) (Winder and Goodacre 2004); the bands around 1700–1500 cm−1 (window 2) derived from proteins and the region from 1450 to 1200 cm−1 (window 3) was mainly due to the carboxylic groups of proteins, free amino acids, and polysaccharides wherein wavenumber 1250–1200 cm−1 is characteristic for the phosphate stretching mode due to the nucleic acids and phospholipids (San-Blas et al. 2012; Winder and Goodacre 2004). In addition to proteins, lipids, and nucleic acids, carbo- hydrates are also present, with a typical region of 1200– 900 cm−1 wavenumber (window 4). For the analyzed strains, the reproducibility of the typing method and the clonal diver- sity of the bacterial strains were examined based on the mean value of D indexes evaluated for all pairs of spectra within a given strain. Reproducibility was measured for the five work- ing ranges (windows 1, 2, 3, 4, and total 4000–900 cm−1 wavenumber) (Table 2). The total D value among tested strains was relatively low (0.9–4.2), except paired isolates (21.7–24.5). PA strains diversity The highest phenotypic clonal variations were noted for the non-CF0038 strain in the lipid window (145.9) and for mucoid strains in the carbohydrates and carboxylic Table 1 Morphological diversity of selected six PA strains tested by the following features: mucoidy, production of specific type IV pili– twitching motility, and biofilm-forming ability, as the elements consid- ered in term of phage susceptibility variation ered in term of phage susceptibility variation Strain Mucoidy Twitching motility zone diameter (mm) Biofilm formation OD (590 nm) PAO1 Negative (stable) 4.6±0.7 1.14±0.23 Non-CF0038 Negative (stable) 30.4±2.5 0.67±0.1 CF217 Negative (stable) 17.2±1.6 1.92±0.56 CF708 Negative (stable) 1.45±0.9 0.15±0.04 CF532 Negative (stable) 3.8±0.6 0.28±0.09 CF832 Positive (stable) 2.9±0.7 0.04±0.02 Appl Microbiol Biotechnol (2015) 99:6021–6033 6027 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage PAO1 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage nonCF0038 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF217 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF708 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF532 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF832 Passage 1 Passage 2 Passage 3 Passage 4 Fig. 4 PA strains sensitivity variation to selected bacteriophages during four passages. The strain sensitivity denotes confluent clear lysis (3), confluent opaque lysis (2), single resistant colonies on plaque (1), and resistance to phage (0) 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage PAO1 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage nonCF0038 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF217 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF708 Passage 1 Passage 2 Passage 3 Passage 4 Fig. 4 PA strains sensitivity variation to selected bacteriophages during four passages. PA strains diversity Variability of the D value (above 10) are the crucial differences in colony chemical profile were found in two regions: 3000–2800 cm−1 (window 1) and 1200– 900 cm−1 (window 5) for lipids and carbohydrates, respective- ly (Table 2). We observed that the variation in phage suscep- tibility in vitro, measured in four passages treatment, was not related with high D values. and 40 % of infected larvae, respectively, after 3 days of in- fection). These isolates exhibited lower virulence reflected by delay in larvae killing and a slow rate of growth. The lethal doses of 10 CFU for PAO1, non-CF0038 and CF217, and 105 CFU for the remaining strains were chosen for further experiments. The phage treatment results were observed at 8, 24, 48, and 72 h after injection (Fig. 6). All experiments were controlled by observation of uninfected larvae, sham-infected larvae, lar- vae receiving phage lysate only (100 % of larvae survival), and infected but phage untreated larvae. To determine whether the effects of phage therapy were associated with a nonspecif- ic immune activation response, UV-inactivated phages at the same inoculum were used to assess their ability to rescue infected larvae. The efficacy of UV inactivation was assessed if no viable phage was detected by in vitro plating. The inactivated phages were used to treat larvae infected with a lethal dose of PA strains, and no differences in larvae mortality were noted in comparison to control infected caterpillars. This confirmed that larval survival was entirely due to phage lytic activity rather than to host immune stimulation. PA strains diversity The strain sensitivity denotes confluent clear lysis (3), confluent opaque lysis (2), single resistant colonies on plaque (1), and resistance to phage (0) 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF532 Passage 1 Passage 2 Passage 3 Passage 4 0 1 2 3 PA5oct KT28 Phage mix Strain sensivity Bacteriophage CF832 Passage 1 Passage 2 Passage 3 Passage 4 incubation time, sample preparation, and spectrum measure- ment; thus, the appropriate spectral normalization should be included according to Naumann (2000), where mean D values up to 10 are considered as standard when analyzing the sam- ples prepared from independently grown cultures and all spec- tral ranges are analyzed. The obtained results indicated that groups spectra (D=70.9 and D=72.8). The most homoge- neous culture was the laboratory PAO1 strain, with the D value varying from 0.9 to 14.7 in four window spectra. Variability of the D value strongly depends on the standardi- zation of sample preparation procedures, spectral data acqui- sition parameters, medium preparation, growth temperature, Appl Microbiol Biotechnol (2015) 99:6021–6033 6028 Table 2 Differentiation index for analyzing biochemical clonal variations within given P. aeruginosa strains Strain 4000–900 cm−1 Total spectrum 1200–900 cm−1 Window 4 (carbohydrates) 1500–1200 cm−1 Window 3 (carboxylic groups) 1700–1500 cm−1 Window 2 (proteins) 3000–2800 cm−1 Window 1 (lipids) PAO1 0.9 7.6 6.4 0.3 14.7 Non-CF0038 3.0 17.8 9.1 1.0 145.9 CF217 1.4 9.9 7.7 1.0 27.8 CF708 4.2 32.2 16.1 3.7 23.5 CF532 21.7 36.4 52.9 10.1 111.2 CF832 24.5 70.9 72.8 9.2 90.1 The appropriate spectral normalization according to Naumann (2000) consider D values up to 10 as standard. Variability of the D value (above 10) are italicized e 2 Differentiation index for analyzing biochemical clonal variations within given P. aeruginosa strains The appropriate spectral normalization according to Naumann (2000) consider D values up to 10 as standard. Variability of the D value (above 10) are italicized The appropriate spectral normalization according to Naumann (2000) consider D values up to 10 as standard. Variabil italicized zation according to Naumann (2000) consider D values up to 10 as standard. Variability of the D value (above 10) are zation according to Naumann (2000) consider D values up to 10 as standard. Survival of infected larvae was followed over a period of 4 days. Three independent experiments containing ten larvae per each were pooled. The caterpillar survival rate is presented as a percentage in comparison to 100 % survival rate of uninfected larvae In vivo efficacy of phage treatment To verify the in vitro efficacy of two phages in relation to PA strains diversity, the following experiments were done in vivo on the wax moth larvae model. Prior to the appropriate exper- iments, the virulence of six chosen PA strains was determined. P. aeruginosa is highly virulent in this model, being their natural pathogen; thus, the lethal doses are very low. The wax moth larvae were injected with serial bacterial inocula (from 10 to 108 CFU) and incubated for 4 days. The caterpillar mortality rate was checked at 8, 24, 48, and 72 h after infection (Table 3). The experiments were controlled by a group of uninfected larvae and a group of larvae that received a control injection of saline where a 100 % survival rate was obtained in both groups. We found that PAO1, non-CF0038, and CF217 PA isolates were lethal even at the dose of 10 CFU where over 90 % larvae mortality was noted after 1 day of infection. The CF532, CF832, and CF708 isolates were significantly less virulent in comparison to the most virulent PAO1 (p<0.0001) and even at very high concentrations as 105 CFU dose the delay of killing was observed (100, 90, The phage application showed a significant impact on Galleria larval rescue from lethal infection (Fig. 6). Phage KT28 was the most potent whereas PA5oct was the least ef- fective preparation considering both non-CF Pseudomonas strains (p<0.0001). The antibacterial activity of the former against non-CF strains showed rescuing around 20 % of larvae even 2 days after lethal dose application. Moreover, KT28 Table 3 The virulence of PA strains tested in Galleria model PA01 (10 CFU) Non-CF0038 (10 CFU) CF217 (10 CFU) CF708 (105 CFU) CF532 (105 CFU) CF832 (105 CFU) 8 h 80 % 90 % 77 % 100 % 100 % 100 % 24 h 0 % 10 % 6 % 100 % 90 % 100 % 48 h 0 % 0 % 0 % 90 % 40 % 40 % 72 h 0 % 0 % 0 % 60 % 0 % 10 % 96 h 0 % 0 % 0 % 40 % 0 % 0 % Survival of infected larvae was followed over a period of 4 days. Three independent experiments containing ten larvae per each were pooled. In vivo efficacy of phage treatment The Table 3 The virulence of PA strains tested in Galleria model Survival of infected larvae was followed over a period of 4 days. Three independent experiments containing ten larvae per each were pooled. The caterpillar survival rate is presented as a percentage in comparison to 100 % survival rate of uninfected larvae Appl Microbiol Biotechnol (2015) 99:6021–6033 6029 preparation was also more effective against CF708 in compar- ison to PA5oct (p<0.07). The application of single preparation of KT28 or PA5oct to cure infection of phage resistant strain CF217 was not effective, whereas a mixture of both lysates in the final MOI 100 increased the survival rate of Galleria initially infected with CF217 by up to 30 and 20 % at 24 and 48 h, respectively (Fig. 6). However, the protective activ- ity of the phage cocktail against noncystic fibrosis Pseudomonas infection in larvae was lower than for single phage application (p<0.05 for mix vs PA5oct against PAO1, Fig. 6 Antibacterial activity of phages (MOI 100) in the treatment of infected Galleria larvae by PA strains. Survival of infected larvae was followed over a period of 3 days. Three independent experiments containing ten larvae per each were pooled. Statistical analysis was calculated for pairwise comparisons between infected larvae and phage- treated infected larvae using Mantel-Cox test Appl Microbiol Biotechnol (2015) 99:6021–6033 6029 containing ten larvae per each were pooled. Statistical analysis was calculated for pairwise comparisons between infected larvae and phage- treated infected larvae using Mantel-Cox test Fig. 6 Antibacterial activity of phages (MOI 100) in the treatment of infected Galleria larvae by PA strains. Survival of infected larvae was followed over a period of 3 days. Three independent experiments initially infected with CF217 by up to 30 and 20 % at 24 and 48 h, respectively (Fig. 6). However, the protective activ- ity of the phage cocktail against noncystic fibrosis Pseudomonas infection in larvae was lower than for single phage application (p<0.05 for mix vs PA5oct against PAO1, preparation was also more effective against CF708 in compar- ison to PA5oct (p<0.07). Discussion Recently, phages and phage-encoded proteins have been pro- posed as natural food preservatives and antimicrobial agents to fight bacterial infections in humans, animals, or crops of agricultural importance (Drulis-Kawa et al. 2012; Glonti et al. 2010). Phage therapy is promising in the eradication of drug- resistant pathogens such as bacteria-colonizing patients suffer- ing from cystic fibrosis (Alemayehu et al. 2012; Carmody et al. 2010; Debarbieux et al. 2010; Essoh et al. 2013; Morello et al. 2011). Recent reports of curative and preventive bacteriophage treatments in a mouse lung infection model, where lytic phages were administered in one single dose, suc- cessfully rescued mice from lethal infections caused by Burkholderia cenocepacia (Carmody et al. 2010) and P. aeruginosa (Morello et al. 2011). Special interest is focused on P. aeruginosa infections because of strong clonal diversity of these bacteria which can result in treatment issues. For these reasons, the application of lytic phages was tested in this study on selected PA isolates to determine their efficacy in vitro and in vivo in comparison to bacterial diversity of particular bio- logical features such as mucoidy, twitching motility, the inten- sity of biofilm formation, and biochemical composition. Although strong heterogeneity connected with in vitro phage susceptibility was noted, two myovirus phages with a broad spectrum of activity were selected among 28 environmental phages. The KT28 phage was classified as PB1-like viruses and a novel giant phage PA5oct belonged to the family Myoviridae (Drulis-Kawa et al. 2014). Phage lytic activity tested in vitro in the classical agar method was compared with the Galleria mellonella larvae in vivo model. This model is a very easy and convenient tool for bacterial virulence determination. Some P. aeruginosa strains are highly pathogenic in insects, such as G. mellonella (Fancello et al. 2011; Miriagou et al. 2010) and D. melanogaster (Barrow and Soothill 1997), when inoc- ulated directly into the hemolymph. In our study, the results obtained with the G. mellonella model generally correlated with data obtained from in vitro assays, but only for non-CF strains. The increased larval survival rates in the presence of PB1-like phage, compared to the giant PA5oct, could suggest the importance of phage size and phage generation time rate (KT28<PA5oct) in the therapeutic results. This needs to be further examined with a range of giant phage isolates. An unexpected feature was seen in the application of phage cock- tails. In vivo efficacy of phage treatment The application of single preparation of KT28 or PA5oct to cure infection of phage resistant strain CF217 was not effective, whereas a mixture of both lysates in the final MOI 100 increased the survival rate of Galleria Appl Microbiol Biotechnol (2015) 99:6021–6033 6030 peptidoglycan, teichoic acids, outer membrane proteins, oli- gosaccharides, capsule, and type IV fimbriae for the adhesion process (Abedon 2006; Drulis-Kawa et al. 2012; Guttman et al. 2005; Hanlon 2007; Leclerc et al. 2000; Lenski 1988; Matsuzaki et al. 2005; Young and Wang 2006). The specificity of interaction between phage attachment structures and host cell surface receptors mostly influences the bacterial host range (Drulis-Kawa et al. 2012; Sulakvelidze et al. 2001; Weinbauer 2004). Our previous work done on PAO1 mutants (paper in preparation) allowed the determination of the bacte- rial receptor recognized by KT28 phage (LPS elements) and PA5oct phage (type IV fimbriae and probably LPS as well). Examining our selected PA isolates, we conclude that the PAO1 strain, as the most susceptible to tested phages, was also clonally homogeneous in biochemical analysis done by FTIR and did not produce biofilm intensively. Therefore, this type of host seems to be the best target for effective phage attack. The non-CF PA strain (0038) exhibited strong twitching motility and low activity of biofilm formation. Although this strain showed strong clonal variation in lipid composition, the in vitro susceptibility to phages was relative- ly homogeneous. Lipid composition should have no signifi- cant influence on the first step of viral infection, adhesion, because phages usually utilize carbohydrate or protein as re- ceptors (Abedon 2006; Drulis-Kawa et al. 2012; Guttman et al. 2005; Hanlon 2007; Leclerc et al. 2000; Lenski 1988; Matsuzaki et al. 2005; Young and Wang 2006). p<0.0001 for mix vs KT28 against PAO1 and NS for mix vs PA5oct against non-CF0038, p<0.0001 for mix vs KT28 against non-CF0038). p<0.0001 for mix vs KT28 against PAO1 and NS for mix vs PA5oct against non-CF0038, p<0.0001 for mix vs KT28 against non-CF0038). Unexpected results were obtained for weakly virulent CF isolates (CF708, CF532, CF832). The chosen lethal dose for these isolates was much higher than for previous strains (105 vs 10 CFU); thus, the phage inoculum was also higher to keep MOI value of 100. The application of phage preparation both as single virus or cocktail did not protect larvae from death with one exception (action of KT28 on CF708). In vivo efficacy of phage treatment The possible explanation of such phenomenon could be the release of toxic compound during massive bacterial cell lysis after phage propagation, which may cause an increase in larvae mortality in comparison to not treated caterpillars. Discussion In contrast, in the in vivo study, the cocktail was more efficient in the protection of larvae against PA CF217 infection than the application of individual phage, implying an additive effect of mixed preparation in the case of resistant strains (p<0.0001 for mix vs PA5oct against CF217, p<0.0001 for mix vs KT28 against CF217). The strong clonal diversity and loss of viru- lence of CF-associated PA isolates is well known, and our investigation confirms this statement. Slow growth rate, re- duced twitching motility, low ability to biofilm formation, and finally strong clonal diversity in the chemical profile of the three tested CF isolates (CF708, CF532, and CF832) sig- nificantly influenced the phage efficacy both in vitro and in vivo. The CF708 strain and paired CF532/CF832 isolates were variable in terms of clone carbohydrate and carboxylic group composition, which influenced large differences in phage susceptibility among separate passages (confluent clear and opaque lysis or single resistant colonies on plaque) for CF708 and CF832. Generally, the variations in chemical com- position of the culture measured by the FTIR method affected phage efficacy. More complications were noticed in in vivo assay where the application of phage preparation did not cure larvae from infection but even worsened the caterpillar condi- tion (p<0.05 for KT28 against CF708). We suspect that this was due to the massive release of toxic compounds during bacterial cell lysis causing a deterioration of animal health. It is well known that rapid lysis of a big number of cells during phage propagation and the release of LPS from Gram- negative bacteria in a short period of time may cause serious side effects on the host; however, similar effects may occur during bactericidal antibiotic application, as well (Goodridge 2010). The administration of bacteriolytic agents of any kind in the treatment of Pseudomonas infection may result in se- vere consequences as systemic inflammatory response syn- drome; thus, the therapy should be carefully selected. Acknowledgments The authors are grateful to Hans-W. Ackermann for the performance of transmission electron microscope (TEM) micro- graphs and all valuable comments in manuscript preparation. The authors are grateful to Anne Costello for her help with the in vivo model. This study was supported by the National Science Centre research grant no. 2012/04/M/NZ6/00335. Discussion The mixture was less effective in the lysis of phage susceptible PA strains PAO1 and non-CF0038 in comparison to single lytic phage administration. A possible explanation for such phenomenon could be the competition between attacking virions for the adhesion receptors on bacterial cells. If the receptors on PA surface used by phages are present in a high number and are the same, similar, or located in close vicinity with each other, the effective adhesion of particular virions may be hampered or impaired. This would suggest that phages selected for cocktail preparations should be designed The phenotypical diversity among PA strains may influence the variation in phage susceptibility by modifying, masking, or replacing phage receptors required in the first virion- bacterial interactions during viral infection (Abedon 2006; Drulis-Kawa et al. 2012; Guttman et al. 2005; Hanlon 2007; Leclerc et al. 2000; Lenski 1988; Matsuzaki et al. 2005; Young and Wang 2006). Phages can recognize surface com- ponents of a bacterial cell including lipopolysaccharide (LPS), Appl Microbiol Biotechnol (2015) 99:6021–6033 6031 to target different receptors enhancing the host range and al- low synergistic activity of phages. Although standardized methods to generate phage cocktails have been proposed (Drulis-Kawa et al. 2012; Merabishvili et al. 2009), no clear official guidelines exist to date (Drulis-Kawa et al. 2012; Pirnay et al. 2011). isolates is promising alternative but requires additional de- tailed investigations. The simple animal models such as wax moth larvae model could be a convenient tool to evaluate the effectiveness of particular phage preparations against clinical isolates in vivo. Future work will need to consider the effec- tiveness of phages in mixed culture models replicating the Bairway microbiome^ including widely available and well- characterized PA strains from diverse sources. The results obtain for CF isolates in this study were more complicated. The CF217 isolate with the growth rate similar to PAO1 strain was almost totally resistant to in vitro admin- istered phages although it produced type IV pili as a potential phage receptor for PA5oct phage. 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Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial
Annals of oncology
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VThe Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. 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. 1Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust, Cambridge; 2Warwick Clinical Trials Unit, University of Warwick, Coventry; 3Medical Oncology, Mount Vernon Hospital, Northwood; 4Department of Medical Oncology, Christie Hospital, Manchester; 5Royal Marsden Hospital NHS Trust, London; 6Oncology Research, Broomfield Hospital, Chelmsford; 7Oncology Department, Leicester Royal Infirmary, Leicester; 8Sir Bobby Robson Cancer Trials Research Centre, Freeman Hospital, Newcastle upon Tyne; 9Leeds Cancer Centre, St James’s University Hospital, Leeds; 10Weston Park Hospital, Academic Unit of Clinical Oncology, Sheffield; 11Cancer & Palliative Care, St. Helen’s Hospital, St. Helens; 12Oncology Department, Royal Surrey County Hospital, Guildford; 13Rosemere Cancer Centre, Royal Preston Hospital, Preston; 14Clinical Trials Unit, Beatson WOS Cancer Centre, Glasgow; 15Department of Clinical Oncology, Norfolk & Norwich University Hospital, Norwich; 16Guy’s & St. Thomas’ Hospital, Guy’s Cancer Centre, London; 17Velindre Cancer Centre, Cardiff; 18Exeter Oncology Centre, Royal Devon and Exeter Hospital, Exeter; 19St George’s Hospital, Cancer Centre, London; 20Cancer Centre, Queen Elizabeth Hospital, Birmingham; 21Sussex Cancer Centre, Royal Sussex County Hospital, Brighton; 22Cancer Centre, Ninewells Hospital, Dundee; 23Royal Free Hospital, London; 24Castle Hill Hospital, Cottingham; 25Academic Unit of Clinical Oncology, Nottingham University Hospitals NHS Trust, Nottingham; 26CRUK and NIHR Southampton Experimental Cancer Medicine Centre, Southampton University Hospitals NHS Foundation Trust, Southampton; 27Oncology Centre, Cheltenham General Hospital, Cheltenham; 28Oncology Department, James Cook University Hospital, Middlesbrough; 29Oncology Department, Poole Hospital, Dorset; 30Department of Oncology, University of Oxford, Oxford; 31Experimental Cancer Medicine Centre, Oxford; 32Melanoma Focus, Cambridge; 33Oxford NIHR Biomedical Research Centre, Oxford, UK ORIGINAL ARTICLE Thomas’ Hospital, Guy’s Cancer Centre, London; 17Velindre Cancer Centre, Cardiff; 18Exeter Oncology Centre, Royal Devon and Exeter Hospital, Exeter; 19St George’s Hospital, Cancer Centre, London; 20Cancer Centre, Queen Elizabeth Hospital, Birmingham; 21Sussex Cancer Centre, Royal Sussex County Hospital, Brighton; 22Cancer Centre, Ninewells Hospital, Dundee; 23Royal Free Hospital, London; 24Castle Hill Hospital, Cottingham; 25Academic Unit of Clinical Oncology, Nottingham University Hospitals NHS Trust, Nottingham; 26CRUK and NIHR Southampton Experimental Cancer Medicine Centre, Southampton University Hospitals NHS Foundation Trust, Southampton; 27Oncology Centre, Cheltenham General Hospital, Cheltenham; 28Oncology Department, James Cook University Hospital, Middlesbrough; 29Oncology Department, Poole Hospital, Dorset; 30Department of Oncology, University of Oxford, Oxford; 31Experimental Cancer Medicine Centre, Oxford; 32Melanoma Focus, Cambridge; 33Oxford NIHR Biomedical Research Centre, Oxford, UK †All Avast-M Investigators are listed in the Acknowledgements. Background: Bevacizumab is a recombinant humanised monoclonal antibody to vascular endothelial growth factor shown to improve survival in advanced solid cancers. We evaluated the role of adjuvant bevacizumab in melanoma patients at high risk of recurrence. Patients and methods: Patients with resected AJCC stage IIB, IIC and III cutaneous melanoma were randomised to receive either adjuvant bevacizumab (7.5 mg/kg i.v. 3 weekly for 1 year) or standard observation. The primary end point was detection of an 8% difference in 5-year overall survival (OS) rate; secondary end points included disease-free interval (DFI) and distant metastasis-free interval (DMFI). Tumour and blood were analysed for prognostic and predictive markers. Results: Patients (n¼1343) recruited between 2007 and 2012 were predominantly stage III (73%), with median age 56 years (range 18–88 years). With 6.4-year median follow-up, 515 (38%) patients had died [254 (38%) bevacizumab; 261 (39%) observation]; 707 (53%) patients had disease recurrence [336 (50%) bevacizumab, 371 (55%) observation]. OS at 5 years was 64% for both groups [hazard ratio (HR) 0.98; 95% confidence interval (CI) 0.82–1.16, P ¼ 0.78). At 5 years, 51% were disease free on bevacizumab versus 45% on observation (HR 0.85; 95% CI 0.74–0.99, P ¼ 0.03), 58% were distant metastasis free on bevacizumab versus 54% on observation (HR 0.91; 95% CI 0.78–1.07, P ¼ 0.25). Forty four percent of 682 melanomas assessed had a BRAFV600 mutation. In the observation arm, BRAF mutant patients had a trend towards poorer OS compared with BRAF wild-type patients (P ¼ 0.06). BRAF mutation positivity trended towards better OS with bevacizumab (P ¼ 0.21). Conclusions: Adjuvant bevacizumab after resection of high-risk melanoma improves DFI, but not OS. *Correspondence to: Dr Pippa G Corrie, Consultant and Associate Lecturer in Medical Oncology, Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust (Addenbrooke’s Hospital), Hills Road, Cambridge CB2 0QQ, UK. Tel: þ44-01223-274376; E-mail: pippa.corrie@addenbrookes.nhs.uk V C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society for Medical Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), whi reproduction in any medium, provided the original work is properly cited. Annals of Oncology 29: 1843–1852, 2018 doi:10.1093/annonc/mdy229 Published online 13 July 2018 ORIGINAL ARTICLE ORIGINAL ARTICLE ORIGINAL ARTICLE Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial P. G. Corrie1,*, A. Marshall2, P. D. Nathan3, P. Lorigan4, M. Gore5, S. Tahir6, G. Faust7, C. G. Kelly8, M. Marples9, S. J. Danson10, E. Marshall11, S. J. Houston12, R. E. Board13, A. M. Waterston14, J. P. Nobes15, M Harries16 S Kumar17 A Goodman18 A Dalgleish19 A Martin Clavijo20 S Westwell21 R Casasola22 P. G. Corrie1,*, A. Marshall2, P. D. Nathan3, P. Lorigan4, M. Gore5, S. Tahir6, G. Faust7, C. G. Kelly8, 9 10 11 12 13 14 15 G. Corrie1,*, A. Marshall2, P. D. Nathan3, P. Lorigan4, M. Gore5, S. Tahir6, G. Faust7, C. G. Kelly8, g y 9, S. J. Danson10, E. Marshall11, S. J. Houston12, R. E. Board13, A. M. Waterston14, J. P. Nobes15, 6 17 18 19 20 21 22 p M. Harries16, S. Kumar17, A. Goodman18, A. Dalgleish19, A. Martin-Clavijo20, S. Westwell21, R. Casasola22, D. Chao23, A. Maraveyas24, P. M. Patel25, C. H. Ottensmeier26, D. Farrugia27, A. Humphreys28, B. Eccles29, G. Young1, E. O. Barker1, C. Harman1, M. Weiss1, K. A. Myers30,31, A. Chhabra1, S. H. Rodwell32, J. A. Dunn2 & M. R. Middleton33, on behalf of the AVAST-M Investigators† 1Cambridge Cancer Centre, Cambridge University Hospitals NHS Foundation Trust, Cambridge; 2Warwick Clinical Trials Unit, University of Warwick, Coventry; 3Medical Oncology, Mount Vernon Hospital, Northwood; 4Department of Medical Oncology, Christie Hospital, Manchester; 5Royal Marsden Hospital NHS Trust, London; 6Oncology Research, Broomfield Hospital, Chelmsford; 7Oncology Department, Leicester Royal Infirmary, Leicester; 8Sir Bobby Robson Cancer Trials Research Centre, Freeman Hospital, Newcastle upon Tyne; 9Leeds Cancer Centre, St James’s University Hospital, Leeds; 10Weston Park Hospital, Academic Unit of Clinical Oncology, Sheffield; 11Cancer & Palliative Care, St. Helen’s Hospital, St. Helens; 12Oncology Department, Royal Surrey County Hospital, Guildford; 13Rosemere Cancer Centre, Royal Preston Hospital, Preston; 14Clinical Trials Unit, Beatson WOS Cancer Centre, Glasgow; 15Department of Clinical Oncology, Norfolk & Norwich University Hospital, Norwich; 16Guy’s & St. Introduction 85% power and a 5% significance level, equating to an HR of 0.80. OS was defined as the time from date of randomisation until date of death from any cause, or censored at the last known date alive. Analysis was follow-up driven and pre-planned when all patients had been on study for 5 years. Angiogenesis is a host-dependent hallmark of cancer [1] and vas- cular endothelial growth factor (VEGF) is a key driver of angio- genesis [2]. VEGF is over-expressed in melanoma and high levels have been reported to be associated with poorer outcome [3–6]. Bevacizumab (AvastinV R, F. Hoffman-La Roche AG, Basel, Switzerland) is a recombinant humanised monoclonal antibody to VEGF licensed for treatment of several common cancers, with modest activity reported in advanced melanoma [7]. Since VEGF is a relevant target in melanoma, we carried out a UK multi- centre, open-label, randomised controlled phase III trial of adju- vant bevacizumab versus standard surveillance in patients with resected cutaneous melanoma at high risk of recurrence. Secondary end points were DFI, distant metastasis-free interval (DMFI), safety, toxicity and health-related quality of life (QoL). Adverse events were only collected during treatment and were reported previously [8]. Tertiary end points were to evaluate biological predictive and prog- nostic markers. DFI was defined as the time from date of randomisation until date of first tumour recurrence (including distant and locoregional recurrence), or date of death due to melanoma. DMFI was defined as the time from date of randomisation until date of first distant recurrent dis- ease, or date of death due to melanoma. Survival from recurrence was defined as the time between the date of first tumour progression (in any site) and the date of death. Kaplan–Meier survival curves were con- structed and a Cox proportional hazard model was used to obtain HRs and associated 95% CIs. Multivariable Cox regression models were used to adjust the treatment effect for stratification variables, to evaluate inde- pendent prognostic factors of OS and DFI and to assess treatment inter- actions. EORTC-QLQ-C30 QoL data were analysed by standardised area under the curve (AUC) and compared across trial arms using Wilcoxon rank sum tests. Mixed-effect models were used to assess whether VEGF and VEGFR1 levels changed over time or differed across trial arms. LDH levels measured over time were fitted as time-dependent continuous covariates in a Cox regression model. Results Between 18 July 2007 and 29 March 2012, 1343 patients were randomised to either the bevacizumab (N ¼ 671) or observation (N ¼ 672) arms. Seven hundred fifty-three (56%) patients were male, their median age was 56 years (range 18–88 years), 364 (27%) patients had stage II melanoma, 195 (14%) had stage IIIA and 784 (59%) had stage IIIB/C disease. Sentinel lymph node bi- opsy (SLNB) was not mandated and 32% of patients in each arm underwent SLNB. Other baseline characteristics were similar be- tween groups and were reported in full previously [8]. Six hun- dred eight two (51%) patients’ tumours were assessed for BRAF and NRAS mutation status; BRAF V600 and NRAS mutations were detected in 303 (44%) and 134 (20%) tumours tested. Eligible patients were randomly assigned to adjuvant bevacizumab (7.5 mg/kg i.v. infusion once every 3 weeks for 1 calendar year) or surveil- lance in a 1 : 1 ratio, stratified by primary tumour Breslow thickness, N stage, primary tumour ulceration status and patient sex. Randomisation occurred within 12 weeks of surgical resection and was carried out cen- trally using a computer minimisation algorithm held at the Warwick Clinical Trials Unit. This was an open-label trial. Biomarker analyses With a median follow-up of 6.4 years, 515 (38%) patients had died: 254 (38%) of patients in the bevacizumab arm, 261 (39%) in the observation arm, 92% from metastatic melanoma on both arms. Seven hundred seven (53%) patients had melanoma recur- rence: 336 (50%) in the bevacizumab arm, 371 (55%) in the ob- servation arm. Of the 707 patients who had a recurrence, 117 (16%) patients had locoregional recurrence only, 359 (51%) had distant recurrence only and 231 (33%) had both locoregional and distant recurrence. One hundred twelve (16%) received an im- mune checkpoint inhibitor or targeted therapy as treatment for recurrence, totalling 55 (16%) on the bevacizumab arm and 57 (15%) on the observation arm (Table 1). At trial entry, plasma lactate dehydrogenase (LDH) was measured by local hospital laboratories for all patients. A patient was classed as having raised LDH if the value was above the upper level of normal (ULN) for their hospital. LDH was also measured centrally in plasma at baseline (pre-randomisation), 3 and 12 months from trial entry. VEGF and sol- uble VEGF receptor-1 (VEGFR1) were measured centrally by ELISA in both plasma and serum samples at baseline and then at 3, 12 and 24 months in exploratory patient cohorts. BRAF and NRAS mutation sta- tus were determined in archival tumour tissue using accredited methods. ORIGINAL ARTICLE BRAF mutation status may predict for poorer OS untreated and potential benefit from bevacizumab. Annals of Oncology Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64 Key words: melanoma, bevacizumab, adjuvant therapy g Clinical Trial Information: ISRCTN 81261306; EudraCT Number: 2006-005505-64 Key words: melanoma, bevacizumab, adjuvant therapy g Introduction The interim analysis of the AVAST-M trial when 1343 patients had been recruited and followed for more than 1 year showed a significant improvement in disease-free interval (DFI) with adju- vant bevacizumab [hazard ratio (HR) 0.83 (95% confidence interval [CI] 0.70–0.98), P ¼ 0.03] [8], which was well tolerated. We report the analysis of the primary overall survival (OS) end point, mandated when all surviving patients had been on study for at least 5 years. Two-sided P values and 95% CIs are reported. All analyses were carried out on an intention-to-treat basis using the SAS statistical package. Methods The study design, eligibility criteria, stratification variables and treatment schedules have been described previously in detail [8]. Briefly, patients at least 16-year old with histological confirmation of completely resected AJCC 7th edition stage IIB, IIC or IIIA–C cutaneous melanoma were eli- gible for the trial. Written informed consent was obtained for all patients. Multicentre Research Ethics Committee and regulatory approvals were obtained. Patients were followed up at least annually for 10 years after randomisation. Statistical analysis A high percentage (89%) of QoL forms were completed. There was no difference in overall QoL over the 5 years for the two trial arms: median standardised AUC for the QLQ-C30 global health scale was 81.7% [interquartile range (IQR) 69.8%–90.7%] for patients on the bevacizumab arm and 81.9% (IQR 68.6%– 91.7%) on the observation arm (P ¼ 0.52). 91.7%) on the observation arm (P ¼ 0.52). In the observation arm, BRAF mutant melanoma patients had poorer OS compared with BRAF wild-type melanomas (P ¼ 0.06, Figure 3A). Overall, this effect was similar after adjustment for disease stage, ECOG performance status, primary melanoma Breslow thickness and sex (P ¼ 0.08, Table 2). A trend for improved OS with bevacizumab was only evident for the patients with BRAF mutant melanomas (HR ¼ 0.80; CI 0.57–1.13; P ¼ 0.21, Figure 3C) and not seen in the patients with BRAF wild- type melanomas (HR ¼ 1.17; CI 0.82–1.61; P ¼ 0.34, Figure 3E). BRAF mutant patients received more checkpoint inhibitors/tar- geted therapy at recurrence (22% versus 9%, supplementary Table S1, available at Annals of Oncology online), but the benefit from bevacizumab was evident for DFI as well as OS (Figure 3D). At trial entry 179 (13%) patients had plasma LDH levels above the hospital reported ULN. Baseline LDH was not found to be prognostic of DFI (HR ¼ 1.01, CI 0.81–1.25, P ¼ 0.97), DMFI (HR ¼ 1.10, CI 0.88–1.39, P ¼ 0.40) or OS (HR ¼ 1.05; CI 0.81– 1.35; P ¼ 0.73). LDH measurements across three time-points— baseline, 3 months and 12 months—were also assessed. After In the observation arm, BRAF mutant melanoma patients had poorer OS compared with BRAF wild-type melanomas (P ¼ 0.06, Figure 3A). Overall, this effect was similar after adjustment for disease stage, ECOG performance status, primary melanoma Breslow thickness and sex (P ¼ 0.08, Table 2). A trend for improved OS with bevacizumab was only evident for the patients with BRAF mutant melanomas (HR ¼ 0.80; CI 0.57–1.13; P ¼ 0.21, Figure 3C) and not seen in the patients with BRAF wild- type melanomas (HR ¼ 1.17; CI 0.82–1.61; P ¼ 0.34, Figure 3E). BRAF mutant patients received more checkpoint inhibitors/tar- geted therapy at recurrence (22% versus 9%, supplementary Table S1, available at Annals of Oncology online), but the benefit from bevacizumab was evident for DFI as well as OS (Figure 3D). Statistical analysis Patients (n ¼ 1320; 660 patients per arm) were required to detect an 8% increase in the 5-year OS rate (primary end point) from 40% to 48% with Volume 29 | Issue 8 | 2018 1844 | Corrie et al. Original article Table 1. Details of melanoma recurrence and associated treatment of recurrence Bevacizumab Observation Total (N 5 671) (N 5 672) (N 5 1343) N (%) N (%) N (%) Patients with any recurrence 336 (50%) 371 (55%) 707 (53%) Locoregional only 54 (16%) 63 (17%) 117 (16%) Distant only 169 (50%) 190 (51%) 359 (51%) Both locoregional and distant recurrence 113 (34%) 118 (32%) 231 (33%) Treatment for any recurrence Immune checkpoint inhibitors/targeted therapya 55 (16%) 57 (15%) 112 (16%) Vemurafenib 27 34 61 Ipilimumab 19 17 36 Dabrafenib þ/ trametinib 16 8 24 Ipilimumab þ nivolumab 2 1 5 Pembrolizumab 2 2 4 Pazopanib 0 1 1 Vandetanib 1 0 1 Blinded ipilimumab, nivolumab or ipilimumabþnivolumab 0 2 2 Other systemic therapy 79 (24%) 97 (26%) 176 (25%) Given as part of a clinical trial 9 19 28 Dacarbazine 56 59 115 Other cytotoxic chemotherapy 11 12 23 Other immunotherapy 3 5 8 Other biological agent 0 2 2 Surgery only 89 (26%) 119 (32%) 208 (29%) Other (including radiotherapy) 66 (20%) 62 (17%) 128 (18%) None 47 (14%) 36 (10%) 83 (12%) aPatients could receive more than one line of treatment for recurrence; 98% patients receiving systemic therapy had distant metastatic disease. Annals of Oncology Original article Annals of Oncology aPatients could receive more than one line of treatment for recurrence; 98% patients receiving systemic therapy had distant metastatic disease. aPatients could receive more than one line of treatment for recurrence; 98% patients receiving systemic therapy had distant metastatic disease. aPatients could receive more than one line of treatment for recurrence; 98% patients receiving systemic therapy had distant metastatic disease. There was no significant difference in OS between trial arms (HR for bevacizumab ¼ 0.98; CI 0.82–1.16; P ¼ 0.78, Figure 1A). The 5-year OS rate was 64% for both arms (CI 61%–68% for bev- acizumab, 60%–67% for observation). Multivariate analysis identified disease stage, ECOG performance status, primary mel- anoma Breslow thickness and sex as independently prognostic of OS; trial arm remained non-significant (P ¼ 0.92; Table 2). There was no statistically significant interaction between any of these variables and trial arm (Figure 2). Statistical analysis The 5-year DMFI rate was 58% (CI 54%–62%) for the bevaci- zumab arm and 54% (CI 50%–58%) for the observation arm, but this was not statistically significantly different (HR ¼ 0.91; CI 0.78–1.07; P ¼ 0.25, Figure 1B). The median DMFI for the bevaci- zumab arm was not reached (CI 7.3 years to limit not reached) and 9.6 years (CI 5.5–9.6 years) for the observation arm. The significant improvement in DFI for those on the bevacizu- mab arm reported at the interim analysis was maintained over time (HR ¼ 0.85; CI 0.74–0.99; P ¼ 0.03, Figure 1C) and persisted after adjustment for the stratification variables (HR ¼ 0.86; CI 0.74–0.99; P ¼ 0.04). Patients receiving bevacizumab had a higher 5-year DFI rate (51%; CI 47%–55%) compared with the observa- tion arm (45%; CI 42%–49%). The median DFI for patients in the bevacizumab arm was 63 months (CI, 44 months to limit not reached) and 37 months (CI 30–50 months) for those in the ob- servation arm. At trial entry 179 (13%) patients had plasma LDH levels above the hospital reported ULN. Baseline LDH was not found to be prognostic of DFI (HR ¼ 1.01, CI 0.81–1.25, P ¼ 0.97), DMFI (HR ¼ 1.10, CI 0.88–1.39, P ¼ 0.40) or OS (HR ¼ 1.05; CI 0.81– 1.35; P ¼ 0.73). LDH measurements across three time-points— baseline, 3 months and 12 months—were also assessed. Statistical analysis After Volume 29 | Issue 8 | 2018 doi:10.1093/annonc/mdy229 | 1845 0 25 50 75 100 A B C 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Percentage Surviving Years from entry Bevacizumab Observation 0 25 50 75 100 Percentage distant metastasis free Years from entry Bevacizumab Observation Numbers atrisk: 25 50 75 100 Percentage disease free Bevacizumab Observation 671 624 535 471 428 372 250 148 74 672 623 538 475 431 367 249 140 71 Bevacizumab 671 503 395 366 336 295 201 122 66 Observation 672 469 378 332 307 269 188 108 59 Numbers atrisk: Bevacizumab Observation nal article Annals of Oncology 0 25 50 75 100 A 0 1 2 3 4 5 6 7 8 Percentage Surviving Years from entry Bevacizumab Observation 671 624 535 471 428 372 250 148 74 672 623 538 475 431 367 249 140 71 Numbers atrisk: Bevacizumab Observation article Annals of Oncolog Annals of Oncology 0 25 50 75 B C 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Percentage Surviving Years from entry Bevacizumab Observation 0 25 50 75 100 Percentage distant metastasis free Years from entry Bevacizumab Observation Numbers atrisk: 0 25 50 75 100 Percentage disease free Years from entry Bevacizumab Observation Numbers atrisk: 671 624 535 471 428 372 250 148 74 672 623 538 475 431 367 249 140 71 Bevacizumab 671 503 395 366 336 295 201 122 66 Observation 672 469 378 332 307 269 188 108 59 Bevacizumab 671 497 388 359 329 286 192 113 57 Observation 672 462 370 322 296 258 176 95 46 Numbers atrisk: Bevacizumab Observation rall survival (A), distant metastasis-free interval (B) and disease-free interval (C), by trial arm. Statistical analysis Neither baseline plasma nor serum VEGF w prognostic factors for OS (HR ¼ 1.02, CI 0.95–1.10 per 50 increase P ¼ 0 53 for plasma; HR ¼ 1 03 CI 0 98–1 07 per Gender Events/Patients Treatment Treatment events *Hazard Ratio & CI (Treatment : Obs) *HR & CI (Treatment : Obs) Obs (O-E) Var Disease stage Interaction between 2 groups χ2 1= 0.3; P=.59 Heterogeneity between 3 groups χ2 2= 1.7; P=.42 Heterogeneity between 3 groups χ2 2= 3.4; P=.18 Heterogeneity between 4 groups χ2 3= 2.7; P=.44 Interaction between 2 groups χ2 1= 2.6; P=.10 Interaction between 2 groups χ2 1= 2.9; P=.09 Ulceration II III(N1a & N2a) III(Other N) Unknown Breslow Thickness Unknown Performance status Baseline LDH ECOG=0 <=ULN >ULN ECOG=1 Male Present Absent <=2mm 2-4mm >4mm Female Stratified 159/377 157/376 1.01 (0.81, 1.26) 0.92 (0.70, 1.21) 0.98 (0.82, 1.16) 1.21 (0.84, 1.73) 0.85 (0.54, 1.33) 0.95 (0.77, 1.19) 0.99 (0.83, 1.18) 1.12 (0.86, 1.46) 0.81 (0.62, 1.05) 1.13 (0.72, 1.77) 0.97 (0.82, 1.16) 0.94 (0.68, 1.32) 0.80 (0.58, 1.11) 1.14 (0.86, 1.52) 1.03 (0.52, 2.07) 0.93 (0.77, 1.13) 1.40 (0.89, 2.19) 0.99 (0.83, 1.18) 0.95 (0.78, 1.14) 1.47 (0.92, 2.36) 1.01 (0.85, 1.20) 0.98 (0.82, 1.16) 0.97 (0.82, 1.16) (P=.92) (P=.76) (P=.77) (P=.91) (P=.94) (P=.78) (P=.78) 1.0 79.0 49.7 128.7 –4.2 –3.1 5.7 29.7 19.2 79.7 128.6 54.0 55.7 19.0 128.7 35.0 37.2 48.5 8.0 128.7 109.0 128.0 108.7 17.3 126.0 128.7 0.0 0.5 Treatment better Obs better 1.0 1.5 2.0 19.1 –3.1 –3.7 –1.1 6.1 –11.9 –3.5 –2.0 –8.1 6.6 0.3 –3.3 –7.6 6.4 –1.3 –5.9 –3.1 6.7 0.8 2.3 104/296 261/672 (41.8%) (35.1%) (38.8%) 254/671 66/187 53/177 (42.2%) (32.3%) (37.9%) (35.3%) (29.9%) (29.6%) (46.7%) (38.8%) 40/135 168/360 37/141 151/343 254/671 114/262 102/256 124/323 99/310 41/99 35/93 261/672 254/671 67/198 73/201 81/202 91/217 16/52 261/672 68/203 103/221 254/671 213/602 223/593 38/79 261/672 40/67 253/669 16/49 (43.5%) (39.8%) (38.4%) (37.6%) (38.8%) (31.9%) (41.4%) (37.9%) (33.8%) (36.3%) (40.1%) (41.9%) (30.8%) (38.8%) (33.5%) (46.6%) (32.7%) (37.9%) (35.4%) (37.6%) (48.1%) (38.8%) (59.7%) (37.8%) 214/568 221/563 253/654 32/91 38/88 252/656 254/671 261/672 (37.7%) (39.3%) (35.2%) (38.7%) (43.2%) (38.4%) (37.9%) (38.8%) 261/672 (26.2%) (44.0%) (37.9%) 95/294 Stratified Stratified Stratified Stratified Stratified Unstratified * 95% CI 95% CI igure 2. Hazard ratio plot of the treatment effect by prognostic factors for overall survival. Statistical analysis B 0 1 2 3 4 5 6 7 8 0 25 50 75 100 Percentage distant metastasis free Years from entry Bevacizumab Observation Numbers atrisk: Bevacizumab 671 503 395 366 336 295 201 122 66 B C 0 1 2 3 4 5 6 7 8 0 25 50 75 100 Percentage distant metastasis free Years from entry Bevacizumab Observation Numbers atrisk: 50 75 100 entage disease free Bevacizumab Observation Bevacizumab 671 503 395 366 336 295 201 122 66 Observation 672 469 378 332 307 269 188 108 59 B C 0 1 2 3 4 5 6 7 8 0 25 50 75 100 Percentage disease free Years from entry Bevacizumab Observation Numbers atrisk: Bevacizumab 671 497 388 359 329 286 192 113 57 Observation 672 462 370 322 296 258 176 95 46 ll l d f l d d f l b l Figure 1. Overall survival (A), distant metastasis-free interval (B) and disease-free interval (C), by trial arm. Volume 29 | Issue 8 | 2018 1846 | Corrie et al. Annals of Oncology tting LDH as a time-dependent continuous covariate, LDH was ill not found to be prognostic of DFI (HR ¼ 1.00, CI 0.96–1.03 er 50 unit increase, P ¼ 0.81), DMFI (HR ¼ 1.01, CI 0.97–1.05 er 50 unit increase, P ¼ 0.56) or OS (HR ¼ 1.02, CI 0.98–1.06 er 50 unit increase P ¼ 0 36) measurements at baseline and serially over the 2 years from domisation (supplementary Figure S1, available at Annal Oncology online). Statistical analysis nnals of Oncology Original artic Gender Events/Patients Treatment Treatment events *Ha (Tr Obs (O-E) Var Disease stage Interaction between 2 groups χ2 1= 0.3; P=.59 Heterogeneity between 3 groups χ2 2= 1.7; P=.42 Heterogeneity between 3 groups χ2 2= 3.4; P=.18 Heterogeneity between 4 groups χ2 3= 2.7; P=.44 Interaction between 2 groups χ2 1= 2.6; P=.10 Interaction between 2 groups χ2 1= 2.9; P=.09 Ulceration II III(N1a & N2a) III(Other N) Unknown Breslow Thickness Unknown Performance status Baseline LDH ECOG=0 <=ULN >ULN ECOG=1 Male Present Absent <=2mm 2-4mm >4mm Female Stratified 159/377 157/376 1.0 79.0 49.7 128.7 –4.2 –3.1 5.7 29.7 19.2 79.7 128.6 54.0 55.7 19.0 128.7 35.0 37.2 48.5 8.0 128.7 109.0 128.0 108.7 17.3 126.0 128.7 0.0 0.5 Treatment bett 19.1 –3.1 –3.7 –1.1 6.1 –11.9 –3.5 –2.0 –8.1 6.6 0.3 –3.3 –7.6 6.4 –1.3 –5.9 –3.1 6.7 0.8 2.3 104/296 261/672 (41.8%) (35.1%) (38.8%) 254/671 66/187 53/177 (42.2%) (32.3%) (37.9%) (35.3%) (29.9%) (29.6%) (46.7%) (38.8%) 40/135 168/360 37/141 151/343 254/671 114/262 102/256 124/323 99/310 41/99 35/93 261/672 254/671 67/198 73/201 81/202 91/217 16/52 261/672 68/203 103/221 254/671 213/602 223/593 38/79 261/672 40/67 253/669 16/49 (43.5%) (39.8%) (38.4%) (37.6%) (38.8%) (31.9%) (41.4%) (37.9%) (33.8%) (36.3%) (40.1%) (41.9%) (30.8%) (38.8%) (33.5%) (46.6%) (32.7%) (37.9%) (35.4%) (37.6%) (48.1%) (38.8%) (59.7%) (37.8%) 214/568 221/563 253/654 32/91 38/88 252/656 254/671 261/672 (37.7%) (39.3%) (35.2%) (38.7%) (43.2%) (38.4%) (37.9%) (38.8%) 261/672 (26.2%) (44.0%) (37.9%) 95/294 Stratified Stratified Stratified Stratified Stratified Unstratified * 95% CI 95% CI gure 2. Hazard ratio plot of the treatment effect by prognostic factors for overall survival. nnals of Oncology 0.80 (0.58, 1.11) 1.14 (0.86, 1.52) 1.03 (0.52, 2.07) 0.93 (0.77, 1.13) 1.40 (0.89, 2.19) 0.99 (0.83, 1.18) 0.95 (0.78, 1.14) 1.47 (0.92, 2.36) 1.01 (0.85, 1.20) 0.98 (0.82, 1.16) 0.97 (0.82, 1.16) (P=.77) (P=.91) (P=.94) (P=.78) 37.2 48.5 8.0 128.7 109.0 128.0 108.7 17.3 126.0 128.7 0.0 0.5 Treatment better Obs better 1.0 1.5 2.0 19.1 Figure 2. Hazard ratio plot of the treatment effect by prognostic factors for overall survival. fitting LDH as a time-dependent continuous covariate, LDH was still not found to be prognostic of DFI (HR ¼ 1.00, CI 0.96–1.03 per 50 unit increase, P ¼ 0.81), DMFI (HR ¼ 1.01, CI 0.97–1.05 per 50 unit increase, P ¼ 0.56) or OS (HR ¼ 1.02, CI 0.98–1.06 per 50 unit increase, P ¼ 0.36). Statistical analysis measurements at baseline and serially over the 2 years from ran- domisation (supplementary Figure S1, available at Annals of Oncology online). Neither baseline plasma nor serum VEGF were prognostic factors for OS (HR ¼ 1.02, CI 0.95–1.10 per 50 unit increase, P ¼ 0.53 for plasma; HR ¼ 1.03, CI 0.98–1.07 per 50 unit increase, P ¼ 0.21 for serum). Serum, but not plasma, VEGF levels significantly fell over time in the bevacizumab-treated measurements at baseline and serially over the 2 years from ran- domisation (supplementary Figure S1, available at Annals of Oncology online). Neither baseline plasma nor serum VEGF were prognostic factors for OS (HR ¼ 1.02, CI 0.95–1.10 per 50 unit increase, P ¼ 0.53 for plasma; HR ¼ 1.03, CI 0.98–1.07 per 50 unit increase, P ¼ 0.21 for serum). Serum, but not plasma, VEGF levels significantly fell over time in the bevacizumab-treated Patients (N ¼ 414; 198 in the bevacizumab arm, 216 in the ob- servation arm) had VEGF and VEGFR1 plasma and serum doi:10.1093/annonc/mdy229 | 1847 Volume 29 | Issue 8 | 2018 Original article Annals of Oncology Annals of Oncology Table 2. Statistical analysis Multivariate analysis for overall survival for all trial patients and for the subgroup of patients for whom BRAF mutation status was assessed All trial patients BRAF mutation status assessed All trial patients Deaths Hazard ratio (95% CI) BRAF mutant BRAF WT Deaths Hazard ratio (95% CI) N (%) N (%) N (%) N (%) N (%) Total 1343 303 379 Sex P ¼ 0.003 P ¼ 0.19 Male 753 (56%) 316 (42%) 1.31 (1.10-1.57) 156 (51%) 230 (61%) 167 (43%) 1.18 (0.92-1.51) Females 590 (44%) 199 (34%) 1.00 147 (49%) 149 (39%) 113 (38%) 1.00 Breslow thickness of primary melanoma P ¼ 0.0003 P ¼ 0.004 <2.0 mm 399 (30%) 140 (35%) 1.00 126 (42%) 87 (23%) 83 (39%) 1.00 >2–4 mm 405 (30%) 149 (37%) 1.12 (0.89-1.42) 94 (31%) 108 (29%) 81 (40%) 1.16 (0.85-1.59) >4 mm 438 (33%) 194 (44%) 1.53 (1.19-1.96) 65 (21%) 153 (40%) 101 (46%) 1.63 (1.16-2.27) Unknown 101 (7%) 32 (32%) 0.75 (0.51-1.10) 18 (6%) 31 (8%) 15 (31%) 0.67 (0.38-1.17) AJCC disease stagea P < 0.0001 P < 0.0001 II 364 (27%) 119 (33%) 1.00 52 (17%) 117 (31%) 55 (33%) 1.00 IIIA 195 (15%) 41 (21%) 0.78 (0.53-1.48) 56 (19%) 29 (7%) 23 (27%) 1.00 (0.59-1.70) IIIB 495 (37%) 210 (42%) 1.89 (1.47-2.44) 130 (43%) 147 (39%) 127 (46%) 2.18 (1.53-3.12) IIIC 289 (21%) 145 (50%) 2.27 (1.74-2.96) 65 (21%) 86 (23%) 75 (50%) 2.40 (1.65-3.51) ECOG performance status P < 0.0001 P ¼ 0.001 0 1195 (89%) 436 (36%) 1.00 269 (89%) 345 (91%) 240 (39%) 1.00 1 146 (11%) 78 (53%) 1.64 (1.29-2.10) 34 (11%) 33 (9%) 39 (58%) 1.75 (1.24-2.46) Trial arm P ¼ 0.92 P ¼ 0.83 Bevacizumab 671 (50%) 254 (38%) 1.01 (0.85-1.20) 132 (44%) 184 (49%) 128 (41%) 1.03 (0.81-1.30) Observation 672 (50%) 261 (39%) 1.00 171 (56%) 195 (51%) 152 (42%) 1.00 BRAF status P ¼ 0.08 BRAF mutant 303 (100%) 0 129 (43%) 1.24 (0.97-1.59) BRAF WT 0 379 (100%) 151 (40%) 1.00 aAJCC 7th edition. patients compared with observation (P < 0.0001 and P ¼ 0.58, re- spectively). Neither baseline plasma nor serum VEGFR1 were prognostic factors for OS (HR ¼ 0.85, CI 0.58–1.25 per 50 unit increase, P ¼ 0.41 for plasma; HR ¼ 0.86, CI 0.60–1.22 per 50 unit increase, P ¼ 0.40 for serum). Plasma VEGFR1 levels increased during bevacizumab treatment compared with obser- vation (P < 0.001). Statistical analysis However, VEGFR1 serum results did not vary over time (P ¼ 0.75) or by trial arm (P ¼ 0.92). standard adjuvant therapy after resection of melanoma at high risk of recurrence. The 64% 5-year OS rate for both observation and treatment arms of the AVAST-M trial was notably higher than predicted when the trial was designed. The original statistical premise was based on the results of the UK AIM High trial, which recruited patients with similar demographics between 1995 and 2000 [10]. Since then, improvements in healthcare and more accurate staging have contributed to an upward trend in melanoma pa- tient survival [11]. The step change is evident in observation arms of other adjuvant melanoma trials: EORTC 18991 recruited stage III patients only between 2000 and 2003 and had a 7-year OS of 46% [12], while the 5-year OS rate in the EORTC 18071 trial which recruited similar patients between 2008 and 2011 was 54% [13]. The AVAST-M observation arm carried out even better, although one quarter of patients had lower risk stage II disease. Discussion AVAST-M represents the largest trial in a melanoma patient population evaluating angiogenesis inhibition. This survival analysis was pre-planned when all patients had been on study for 5 years. With longer follow-up, the trial has confirmed the interim finding that adjuvant bevacizumab improved DFI [8]. The HR of 0.85 favouring bevacizumab is comparable to the event-free survival HR of 0.86 reported for adjuvant interferon in a recent meta-analysis [9]. However, while for ad- juvant interferon this HR translated into a small OS benefit, this was not the case for bevacizumab. The conditional power for futility of the primary outcome of OS was less than 10%. Therefore adjuvant bevacizumab cannot be recommended as a During the time that AVAST-M was recruiting, MAP kinase inhibitors and immune checkpoint inhibitors were approved for treatment of metastatic melanoma and are now standard of care. Only 16% of patients taking part in AVAST-M received these drugs at recurrence and the proportions were equal between the two trial arms, so we can be confident that treatment at recur- rence cannot explain the lack of survival benefit from Volume 29 | Issue 8 | 2018 1848 | Corrie et al. Discussion 100 A B C D E F 75 BRAF WT BRAF WT BRAF mutant BRAF mutant 50 Events 101 (59%) 113 (58%) 40% 43% 1.09 (0.83-1.42) 5 year DFI rate HR (95% CI) BRAF WT BRAF mutant Events 101 (59%) 113 (58%) 40% 43% 1.09 (0.83-1.42) 69 (52%) 101 (59%) 48% 40% 0.81 (0.60-1.10) 0.80 (0.57-1.13) 5 year DFI rate HR (95% CI) Events 5 year DFI rate HR (95% CI) Percentage surviving 25 0 Numbers at risk: 195 182 159 148 133 118 89 50 25 23 42 64 84 97 107 133 155 171 132 132 171 95 104 87 74 67 75 61 69 55 61 42 19 35 11 20 50 118 101 90 107 81 97 74 53 64 25 42 15 23 84 133 155 171 195 139 104 112 92 81 69 76 56 50 27 12 20 35 61 75 87 171 BRAF WT BRAF mutant Numbers at risk: Bevacizumab Observation Bevacizumab Deaths HR (95% CI) 5 year OS rate Observation Numbers at risk: Bevacizumab Observation Numbers at risk: Bevacizumab Bevacizumab Observation Numbers at risk: BRAF WT BRAF mutant 100 75 50 Percentage surviving 25 0 0 1 2 3 4 5 Years from entry 6 7 8 0 1 2 3 4 5 Years from entry 6 7 8 100 75 50 Percentage surviving 25 0 100 75 50 Percentage surviving 25 0 100 75 50 Percentage disease free 25 0 100 75 50 Percentage disease free 25 0 0 1 2 3 4 5 Years from entry 6 7 8 0 184 172 147 131 118 133 103 79 89 56 25 25 50 118 148 159 182 195 Numbers at risk: Bevacizumab Observation 184 195 136 104 112 98 92 90 81 81 59 56 44 27 20 12 76 139 1 2 3 4 5 Years from entry 6 7 8 0 1 2 3 4 5 Years from entry 6 7 8 0 1 2 3 4 5 Years from entry 6 7 8 BRAF WT BRAF mutant Bevacizumab Observation Bevacizumab Observation Bevacizumab Observation Observation Bevacizumab Observation 63% 50 (38%) 261 (46%) 55% Events 5 year DFI rate HR (95% CI) Deaths 78 (42%) 73 (37%) 1.17 (0.85-1.61) 62% 65% 5 year OS rate HR (95% CI) Bevacizumab 96 (52%) 113 (58%) 50% 43% 0.86 (0.65-1.12) Observation Bevacizumab Observation Figure 3. Discussion Overall survival (A) and disease-free interval (B) by BRAF status for the observation arm patients only; Overall survival and disease- ree interval by trial arm for BRAF mutant patients (C and D); Overall survival and disease-free interval by trial arm for BRAF wild type patients E and F). Discussion Annals of Oncology Original article B BRAF WT BRAF mutant Events 101 (59%) 113 (58%) 40% 43% 1.09 (0.83-1.42) 5 year DFI rate HR (95% CI) 195 139 104 112 92 81 69 76 56 50 27 12 20 35 61 75 87 171 Numbers at risk: BRAF WT BRAF mutant 100 75 50 Percentage surviving 25 0 0 1 2 3 4 5 Years from entry 6 7 8 BRAF WT BRAF mutant Original article Annals of Oncology 100 A 75 BRAF WT BRAF WT BRAF mutant BRAF mutant 50 Events 101 (59%) 113 (58%) 40% 43% 1.09 (0.83-1.42) 5 year DFI rate HR (95% CI) Percentage surviving 25 0 Numbers at risk: 195 182 159 148 133 118 89 50 25 23 42 64 84 97 107 133 155 171 BRAF WT BRAF mutant 0 1 2 3 4 5 Years from entry 6 7 8 Annals of Oncology B A D F 69 (52%) 101 (59%) 48% 40% 0.81 (0.60-1.10) Events 5 year DFI rate HR (95% CI) 132 171 95 104 87 74 67 75 61 69 55 61 42 19 35 11 20 50 195 139 104 112 92 81 69 76 56 50 27 12 20 35 61 75 87 171 Numbers at risk: Bevacizumab Bevacizumab Observation Numbers at risk: BRAF WT BRAF mutant P 25 0 0 1 2 3 4 5 Years from entry 6 7 8 100 100 75 50 Percentage disease free 25 0 0 1 2 3 4 5 Years from entry 6 7 8 BRAF mutant Bevacizumab Observation Observation Bevacizumab Observation D 69 (52%) 101 (59%) 48% 40% 0.81 (0.60-1.10) Events 5 year DFI rate HR (95% CI) 132 171 95 104 87 74 67 75 61 69 55 61 42 19 35 11 20 50 Numbers at risk: Bevacizumab Bevacizumab Observation 100 75 50 Percentage disease free 25 0 0 1 2 3 4 5 Years from entry 6 7 8 Bevacizumab Observation Observation C 0.80 (0.57-1.13) 132 118 101 90 107 81 97 74 53 64 25 42 15 23 84 133 155 171 Numbers at risk: Bevacizumab Observation Bevacizumab Deaths HR (95% CI) 5 year OS rate Observation 100 75 50 Percentage surviving 25 0 0 1 2 3 4 5 Years from entry 6 7 8 Bevacizumab Observation 63% 50 (38%) 261 (46%) 55% D C F 100 75 50 Percentage disease free 25 0 Numbers at risk: Bevacizumab Observation 184 195 136 104 112 98 92 90 81 81 59 56 44 27 20 12 76 139 0 1 2 3 4 5 Years from entry 6 7 8 Bevacizumab Observation Events 5 year DFI rate HR (95% CI) Bevacizumab 96 (52%) 113 (58%) 50% 43% 0.86 (0.65-1.12) Observation E Numbers at risk: Bevacizumab Observation 100 75 50 Percentage surviving 25 0 0 184 172 147 131 118 133 103 79 89 56 25 25 50 118 148 159 182 195 1 2 3 4 5 Years from entry 6 7 8 Bevacizumab Observation Deaths 78 (42%) 73 (37%) 1.17 (0.85-1.61) 62% 65% 5 year OS rate HR (95% CI) Bevacizumab Observation F E Figure 3. Annals of Oncology Ernest Marshall, Nottingham University Hospital—Prof Poulam Patel, Southampton General Hospital—Prof Christian Ottensmeier, Cheltenham General Hospital—Dr David Farrugia, James Cook University Hospital—Dr Alison Humphreys, Poole Hospital—Dr Bryony Eccles, Dr Renata Dega, Bristol Haematology & Oncology Centre—Dr Chris Herbert, Dr Christopher Price, City General Hospital—Dr Murray Brunt, University Hospital, Coventry—Dr Martin Scott-Brown, Dr Joanna Hamilton, Western General Hospital—Dr Richard Larry Hayward, Dr John Smyth, Royal Derby Hospital—Dr Pamela Woodings, Salisbury District Hospital—Dr Neena Nayak, Dr Lorna Burrows, St. Bartholomew’s Hospital—Dr Virginia Wolstenholme, Singleton Hospital—Prof John Wagstaff, Aberdeen Royal Infirmary—Dr Marianne Nicolson, Yeovil District Hospital— Dr Andrew Wilson, Dr Clare Barlow, Ipswich Hospital—Dr Christopher Scrase, Dr Timothy Podd, Charing Cross Hospital—Dr Michael Gonzalez, Dr John Stewart, Derriford Hospital—Dr Martin Highley, Dr Virginia Wolstenholme, New Cross Hospital—Dr Simon Grumett, Torbay Hospital— Dr Andrew Goodman, Royal Cornwall Hospital—Dr Toby Talbot, Kent & Canterbury Hospital East Kent—Dr Kannon Nathan, Dr Robert Coltart, Warwick Hospital—Dr. Bruce Gee, The Royal Marsden Hospital Sutton Branch—Prof Martin Gore, Gloucestershire Royal Hospital—Dr David Farrugia, Selly Oak Hospital—Dr Agustin Martin-Clavijo, Dr Jerry Marsden, Worcestershire Royal Hospital—Dr Christopher Price, Dr David Farrugia, Queen Elizabeth Queen Mother Hospital East Kent—Dr Kannon Nathan, Dr Robert Coltart, William Harvey Hospital East Kent—Dr Kannon Nathan, Dr Robert Coltart according to BRAF mutation status reported for patient cohorts after resection of primary melanomas have been inconsistent [17–19]. In our study, we saw a trend in poorer OS for BRAF mutant patients compared with BRAF wild-type patients, al- though this did not reach statistical significance. We also identi- fied a trend towards enhanced OS from adjuvant bevacizumab limited only to the subgroup of patients with BRAF mutated tumours. BRAF mutation status was recently reported to de- scribe populations with differing OS after immune checkpoint inhibitors [20]. BRAF V600E is pro-angiogenic in several human tumour models [21, 22], while VEGF has wider regula- tory function beyond angiogenesis, including on immune cells [23–25]. Exploratory studies combining bevacizumab with ipili- mumab [26] or atezolizumab [27, 28] have reported early effi- cacy signals. Our findings raise the hypothesis that combining bevacizumab with adjuvant immune checkpoint inhibitors may benefit high-risk BRAF mutant melanoma patients, who in our study had a poorer prognosis than patients with tumours lack- ing the mutation. Funding This work was supported by Cancer Research UK (grant ref. C7535/A6408 and C2195/A8466). Bevacizumab was provided free of charge for all patients throughout the trial by Roche Products Ltd. The sponsor was Cambridge University Hospitals NHS Foundation Trust (Cambridge, UK). Acknowledgements We are grateful to all the patients who participated in the AVAST-M trial, all investigators and their teams from the par- ticipating sites, members of the independent data and safety monitoring committee and steering committee and the National Cancer Research Institute Clinical Studies Group for overall support. National Institute for Health Research funding to the Clinical Research Networks, Biomedical Research Centres and Experimental Cancer Medicine Centres contrib- uted to the undertaking of this trial at various sites. Complete list of AVAST-M participating sites and Principal Investigators past and present: Mount Vernon Hospital—Dr Paul Nathan, The Christie—Prof Paul Lorigan, Broomfield Hospital—Mr Peter Dziewulski, Dr Sonja Holikova, Dr Udaiveer Panwar, Prof Saad Tahir, Leicester Royal Infirmary—Dr Guy Faust, Dr Anne Thomas, Addenbrooke’s Hospital—Dr Pippa Corrie, Dr Bhawna Sirohi, Northern Centre for Cancer Care—Dr Charles Kelly, Churchill Hospital—Prof Mark Middleton, St James’s University Hospital—Dr Maria Marples, Weston Park Hospital—Prof Sarah Danson, Dr James Lester, St Helen’s Hospital—Dr Ernest Marshall, Royal Surrey County Hospital—Dr Mazhar Ajaz, Dr Stephen Houston, Royal Preston Hospital—Dr Ruth Board, Dr David Eaton, Beatson WOS Cancer Centre—Dr Ashita Waterston, Norfolk & Norwich University Hospital—Dr Jenny Nobes, Dr Suat Loo, Dr Gill Gray, Dr Helen Stubbings, The Royal Marsden Hospital Chelsea Branch—Prof Martin Gore, Guy’s and St Thomas’ Hospital, MG is funded by the NIHR Biomedical Research Centre at the Royal Marsden Hospital and Institute of Cancer Research—Dr Mark Harries, Velindre Hospital—Dr Satish Kumar, Royal Devon and Exeter Hospital—Dr Andrew Goodman, St George’s Hospital—Prof Angus Dalgleish, Queen Elizabeth Hospital—Dr Agustin Martin-Clavijo, Dr Jerry Marsden, Royal Sussex County Hospital—Dr Sarah Westwell, Ninewells Hospital—Dr Richard Casasola, Royal Free Hospital—Dr David Chao, Castle Hill Hospital—Prof Anthony Maraveyas, Clatterbridge Oncology Centre—Dr Discussion Overall survival (A) and disease-free interval (B) by BRAF status for the observation arm patients only; Overall survival and disease- free interval by trial arm for BRAF mutant patients (C and D); Overall survival and disease-free interval by trial arm for BRAF wild type patients (E and F). bevacizumab reported here. Central gene mutation testing for just over half of recruited patients identified BRAF and NRAS mutation rates of 44% and 20%, respectively. These proportions reflect those in metastatic melanoma populations, suggesting sta- bility over time. melanoma patient cohort conducted to date, but has not identi- fied any immediate clinical value in measuring VEGF or VEGFR1 after melanoma surgery. Other circulating factors associated with angiogenesis [14] could be considered in future melanoma trials evaluating angiogenesis inhibitors. The most common melanoma genetic mutation, BRAF, is a near-perfect biomarker predictive of sensitivity to BRAF tar- geted therapies in both advanced and high-risk resected melan- oma [15]. Its role as a prognostic marker in each of these disease stages is, however, controversial [16]. Survival differences We used this large-scale adjuvant trial to explore potential prognostic and predictive biomarkers. Although LDH may be of prognostic value in metastatic disease, this was not the case after melanoma resection. Our study represents the most comprehen- sive analysis of angiogenesis biomarkers associated with a Volume 29 | Issue 8 | 2018 doi:10.1093/annonc/mdy229 | 1849 Annals of Oncology References 1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144(5): 646–674. 2. Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti- tumour activity. Nat Rev Cancer 2008; 8(8): 579–591. 3. Salven P, Heikkila P, Joensuu H. Enhanced expression of vascular endo- thelial growth factor in metastatic melanoma. Br J Cancer 1997; 76(7): 930–934. 4. Ugurel S, Rappl G, Tilgen W, Reinhold U. Increased serum concentra- tion of angiogenic factors in malignant melanoma patients correlates with tumour progression and survival. J Clin Oncol 2001; 19(2): 577–583. 5. Ascierto PA, Leonardi E, Ottaiano A et al. Prognostic value of serum VEGF in melanoma patients: a pilot study. Anticancer Res 2004; 24(6): 4255–4258. 6. Scheri R, Morton D, Essner R et al. Molecular profiling of melanoma intransit metastases identifies VEGF as a therapeutic target. Melanoma Res. 2006; 16(Suppl 1): S16–S18. 7. Kim KB, Sosman JA, Fruehauf JP et al. BEAM: a randomized phase II study evaluating the activity of bevacizumab in combination with carbo- platin plus paclitaxel in patients with previously untreated advanced mel- anoma. J Clin Oncol 2012; 30(1): 34–41. 8. Corrie PG, Marshall A, Dunn JA et al. Adjuvant bevacizumab in patients with melanoma at high risk of recurrence (AVAST-M): preplanned in- terim results from a multicentre, open-label, randomised controlled phase 3 study. Lancet Oncol.2014; 15(6): 620–630. 9. Ives NJ, Suciu S, Eggermont AMM et al. Adjuvant interferon-a for the treatment of high-risk melanoma: an individual patient data meta-ana- lysis. Eur J Cancer 2017; 82: 171–183. 10. Hancock BW, Wheatley K, Harris S et al. Adjuvant interferon in high- risk melanoma: the AIM HIGH study—United Kingdom Coordinating Committee on Cancer Research randomized study of adjuvant low-dose extended-duration interferon alfa-2a in high-risk resected malignant melanoma. J Clin Oncol 2004; 22(1): 53–61. 11. Gershenwald JE, Scolyer RA, Hess KR et al. Melanoma staging: evidence- based changes in the American Joint Committee on Cancer eighth edi- tion cancer staging manual. CA Cancer J Clin 2017; 67(6): 472–492. tion cancer staging manual. CA Cancer J Clin 2017; 67(6): 4 12. Eggermont AM1, Suciu S, Testori A et al. Long-term results of the randomized phase III trial EORTC 18991 of adjuvant therapy with pegy- lated interferon alfa-2b versus observation in resected stage III melan- oma. J Clin Oncol 2012; 30(31): 3810–3818. 13. Eggermont AMM, Chiarion-Sileni V, Grob JJ et al. Prolonged survival in stage III melanoma with Ipilimumab adjuvant therapy. Disclosures The following represents disclosure information provided by authors of this manuscript. Relationships are self-held unless noted. I ¼ Immediate Family Member, Inst ¼ My Institution. Relationships may not relate to the subject matter of this article. PC reports Honoraria: Novartis, Merck Sharp & Dohme, Consulting or Advisory Role: Celgene, Novartis, Bristol-Myers Squibb, Merck Sharp & Dohme, Incyte, Speakers’ Bureau: Merck Sharp & Dohme, Novartis, Research Funding: Novartis, Travel, Accommodations, Expenses: Bristol-Myers Squibb, Merck Sharp & Dohme. A Marshall reports Research Funding: Bayer AG (Inst), PN reports Consulting or Advisory Role: AstraZeneca, Bristol-Myers Squibb, MSD, Immunocore, Pfizer, Pierre Fabre, Novartis, GlaxoSmithKline, Ipsen, Speakers’ Bureau: Bristol-Myers Squibb, Novartis, Travel, Volume 29 | Issue 8 | 2018 1850 | Corrie et al. Annals of Oncology Accommodations, Expenses: Bristol-Myers Squibb, MSD. PL reports Consulting or Advisory Role: Merck Sharp & Dohme, Bristol-Myers Squibb, Novartis, Amgen, GlaxoSmithKline, NeraCare GmbH, Pierre Fabre, Speakers’ Bureau: Merck Sharp & Dohme, Novartis, Bristol-Myers Squibb, Roche, Travel, Accommodations, Expenses: Merck Sharp & Dohme, Bristol- Myers Squibb. ST reports Honoraria: Roche, Travel, Accommodations, Expenses: PharmaMar. GF reports Consulting or Advisory Role: Novartis, Ipsen, Roche, BMS, Janssen, Speakers’ Bureau: Pfizer, Janssen, Novartis, BMS, Ipsen, Travel, Accommodations, Expenses: Janssen, Ipsen, Astellas, BMS. CK reports Honoraria: MSD, Consulting or Advisory Role: MSD, Research Funding: BMS (Inst), Travel, Accommodations, Expenses: MSD. MM reports Consulting or Advisory Role: Novartis, Research Funding: Amgen (Inst), Roche (Inst). SJD reports Honoraria: Amgen, BMS, Consulting or Advisory Role: Incanthera (Inst), Research Funding: BMS (Inst), MSD (Inst), Lilly (Inst), Bayer (Inst), Amgen (Inst), Boehringer (Inst), Sieira (Inst), Travel, Accommodations, Expenses: MSD, BMS, Abbvie. RB reports Consulting or Advisory Role: Roche, MSD, BMS. MH reports Honoraria: Genomic Health, Roche, Amgen, Speakers’ Bureau: Roche, MSD Oncology, Eisai, Research Funding: Amgen, Travel, Accommodations, Expenses: Roche, GlaxoSmithKline. AW reports Speakers’ Bueau MSD, BMS, Research Funding: MSD (Inst), BMS (Inst), Travel, Accommodations, Expenses: MSD, BMS. SK reports Consulting or Advisory Role: BMS, Roche, MSD, Speakers’ Bureau: BMS, Travel, Accommodations, Expenses: BMS, MSD, Ipsen. AG reports Honoraria: Roche, Genomic Health. AD reports Stock or Other Ownership: Celgene, Honoraria: Novartis, Consulting or Advisory Role: Novartis, Research Funding: Celgene (Inst), Immodulon (Inst), LDH Pharma (Inst), Jay Pharma (Inst), Patents, Royalties, Other Intellectual Property: Celgene, LDN Pharma, Jay Pharma. AMC reports Honoraria: LEO Pharma, Celgene, Novartis Pharmaceutical UK Ltd, Almirall, Almirall (I), Chiesi (I), Pfizer (I), Novartis (I), GlaxoSmithKline (I), Research Funding: LEO Pharma (Inst), Abbvie (Inst), Novartis (Inst), Roche/Genentech (Inst), Chugai Pharma (Inst), Travel, Accommodations, Expenses: LEO Pharma, Celgene. DC reports Consulting or Advisory Role: Incyte, Novartis, Merck, BMS, Research Funding: Merck (Inst), BMS (Inst), Novartis (Inst), Travel, Accommodations, Expenses: Merck. AM reports Honoraria: BMS, Pfizer, Bayer, Incyte, Roche, Consulting or Advisory Role: BMS, Bayer, Speakers’ Bureau: BMS, LEO, Travel, Accommodations, Expenses: Bayer, BMS, Roche, Merck. PP reports Consulting or Advisory Role: Novartis, Ipsen (Inst), Travel, Accommodations, Expenses: Pfizer, Bristol-Myers Squibb, Merck Sharp & Dohme, Ipsen. Annals of Oncology CHO reports Consulting or Advisory Role: Bristol-Myers Squibb, Merck Sharp & Dohme, Immactics, Speakers’ Bureau: Bristol-Myers Squibb, Merck Sharp & Dohme, Roche, Research Funding: Bristol- Myers Squibb (Inst), Verastem (Inst), Merck Sharp & Dohme (Inst), Inovio Pharmaceuticals (Inst), BioNTech AG (Inst), Serametrix (Inst), Touchlight Genetics (Inst), Delcath Systems (I t) T l A d ti E B i t l M Merck (Inst), Novartis (Inst), Pfizer (Inst), Boehringer Ingelheim (Inst), GlaxoSmithKline (Inst), Janssen (Inst), Orion (Inst), EUSA Pharma (Inst), Travel, Accommodations, Expenses: Bristol-Myers Squibb. KAM reports Employment: Porton Biopharma. MRM reports Honoraria: Amgen, Roche, Consulting or Advisory Role: Merck, CytomX Therapeutics, RigonTEC, Bristol-Myers Squibb, Newlink Genetics, Novartis, Research Funding: Immunocore (Inst), Novartis (Inst), AstraZeneca (Inst), Roche (Inst), Amgen (Inst), Millennium (Inst), Bristol-Myers Squibb (Inst), Vertec (Inst), Merck (Inst), Pfizer (Inst), RigonTEC (Inst), Replimune (Inst), Array BioPharma (Inst), TC Biopharm (Inst), Regeneron (Inst), Travel, Accommodations, Expenses: Merck. All remaining authors have declared no conflicts of interest. g Annals of Oncology Annals of Oncology 15. Long GV, Hauschild A, Santinami V et al. Adjuvant dabrafenib plus tra- metinib in stage III BRAF-mutated melanoma. N Engl J Med. 2017; 377: 1813–1823. 22. Durante C, Tallini G, Puxeddu E et al. BRAF(V600E) mutation and ex- pression of proangiogenic molecular markers in papillary thyroid carci- nomas. Eur J Endocrinol 2011; 165(3): 455–463. 23. Ohm JE, Carbone DP. VEGF as a mediator of tumor-associated im- munodeficiency. Immunol Res 2001; 23(2–3): 263–272. 16. Bhatia P, Friedlander P, Zakaria EA, Kandil E. Impact of BRAF mutation status in the prognosis of cutaneous melanoma: an area of ongoing re- search. Ann Transl Med 2015; 3(2): 24–31. 24. Kandalaft LE, Motz GT, Busch J, Coukos G. Angiogenesis and the tumor vasculature as antitumor immune modulators: the role of vascular endo- thelial growth factor and endothelin. Curr Top Microbiol Immunol 2011; 344: 129–148. 17. Thomas NE, Edmiston SN, Alexander A et al. Association between NRAS and BRAF mutational status and melanoma-specific survival among patients with higher-risk primary melanoma. JAMA Oncol 2015; 1: 359–368. 25. Li Y-L, Zhao H, Ren X-B. Relationship of VEGF/VEGFR with immune and cancer cells: staggering or forward? Cancer Biol Med 2016; 13(2): 206–214. 18. Rutkowski P, Gos A, Jurkowska M et al. Molecular alterations in clinical stage III cutaneous melanoma: correlation with clinicopathological fea- tures and patient outcome. Oncol Lett 2014; 8(1): 47–54. 26. Hodi FS, Lawrence D, Lezcano C et al. Bevacizumab plus Ipilimumab in patients with metastatic melanoma. Cancer Immunol Res 2014; 2(7): 632–642. 19. Meckbach D, Bauer J, Pflugfelder A et al. Survival according to BRAF- V600 tumor mutations – an analysis of 437 patients with primary melan- oma. PLoS One 2014; 9(1): e86194. 27. Wallin JW, Bendell JC, Funke R et al. Atezolizumab in combination with bevacizumab enhances antigen-specific T-cell migration in metastatic renal cell carcinoma. Nature Comms 2016; doi: 10.1038/ncomms12624. 20. Wolchok JD, Chiarion-Sileni V, Gonzalez R et al. Overall survival with combined nivolumab and Ipilimumab in advanced melanoma. N Engl J Med 2017; 377(14): 1345–1356. 28. McDermott DF, Atkins MB, Motzer RJ et al. A phase II study of atezoli- zumab (atezo) with or without bevacizumab (bev) versus sunitinib (sun) in untreated metastatic renal cell carcinoma (mRCC) patients (pts). J Clin Oncol 2017; 35(Suppl 6): 431. 21. Bottos A, Martini M, Di Nicolantonio F et al. Targeting oncogenic serine/threonine-protein kinase BRAF in cancer cells inhibits angiogen- esis and abrogates hypoxia. References N Engl J Med 2016; 375(19): 1845–1855. 14. Corrie PG, Basu B, Zaki KA. Targeting angiogenesis in melanoma: pros- pects for the future. Ther Adv Med Oncol 2010; 2(6): 367–380. doi:10.1093/annonc/mdy229 | 1851 Volume 29 | Issue 8 | 2018 Annals of Oncology Proc Natl Acad Sci USA 2012; 109(6): E353–E359. Volume 29 | Issue 8 | 2018 1852 | Corrie et al.
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© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  In recent years, the endometriosis has overcome a noteworthy renaissance in the recognition of its potential. In certain patients, a demonstrable malignant progression of ectopic foci leading to development of ovar- ian cancer is seen. The knowledge of endometriosis overthrow background into endometriosis associated ovarian cancer is of paramount importance for selection of patients at risk. The goal of the presented study was to review a malignant potential of the endometriosis and to specify predictive factors of endometriosis progression into ovarian cancer. Altogether 189 patients were included in the study. Conventional cytogenetics as well as measurement of transcriptional activity of CTNNB1 (β-catenin) and HIF1A (HIF1-α) genes were prospectively studied in 60 endometrio- sis patients and 50 control group patients. The retrospective histopathological analysis was performed in 19 endome- triosis associated ovarian cancer patients and 60 patients with histologically confirmed endometriosis. Results:  Five endometriosis patients showed a deviation from normal cytogenetics finding without affecting of their phenotype. In 6 cases of endometriosis associated ovarian cancer ectopic endometrium was not confirmed. The remaining 13 cases demonstrated either benign or atypical endometriosis or even structures of borderline carcinoma. Atypical endometriosis was histologically confirmed in 20% of 60 endometriosis patients. Determination of gene expression (CTNNB1, HIF1A) formed two subgroups. Transcriptionally incipient endometriosis subgroup with insignifi- cant genes expression compared to control group. In transcriptionally evident endometriosis subgroup were genes expressions significantly higher compared to control group (p < 0.01) as well as transcriptionally incipient endome- triosis subgroup (p < 0.05). Conclusions:  Significant structural abnormalities of chromosomes are not included in genetic rigging of endome- triosis patients. Atypical endometriosis represents a histopathologically detectable intermediate of endometriosis progression. Determination of genes expression CTNNB1 and HIF1A helps to allocate risk patients with endometriosis where more precise management is needed. Keywords:  Endometriosis, Endometriosis associated ovarian cancer, Conventional cytogenetics, Atypical endometriosis, CTNNB1, HIF1A Keywords:  Endometriosis, Endometriosis associated ovarian cancer, Conventional cytogenetics, Atypical endometriosis, CTNNB1, HIF1A Varga et al. Journal of Ovarian Research (2022) 15:5 https://doi.org/10.1186/s13048-021-00940-8 Varga et al. Journal of Ovarian Research (2022) 15:5 https://doi.org/10.1186/s13048-021-00940-8 Background In recent years, the endometriosis has overcome a noteworthy renaissance in the recognition of its poten- tial. Today, it is understood as a clinically complex syndrome characterized by chronic hormone-depend- ent inflammation with notable proliferative poten- tial. About 10% of the reproductive age women suffer from this disease and a variety of clinical symptoms are known. In certain patients, a demonstrable malignant *Correspondence: janko.varga@gmail.com *Correspondence: janko.varga@gmail.com 1 Department of Gynaecology and Obstetrics, Faculty of Medicine, P.J. Šafárik University and L. Pasteur University Hospital, Rastislavova 43 Stree 041 90 Košice, Slovakia Full list of author information is available at the end of the article p j g @g 1 Department of Gynaecology and Obstetrics, Faculty of Medicine, P.J. Šafárik University and L. Pasteur University Hospital, Rastislavova 43 Street, 041 90 Košice, Slovakia Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research Page 2 of 13 progression of ectopic foci leading to development of ovarian cancer (OC) is seen.h there assign mainly endometrioid ovarian cancer (EOC) and clear cell ovarian cancer (CCOC). Not every EAOC has histologically proven endometriosis in its structure. Based on this findings, in can be assumed that endome- triosis is a precursor of only certain EAOC. Malignant reversal of endometriosis is an uncommon event, count- ing for less than 1%. Question which endometriotic lesion tends to progress into carcinoma remains unanswered. The large histological variability of OC presupposes more tissues as an initial structure for the process of ovar- ian carcinogenesis [1]. In the ovary itself, ovarian surface epithelium (OSE) constitutes the main structure which either accumulates mutations or undergoes metaplasia to the müllerian epithelium. In both cases, the process of carcinogenesis takes place in cortical inclusion cyst (CIC) after incorporation of pathological tissue. Another source of tissue for OC represents an ectopic müllerian epithelium transported and incorporated into CIC. This is the case of endosalpingiosis as a potential source of serous borderline ovarian tumour [2, 3]. The fallopian tube plays an impor- tant role in process of OC development. Precursors, such as serous tubal intraepithelial carcinoma (STIC) or papillary tubal hyperplasia (PTH) can locally progress or more often are adhered to carcinogenesis more favourable environment of OSE. Finally, retrograde endometrial reflux adhered to the OSE is involved in the development of some carcinomas, type I (Table 1). Inflamed stroma and mutations containing ectopic endometrium have better conditions to progress in more propitious microenvironment of the ovary [4]. This fact confirmed also finding that deep infiltrating endome- triosis although containing the same changes in stroma and epithelium progress into carcinoma sporadically [5]. Common features typical for endometriosis and cancer cells, i.e. to evade apoptosis, ability of stem cells as well as angiogenic potential were described. Haemolysis, a process typical for ectopic endometrium is strongly asso- ciated with oxidation. The compounds included in pro- cess are extracellular free haemoglobin, heme, and iron derivatives. These components were abundantly proven in peritoneal fluid as well as in fluid of endometriomas during menstruation [6]. Oxidation processes in endo- metriosis deposits result in the accumulation of DNA mutations that with the help of immune system lead either to cell death or formation of pathogenic clone of cells [7]. Despite the development of molecular genetic tech- niques, classical – conventional cytogenetics is an essen- tial part of both, basic and advanced genetic testing. By the examination the numerical as well as structural chro- mosomal aberrations are detectable. Based on literature, cytogenetic examinations of endometriosis patients brought often discrepant results. The connections of biologically different tissues have been already confirmed as locus minoris resistentiae to the carcinogenesis initiation in human body (gastro- esophageal or ano-rectal junction). The connection of the fallopian tube and the ovary, tubo-ovarian junction as well as the process of ovulation in its vicinity repre- sent an impeccable interplay to start the formation of pathogenic clone of cells. Based on histopathological criteria a benign (typical) and atypical endometriosis (AE) can be defined, with AE significantly associated in EAOC [8]. Two degrees of atypia were in tissue of AE described. Cellular atypia (cytological) indicating a changes in epithelial layer including hyperchromasia and pleomorphism. Structural atypia (hyperplasia) represent a hyperplastic changes similar to eutopic endometrium (simplex or complex hyperplasia with or without cellular atypia) [9]. Most studies refer AE as a tissue containing both, cellular and Endometriosis associated ovarian cancerh The term endometriosis associated ovarian cancer (EAOC) has didactic dimension and in clinical practice Table 1  Process of ovarian carcinogenesis Initial structure Biological process Final structure OSE Mutation + incorporation into CIC CIC OSE Metaplasia + incorporation into CIC Müllerian CIC Ectopic müllerian epithelium Transport to the ovary Müllerian CIC Endosalpingiosis Transport to the ovary Serous borderline ovarian tumor Fallopian tube epithelium Transport to the ovary Müllerian CIC STIC Local progression Primary fallopian tube carcinoma STIC Transport to the ovary HGSOC PTH Transport to the ovary LGSOC Endometriosis Retrograde reflux EOC, CCOC Table 1  Process of ovarian carcinogenesis Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research Page 3 of 13 Page 3 of 13 submolecular change in ectopic endometrium. Finally, intensive expression of CTNNB1 was even in eutopic endometrium during menstruation observed. structural atypia. However cytological atypia are mostly in cancer free patients seen, whereas structural atypia are found particularly in OC patients [10].fi g Protein hypoxia-inducible factor 1-alpha (HIF-1α) presents a subunit of transcription factor hypoxia- inducible factor 1 (HIF-1), encoded by gene HIF1A. This protein is involved in the processes of cell metab- olism regulation, in particular its response to hypoxia (Fig. 1). Hypoxia represents a condition important for angiogenesis, physiologically seen in embryo or as the part of pathological process in tumour tissue. Hypoxia inhibits HIF-1α degradation leading to its transloca- tion into cell nucleus and subsequent activation of the expression of various genes as well as vascular endothe- lial growth factor (VEGF). The result of these processes is pro-angiogenic potential of the tissue. Thus the main function of HIF-1α is regulation of VEGF, secondly it contributes to the potentiation of tumour-induced immunosuppression. HIF-1α regulation is also per- formed by PI3K which provides an activation of Akt signaling pathway. The PI3K/Akt pathway is an intra- cellular signaling pathway involved in several processes such as cell proliferation, apoptosis, angiogenesis or glucose metabolism. PI3K activation phosphorylates and activates Akt. Akt phosphorylates different sub- strates, including mTOR (mammalian target of rapa- mycin) which activation was in case of OC seen (Fig. 2). In this scenario the decrease in level of mTOR, HIF-1α and VEGF is the goal of the targeted therapy for OC. Many cancers show overactive Akt pathway resulting in apoptosis reduction or proliferation. Endometriosis associated ovarian cancerh Significantly higher expression of HIF1A gene was seen in ectopic endometrium comparing to normal endometrium [17].h p y p Different clinical potential of both atypia confirmed the studies of COX-2, Ki-67 and BAF250a. Immunohis- tochemical COX-2 positivity was significantly higher in benign endometriosis (BE) comparing to AE. The same phenomenon was seen when compared cytological ver- sus structural AE. Four time higher COX-2 expression was in cytological AE confirmed. This conclusions pre- dict cytological AE to reactive changes. Ki-67 positivity was significantly higher in tissue of structural atypia con- firming greater proliferative potential when compared to cytological atypia. Comparable results were also in case of AE versus BE seen. ARID1A mutation phenotypically leading to decrease in protein BAF250a represents an ini- tial genetic change of endometriosis overthrow. BAF250a decrease was confirmed in both, OC as well as AE. When compare structural atypia versus cytological atypia of AE, lower BAF250a expressions were in patients with struc- tural atypia seen [10].i Several studies have confirmed the presence of both, BE and AE in EAOC patients [10–12]. The tissue of EAOC can be also without endometriosis, or with grad- ual transition from BE to AE and borderline carcinoma (BOC). In case of AE the structural atypia are more com- mon [10]. Currently accepted histopathological criteria for AE include features of eosinophilic cytoplasm, large hyperchromatin or pale nuclei with moderate to marked pleomorphism, increased nucleus to cytoplasm ratio and cell aggregation. Catenin beta 1 (CTNNB1) called also β-catenin rep- resents a protein encoded by the gene CTNNB1. It is involved in the process of carcinogenesis of many can- cers. Mainly in the regulation of gene transcription as well as cell adhesion. Activation of the Wnt signaling pathway accompanied by CTNNB1 mutation induces a process of ambient fibrotization [13]. In addition, the potentiation of proliferation as well as an increase in implantation ability or invasion is recorded [14]. CTNNB1 mutation was detected in many cancers includ- ing ovarian [15] or endometrial type as well as in endo- metriosis [16]. The regulation of β-catenin is provided by two processes. It is by β-catenin destructive complex and partially also by adenomatous polyposis coli (APC) protein which is encoded by tumor suppressor APC gene. The APC gene mutation is in several cancer seen, signifi- cantly in colorectal carcinoma. Endometriosis associated ovarian cancerh The relationship between level of HIF-1α and CTNNB1 and the potential of endometriosis maligni- zation was not studied yet.h The goal of the presented study was to review a malig- nant potential of the endometriosis by usage of three methods and to specify predictive factors of endome- triosis progression into OC. Conventional cytogenetics was used to reveal raw genetic changes in endometriosis patients. Histopatho- logical analysis evaluated a presence of AE in endome- triosis patients as well as presence of different type of endometriosis in EAOC patients. By the PCR examina- tion of the mentioned genes we were trying to select a high risk patients with endometriosis where the prob- ability of progression into cancer is significantly seen. Both, CTNNB1 mutation as well as Wnt signaling pathway activation was in ectopic endometrium con- firmed. The strong association between Wnt pathway activation and fibrosis was suggested. Involvement of Wnt activation into this process represents an initial Material and methods Altogether 189 patients were included in the study. Pro- spectively conventional cytogenetics as well as PCR examination of the genes HIF1A and CTNNB1 was Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research (2022) 15:5 Page 4 of 13 Fig. 1  The role of HIF-1α in cell metabolism process of tissue preparation composed from three phases which is well known and has been described in detail: performed in 60 patients with endometriosis. In addition, to compare results of PCR examination both genes were assessed in 50 healthy patients as well. Retrospective his- topathological analysis was done in 79 patients. There were 60 patients with endometriosis and 19 patients with EAOC. 1. Lymphocytes cultivation 2. Preparation of cytogenetic sections 3. Staining of cytogenetic sections Histopathological analysis [eNOS = endothelial nitric oxide synthase, ERK = extracellular signal-regulated kinase, MAPK = mitogen-activated protein kinase, PDGF = platelet-derived growth factor, PGF = placental growth factor, PI3K = phosphatidylinositol-3-kinase, HRE = hypoxia responsive element, GRB7 = Human Growth Factor Receptor Bound Protein 7, NOTCH3 = Human Neurogenic Locus Notch Homolog Protein 3, PGR = Human progesterone receptor, MIEN1 = Migration And Invasion Enhancer 1, ERBB = the human epidermal growth factor receptor, JAK = Janus kinases, STAT = signal transducer and activator of transcription proteins, MMP9 = matrix metallopeptidase 9, CASP = cysteine-aspartic proteases, P4 = progesteron] in selected patients. Thus the patients with endometrio- sis were classified either as a BE or AE. While in EAOC patients the possibilities of histopathological findings were as follows: The experimental group (EG) consisted of 60 patients with histologically confirmed endometriosis where con- ventional cytogenetics was also performed (Table  2). The average age of included patients was 36.9 years (20- 56 years) and the endometriosis patients were free of other diseases. 1. EAOC without endometriosis The control group (CG) was made up of 50 blood donor patients. The inclusion criteria were negative family his- tory of oncological disease in last two generation, nega- tive medical examination, blood findings within reference range including oncomarker CA125 and CEA. Every patient from CG had vaginal ultrasonography with nega- tive result. The average age was 34.2 years (21-46 years).h 2. EAOC with BE 3. EAOC with AE 4. EAOC with histologically proven gradual develop- ment from BE through BOC to EAOC. 5. To avoid a subjective evaluation all the slides in both groups were examined by one pathologist. The main specialization of the pathologist is focused to endo- metriosis and ovarian pathology. h The real time PCR method was used to investigate evidence of expression changes on mRNA levels. Four analyses of each gene, per person, in EG and CG were performed. Total RNA was isolated from peripheral whole venous blood using a RNA blood Mini isola- tion kit (Qiagen). Total RNA was quantified and purity was assessed using the Nanodrop® 3300 (Thermo Histopathological analysis The inflammatory and hypoxic microenvironment in the endometrium regulates the expression of several proteins such as receptors ERBB, NOTCH3 and TGF-β, which trigger a cascade of signalling pathways (JAK/STAT, SMAD and PI3K/AKT/mTOR) finally leading to increase in gene expression VEGF, PDGF, Bcl-XL, MMP9, Ang-2 and Tie-2. The result is culmination in proangiogenic transcriptional responses including proliferation and migration, and inhibition of apoptosis. Increased expression of Ang-2 which competitively binds Tie-2, inhibits Ang-1/Tie-2 signaling and negates its stabilizing effects. The destabilizing effect of Ang-2 on blood vessels and the proliferative and migratory effects of VEGF lead to vascular growth and angiogenesis. [eNOS = endothelial nitric oxide synthase, ERK = extracellular signal-regulated kinase, MAPK = mitogen-activated protein kinase, PDGF = platelet-derived growth factor, PGF = placental growth factor, PI3K = phosphatidylinositol-3-kinase, HRE = hypoxia responsive element, GRB7 = Human Growth Factor Receptor Bound Protein 7, NOTCH3 = Human Neurogenic Locus Notch Homolog Protein 3, PGR = Human progesterone receptor, MIEN1 = Migration And Invasion Enhancer 1, ERBB = the human epidermal growth factor receptor, JAK = Janus kinases, STAT = signal transducer and activator of transcription proteins, MMP9 = matrix metallopeptidase 9, CASP = cysteine-aspartic proteases, P4 = progesteron] Fig. 2  In a hypoxic environment, hydroxylation and degradation of HIF-1α are inhibited. Therefore, HIF-1α can dimerize, enter the nucleus and transcriptionally regulate the expression of its target genes through the transcription factor HRE. This way it regulates a wide range of pathophysiological processes including angiogenesis. The inflammatory and hypoxic microenvironment in the endometrium regulates the expression of several proteins such as receptors ERBB, NOTCH3 and TGF-β, which trigger a cascade of signalling pathways (JAK/STAT, SMAD and PI3K/AKT/mTOR) finally leading to increase in gene expression VEGF, PDGF, Bcl-XL, MMP9, Ang-2 and Tie-2. The result is culmination in proangiogenic transcriptional responses including proliferation and migration, and inhibition of apoptosis. Increased expression of Ang-2 which competitively binds Tie-2, inhibits Ang-1/Tie-2 signaling and negates its stabilizing effects. The destabilizing effect of Ang-2 on blood vessels and the proliferative and migratory effects of VEGF lead to vascular growth and angiogenesis. Histopathological analysis Basic genetic examination, conventional cytogenetics is performed by banding technique which creates a patterns of horizontal bands on the examined chromosomes. The most commonly used technique include G-band forming bright (euchromatin, active regions of chromosomes) and dark (heterochromatin, inactive genes) horizontal bands along chromosomes. By the examination structural and numerical aberration of the chromosomes can be seen. Cytogenetic examination can be performed from periph- eral blood, fetal cells, but also from vital tissue. Altogether 79 patients were retrospectively examined in this section, i.e. 60 patients with endometriosis and 19 with EAOC. During the years 2007-2014 from 178 OC patients were 19 patients categorized as EAOC. Inclusion criteria were histology of EOC or CCOC. The age of the patients was 43-68 years with average age 52.36 years. Five patients were presented with bulky tumor and average diameter more than 11 cm. Excepts in 3 patients the CA125 was examined preoperatively and the average value was 250 IU/ml. All the patients’ characteristics are listed in Table 3. Prospectively 60 reproductive age patients (20-56 years, average age 36.9 years) with histologically proven endo- metriosis were assessed by this method. In all the patients a peripheral blood was for the conventional cytogenetics used. The characteristics, i.e. surgical approach, periop- erative finding, type of surgery as well as other findings are shown in Table 2. Peripheral venous blood (3-5 ml) was after harvesting transported into the laboratory till 60 min. The Retrospectively selected 60 patients with ovarian endo- metriosis were histopathologically analyzed. The average age of the included patients was 33.4 years (19-62 years). All the patients’ characteristics are listed in Table 4. The criteria for diagnosis of AE were already published [18] and this classification was used for definition of AE Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research (2022) 15:5 Page 5 of 13 Fig. 2  In a hypoxic environment, hydroxylation and degradation of HIF-1α are inhibited. Therefore, HIF-1α can dimerize, enter the nucleus and transcriptionally regulate the expression of its target genes through the transcription factor HRE. This way it regulates a wide range of pathophysiological processes including angiogenesis. PCR analysis A prospective PCR analysis of selected genes was done in 110 patients. A prospective PCR analysis of selected genes was done in 110 patients. Page 6 of 13 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research Table 2  Patients for cytogenetic and PCR examination PATIENTS 60 AVERAGE AGE 36.9 years AVERAGE MENARCHE 13.05 years NULLIGRAVIDA 28 patients (47%) INFERTILITY Primary = 5 patients (8%), Secondary = 2 patients (3%), Without = 53 patients (89%) ONCOMARKER AVERAGE CA125 = 83.79 HE4, ROMA, CEA, CA 19-9 = normal finding SURGICAL APPROACH Laparoscopy = 36 (60%), Laparotomy = 20 (33%), Laparoscopy + laparotomy = 4 (7%) PERIOPERATIVE FINDING Endometrioma = 34 (57%), Peritoneal endometriosis = 6 (10%), Endometriosis of sacrouterine ligaments = 6 (10%), Endometrioma + peritoneal endometriosis = 10 (17%), Frozen pelvis = 4 (6%) TYPE OF SURGERY Extirpation of endometriomas = 30 (50%), Adnexectomy = 12 (20%), Extirpation of endometriotic lesion = 8 (13%), Biopsy of endometriotic lesion = 7 (12%), Hysterec- tomy = 3 (5%) 60 36.9 years 13.05 years 28 patients (47%) Primary = 5 patients (8%), Secondary = 2 patients (3%), Without = 53 patients (89%) CA125 = 83.79 HE4, ROMA, CEA, CA 19-9 = normal finding Laparoscopy = 36 (60%), Laparotomy = 20 (33%), Laparoscopy + laparotomy = 4 (7%) Endometrioma = 34 (57%), Peritoneal endometriosis = 6 (10%), Endometriosis of sacrouterine ligaments = 6 (10%), Endometrioma + peritoneal endometriosis = 10 (17%), Frozen pelvis = 4 (6%) Extirpation of endometriomas = 30 (50%), Adnexectomy = 12 (20%), Extirpation of endometriotic lesion = 8 (13%), Biopsy of endometriotic lesion = 7 (12%), Hysterec- tomy = 3 (5%) Table 3  Characteristics of EAOC patients for histopathological analysis PATIENTS 19 AVERAGE AGE 52.36 years PRIMARY DIAGNOSIS Tumour adnex l. sin. = 12 patients Tumour adnex l. dx. = 7 patients ONCOMARKER AVERAGE CA125 = 250 IU/ml, CA 19-9 = 1048 IU/ml TYPE OF SURGERY Radical surgery = 14 patients, Hysterectomy with bilat- eral adnexectomy = 5 patients HISTOLOGY EOC = 12 (63.15%) patients, CCOC = 7 (36.85%) patients OTHER FINDINGS Nulligravida = 5, bulky tumour = 5 Table 3  Characteristics of EAOC patients for histopathological analysis Table 4  Characteristics of endometriosis patients for histopathological analysis PATIENTS 60 AVERAGE AGE 33.4 years PRIMARY DIAGNOSIS Tumour adnex l. dx. = 32 patients Tumour adnex l. sin. Histopathological analysis Altogether 19 (10.67%) of 178 OC patients diagnosed between 2007 and 2014 were confirmed as EAOC. From all EAOC patients 12 of them (63.15%) were EOC and 7 (36.85%) CCOC. PCR analysis Th l 2. absence of chromosome 13 satellite (46,XX, 13 ps-) The analysis of HIF1A and CTNNB1 (mRNA level) in CG patients (n = 50) represented a reference value compared with the findings in the EG patients (n = 60). 3. the association between group D and G chromo- somes – chromosomal configuration with potential of chromosomes withdrawal during next division and forming of numeric aberration. i The patients in EG were regarding the level of gene transcription divided into two subgroups. Transcrip- tionally incipient endometriosis (TIE), with 26 patients showed increased level of gene mRNA for both, HIF1A and CTNNB1 compared to CG however significantly lower than in second group. This group - transcription- ally evident endometriosis (TEE), contained 34 patients from EG where the level of mRNA genes (HIF1A and CTNNB1) were significantly higher compared to the both, CG as well as TIE (Table 5). In the rest of pathological findings – in two patients, the numerical abnormality with manifestation in the mosaic form was confirmed: 1. 45,X(5)/46,XX(45) – in five mitosis the karyotype 45,X was seen. The rest of mitosis was with karyotype 46,XX. In this case Turner mosaic was confirmed. i 2. 46,XX,+mar(2)/46,XX(98) – in two mitoses a marker chromosome from group C was seen. The case was concluded as the mosaic form of X chromosome tri- somy (superfemale) with normal phenotype. HIF1A expressions in TIE group (median 1.21) were increased without significance when compared to CG (median 1.00). However TEE group expressions (median 1.563) were significantly higher compared to CG patients (p < 0.01) as well as TIE patients (p < 0.05) (Fig. 4). By comparison of the results with clinical characteris- tics of the patients the association was not observed. The patients containing a cytogenetic deviations were diag- nosed with not extensive endometriosis. In three of them an ovarian endometrioma and in two of them endometri- osis of sacrouterine ligament was seen. Complete resec- tions were in all patients performed. The similar phenomenon was also in the assessment of CTNNB1 seen. Into TIE group (median 1.094) were selected patients with increased expression comparing to CG (median 1.00) but without statistical significance. While significantly increased expressions compared to CG (p < 0.01) as well as TIE (p < 0.05) were seen in the rest of the patients included into TEE group (median 1.499) (Fig. 5). Conventional cytogenetics From 60 endometriosis patients were 55 identified with normal karyotype 46,XX. 5 patients (8.3%) showed cytogenetic deviation from the physiological finding.h From seven CCOC patients, 3 (42.85%) were free of endometriosis, both BE as well as AE was confirmed in 1 patient (14.28%). Two (28.59%) CCOC patients had except carcinoma cells also BE and BOC in histology finding. i Three of them were concluded as heteromorphisms – variants of human karyotypes without affecting of patients’ phenotype. Such non-aberrant karyotype changes affect those parts of the chromosomes whose length or molecular structure varies within a population. All the deviations were detected in acrocentric chromo- somes – group D (chromosomes 13, 14, 15) and group G (chromosomes 21, 22): i From 60 ovarian endometriosis patients were 12 (20%) patients classified with AE while the rest 48 (80%) showed only BE finding (Fig. 3). 1. chromosome 14 satellite duplication (46,XX, 14pss) PCR analysis = 24 patients Bilateral tumour = 4 patients SURGICAL APPROACH Laparoscopy = 52 patients, laparotomy = 8 patients TYPE OF SURGERY Extirpation of endometrioma = 38 patients, adnexectomy = 19 patients, hysterectomy and bilateral adnexectomy = 3 patients OTHER FINDINGS Nulligravida = 13, CA125 elevation = 9, infertility = 4 Table 4  Characteristics of endometriosis patients for histopathological analysis PATIENTS 60 AVERAGE AGE 33.4 years PRIMARY DIAGNOSIS Tumour adnex l. dx. = 32 patients Tumour adnex l. sin. = 24 patients Bilateral tumour = 4 patients SURGICAL APPROACH Laparoscopy = 52 patients, laparotomy = 8 patients TYPE OF SURGERY Extirpation of endometrioma = 38 patients, adnexectomy = 19 patients, hysterectomy and bilateral adnexectomy = 3 patients OTHER FINDINGS Nulligravida = 13, CA125 elevation = 9, infertility = 4 Table 4  Characteristics of endometriosis patients for histopathological analysis Scientific). Reverse transcription from mRNA to cDNA was achieved using RevertAid Minus First Strand cDNA Synthesis Kit (Merck) with specific reverse primers for each gene. After the definition of reaction conditions for SensiMix™ SYBR® NoROX kit ran the amplification of the specific gene HIF1A and β-catenin for 30 cycles (94 °C 5 min, 94 °C 15 s., 60 °C 20 s. and 72 °C 25 s.), using appropriate primer sequences in the thermocycler Light- Cycler® 480 Instrument II (Roche Life Science). Normali- zation of the results was performed using housekeeping gene HGPRT and GAPDH. Numerical quantification of changes in expression levels was evaluated using the LightCycler® 480 Software, Version 1.5., where were confirmed Ct values corresponded with the midpoint of logarithmic amplification. Relative mRNA concentra- tions were calculated with respect to reference RNAs and inter-class fold changes computed using the ­2-ΔΔCt function. In order to minimize the impact of variability in the experimental data, all samples were measured four times. For the statistical evaluation One-Way ANOVA Student– Newmann–Keuls test was used. Data is presented as mean percent ± SD. Statistical analysis was processed by the program GraphPad INSTAT. Page 7 of 13 Varga et al. Journal of Ovarian Research (2022) 15:5 When we checked for EOC (12 patients), 3 (25%) of them were without endometriosis, 2 (16.66%) with BE, 3 (25%) with AE and finally 4 (33.34%) EOC patients had transition from BE to cancer through BOC in their histology. Discussionh The endometriosis was solidly confirmed as the precur- sor of certain portion of EAOC. Relatively low incidence of endometriosis overthrow makes difficult to define the predictive factors of this process. Tubo-ovarian junction plays a crucial role in process of malignant ovarian trans- formation even in the case of endometriosis. Inflamed stroma together with mutated alleles of the epithelial component is incorporated into CIC where due to the favorable environment a malignant transformation of Overall in 19 EAOC patients the endometriosis was not seen in 6 (31.57%) patients. In 3 patients (15.78%) the tissue of BE was confirmed while AE in 4 EAOC patients (21.08%). In other 6 patients the transition from BE through BOC structures to carcinoma tissue was identified. Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research Page 8 of 13 Page 8 of 13 (2022) 15:5 to the ovaries the highest probability to progress into EAOC. The DNA alterations in endometriomas origi- nate from permanent oxidative stress inside. Significantly higher iron ions concentration in endometriotic cysts was confirmed when compare to non-endometriotic cyst. Higher iron concentration was also in CCOC tissue seen, although the level did not reach the amount measured in endometriotic cysts [19]. Following this parameters the endometriosis patients included in presented study for histopathological examination were only with endo- metriomas. While cytogenetic and experimental PCR study was done in the patients with different types of endometriosis. Fig. 3  Histopathological analysis of EAOC, EOC, CCOC and endometriosis patients The development of genetics in the last decades has sig- nificantly influenced the focus of the study of various dis- eases, including endometriosis. Until 1997, conventional cytogenetics was predominant in genetic studies in endo- metriosis patients. Cytogenetics examinations over the last 20 years yielded discrepant results [20, 21], although the common feature can be defined as the absence of gross chromosome abnormalities. Conventional cytoge- netics did not confirm typical chromosomal structural changes which are common for patient with endome- triosis [22, 23]. Opposite conclusions were obtained by the studies applying fluorescence in  situ hybridization (FISH) and comparative genomic hybridization (CGH). Mainly monosomy, structural aberration or presence of recurrent gene copies increasing clonality potential were confirmed and predominantly chromosomes 1, 16 and 17 were affected [24, 25]. It is well known fact that somatic mutations of chromosome 17 are often seen in process of ovarian carcinogenesis [26]. Discussionh Three patients of presented study with heteromorphisms – variants of human karyo- types had the aberrations without affecting of patients’ phenotype. Although the changes are benign the karyo- type can be instable leading to the problems in meiotic division. All the changes were seen in the group of chro- mosomes D and G. The association of group D and G chromosomes can affect the division leading to numeri- cal aberration. From the presented results it is possible to support the conclusion that usage of conventional cytogenetics did not reveal structural abnormalities in karyotype of endometriosis patients. Fig. 3  Histopathological analysis of EAOC, EOC, CCOC and endometriosis patients Table 5  PCR gene analysis in control and experimental group Group/subgroup Patients Gene expressions (mRNA level) HIF1A CTNNB1 (β-catenin) CG n = 50 minimum – 0.98 maximum – 1.081 median – 1.00 minimum – 0.984 maximum – 1.032 median – 1.00 EG/TIE n = 26 minimum – 1.16 maximum – 1.299 median – 1.21 minimum – 1.086 maximum – 1.126 median – 1.094 EG/TEE n = 34 minimum – 1.484 maximum – 1.613 median – 1.563 minimum – 1.328 maximum – 1.567 median – 1.499 Table 5  PCR gene analysis in control and experimental group Table 5  PCR gene analysis in control and experimental group Group/subgroup Patients Gene expressions (mRNA level) HIF1A CTNNB1 (β catenin The switchover between endometriosis and ovarian cancer through carcinogenesis presupposes common morphological coherence. An extensive effort has been made to define morphological precursor of endometriosis overthrow. The initial concept was presented by Sampson in 1925 [27]. Later on the criteria have been extended by Scott in 1953 [28]. The group of borderline carcinomas defined WHO in 1971. Despite of logical context of the development of BE through mild cell atypia (cytological atypia) as the consequence of the inflammatory process endometriosis occurs [4]. The same microscopic changes are also in deep infiltrated endometriosis seen however malignant overthrow is rare [5]. In this direction ovar- ian endometriomas have due to their close connection Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research Page 9 of 13 Fig. 4  HIF1A expressions in CG, EG/TIE and EG/TEE. *p <0.05 EG/TEE vs. EG/TIE, ** p <0.01 EG/TEE vs. CG Fig. 5  CTNNB1 (β-catenin) expressions in CG, EG/TIE and EG/TEE. *p <0.05 EG/TEE vs. EG/TIE, ** p <0.01 EG/TEE vs. CG Fig. Discussionh 4  HIF1A expressions in CG, EG/TIE and EG/TEE. *p <0.05 EG/TEE vs. EG/TIE, ** p <0.01 EG/TEE vs. CG Fig. 5  CTNNB1 (β-catenin) expressions in CG, EG/TIE and EG/TEE. *p <0.05 EG/TEE vs. EG/TIE, ** p <0.01 EG/TEE vs. CG Fig. 5  CTNNB1 (β-catenin) expressions in CG, EG/TIE and EG/TEE. *p <0.05 EG/TEE vs. EG/TIE, ** p <0.01 EG/TEE vs. CG to structural atypia (hyperplasia) in cases of AE, the cri- teria for definition of AE remain controversial and widely discussed. The chronic inflammatory changes in endo- metriomas lead to metaplastic reaction of the epithelium. The changes are diagnosed as benign and they have no clinical impact. Most probably they represent an early changes starting the process of AE formation. Nowadays widely accepted criteria of AE include structural changes as mentioned earlier (Fig. 6). Generally, not specified endometriosis patients, with all types of endometriosis, the incidence of AE is reported to be 8-10%. Higher inci- dence of AE in endometriosis patients was seen in pre- sented study. 20% of studied histology showed signs of atypia. Reported increased incidence may be caused by types of patients. Due to the close connection with the ovaries only endometriomas were studied and not differ- ent types of endometriosis. Detailed analysis of patients with AE confirmed the patients with long-term history of disease, advanced finding during surgery and higher age in AE patients (39.8) when compare to patients with BE (31.8). The patients with long-term history as well as large endometriomas (> 9 cm) have been already con- firmed at higher risk for progression and are indicated to stricter observation [29, 30]. The frequency of endometriosis overthrow is reported to be 0.3-0.8% [30]. The appearance of AE increases in Varga et al. Journal of Ovarian Research (2022) 15:5 Page 10 of 13 Page 10 of 13 Varga et al. Journal of Ovarian Research EAOC patients confirmed [10]. Following the results, the presence of AE in histology represents a significant risk factor and predisposes to more precise observation of the patient. case of OC patients. Similar phenomenon confirmed Niguez Sevilla with colleagues as well. 8.8% of AE was seen in cancer free patients however in EAOC patients the AE was confirmed in 34.6% [10]. In our study AE was seen in 21.08% of EAOC patients and most of them were with EOC histology (25%), lesser in CCOC patients (14.28%). Discussionh Slightly different results of AE appearance in EOC and CCOC patients were published. 23% of EOC patients and 36% of CCOC patients were with a pres- ence of AE in their histology [19, 30]. Generally the endo- metriosis was seen in 68.43% EAOC patients including benign, atypical type or benign endometriosis with BOC structures. EOC patients revealed 75% cases with endo- metriosis while CCOC patients 57.14%. Wei with colleagues observed the presence of endo- metriosis in EAOC patients in younger women, aver- age 45 years [30]. This average decreases mainly the CCOC patients while EOC patients tend to incease it. When we checked our results the average age of CCOC patients was 49.8 years and EOC 53.8 years. Surpris- ingly the average age in both groups, i.e. EOC and CCOC was lower in endometriosis free patients. The EOC patients without endometriosis were with aver- age age 52.6 years while in patients with endometriosis the average age was 54.2 years. Same phenomenon was in CCOC patients seen. Endometriosis free patients were 47 years old in average while CCOC patients with endometriosis were 52 years old. The patients with EOC histology were captured in earlier stages with more common stepwise development form BE to BOC. On the other hand CCOC patients were diagnosed with more advanced stage with metastases in abdominal cavity. After 1953 many authors described in EAOC the transition starting from benign endometriosis to AE and BOC. When we checked this group, 31.57% EOAC patients in histology showed endometriosis with BOC, in EOC 33.34% and in CCOC 28.59%. This correspondences with literature. EAOC may have in their histology BE, AE or BE with BOC but also it can be without endometrio- sis structures [12]. The stepwise development containing endometriosis as well as structures of BOC was in 25% Fig. 6  The development of atypical endometriosis Fig. 6  The development of atypical endometriosis Page 11 of 13 Page 11 of 13 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research cells leads to the chronic ischemia mainly in central part of the tumours. The result of oxygen depletion is up- regulation of different genes including HIF1A. Finally the VEGF and HIF-1α elevation positively correlate with worse prognosis of OC patients [33]. Therefore the anti- bodies against VEGF are used as target therapy in clinical practice. Discussionh The development of HIF1A expressions showed a similar results. In the group TIE only insignificant HIF1A expressions increase were included. They showed increasing about 9.4% when compared to CG. The signifi- cant elevation comparing to CG as well as TIE was in the rest of patients seen. They were selected into TEE group with 49.9% elevation comparing to CG. The results are comparable with the previous studies [17, 34] although not every patient showed significant increasing. In the analysis of clinical characteristic of the patients was in the TEE group apparently higher amount of advanced stage patients seen. Also patients with suboptimal sur- gery or macroscopic residual disease were mainly in this subgroup detected (Table 6). The role of β-catenin protein in processes of carcino- genesis have been confirmed in many cancers. The muta- tions of CTNNB1 gene were seen in pulmonary cancer, breast cancer, colorectal cancer, but also in endometrial and ovarian cancer, including EAOC [15]. Immunohisto- chemical positivity was confirmed in 61.2% EOC patients but both types of patients, i.e. endometriosis free and EOC with endometriosis showed this result [31]. High incidence, 90%, of CTNNB1 mutation was also in endo- metrioid borderline carcinoma confirmed [32]. These findings suggest that the CTNNB1 mutations may rep- resent an early change in the process of malignization of some ovarian tumors. The activation of Wnt signaling pathway which also includes a CTNNB1 mutation is connected with the ini- tiation of surrounding fibrotization [13], potentiation of proliferation, implantation or invasion [14]. This process was also in endometriosis confirmed [16]. The extent of the consequences can therefore be logically confronted with the extent of the protein defect. Increased expression of CTNNB1 was seen in presented endometriosis patients of EG when compare to CG. When insignificant increase was detected the patients were closed in TIE group. In the second subgroup of EG, i.e. TEE only significantly increased CTNNB1 expres- sions were included. When we checked for clinical char- acteristics of the patients in TEE mainly advanced stage endometriosis patients were seen. Due to the stage of the disease they were often confronted with suboptimal surgery and up to 53% of them had macroscopic residual disease after surgery (Table 6). Taking into account both subgroups we can conclude that CTNNB1 expression correlates with the endometriosis extent in patients. Significant increase in mRNA of both genes HIF1A and CTNNB1 was in TEE seen. Discussionh Those patients revealed mainly extensive preoperative finding. Bilateral endo- metrioma, frozen pelvis and peritoneal endometrio- sis in coincidence with endometriomas were defined as extensive extent. While unilateral endometrioma as well as isolated endometriosis of sacrouterine ligament were defined as unextensive finding. Consent for publication 18. Yamashita Y, Toyokuni S. Endometriosis-associated ovarian cancer: the role of oxidative stress, endometriosis – basic concepts and current research trends, Prof. Koel Chaudhury (Ed). 2012. ISBN:978-95351-0524-4, InTech. All involved patients answered a medical questionnaire and were informed about the usage of their blood for experimental-diagnostic purposes and informed consent was signed. 19. Yamaguchi K, Mandai M, Toyokuni S, et al. Contents of endometriotic cysts, especially the high concentration of free iron, are a possible cause of carcinogenesis in the cysts through the iron-induced persistent oxida- tive stress. Clin Cancer Res. 2008;14:32–40. Funding The study was supported by KEGA grant agency, namely by the grant KEGA- 026 SPU-4/2018. 14. Liang JY, Li CD, Zhang WY. Effects of activating and inhibiting Wnt/ βcatenin signaling pathway on murine model of eutopic endometrium and endometriosis. Zhonghua Yi Xue Za Zhi. 2012;92:1352–6. Acknowledgements 9. Tanase Y, Kawaguchi R, Uchiyama T, et al. Long-term follow up after surgi- cal management for atypical endometriosis: a series of nine cases. Case Rep Oncol. 2019;12:76–83. By this way we would like to acknowledge to all the participants for their interest on this study. Received: 14 January 2021 Accepted: 24 December 2021 Received: 14 January 2021 Accepted: 24 December 2021 Received: 14 January 2021 Accepted: 24 December 2021 Availability of data and materials 15. Lyttle B, Bernardi L, Pavone ME. Ovarian cancer in endometriosis: clinical and molecular aspects. Minerva Ginecol. 2014;66:155–64. The primary data for this study is available from the authors on direct request. 16. Matsuzaki S, Darcha C. Epithelial to mesenchymal transition-like and mesenchymal to epithelial transition-like processes might be involved in the pathogenesis of pelvic endometriosis. Hum Reprod. 2012;27:712–21. Ethics approval and consent to participate 17. Young VJ, Brown JK, Maybin J, et al. Transforming growth factor-β induced Warburg-like metabolic reprogramming may underpin the development of peritoneal endometriosis. J Clin Endocrinol Metab. 2014;99:3450–9. This experiment was approved by the Ethical committee in accordance with the laws and policies of governing authorities. Competing interests The authors declare that they have no competing interest. Abbreviations AE At i l d 4. Suda K, Nakaoka H, Yoshihara K, et al. Clonal expansion and diversification of cancer associated mutations in endometriosis and normal endome- trium. Cell Rep. 2018;24:1777–89. AE: Atypical endometriosis; BE: Benign endometriosis; BOC: Borderline carci- noma; CCOC: Clear cell ovarian cancer; CG: Control group; CGH: Comparative genomic hybridization; CIC: Cortical inclusion cyst; EAOC: Endometriosis associated ovarian cancer; EG: Experimental group; EOC: Endometrioid ovarian cancer; FISH: Fluorescence in situ hybridization; HGSOC: High grade serous ovarian cancer; LGSOC: Low grade serous ovarian cancer; OC: Ovarian cancer; OSE: Ovarian surface epithelium; PTH: Papillary tubal hyperplasia; STIC: Serous tubal intraepithelial carcinoma; TEE: Transcriptionally evident endometriosis; TIE: Transcriptionally incipient endometriosis; VEGF: Vascular endothelial growth factor. 5. Anglesio MS, Papadopoulos N, Ayhan A, et al. Cancer associated muta- tions in endometriosis without cancer. N Engl J Med. 2017;376:1835–48. 6. Yoshimoto C, Iwabuchi T, Shigetomi H, et al. Cyst fluid iron-related com- pounds as useful markers to distinguish malignant transformation from benign endometriotic cysts. Cancer Biomark. 2015;15:493–9. g y 7. Kobayashi H. Potential scenarios leading to ovarian cancer arising from endometriosis. Redox Rep. 2016;21:119–26. 8. Munksgaard PS, Blaakaer J. The association between endometriosis and ovarian cancer: a review of histological, genetic and molecular altera- tions. Gynecol Oncol. 2012;124:164–9. 8. Munksgaard PS, Blaakaer J. The association between endometriosis and ovarian cancer: a review of histological, genetic and molecular altera- tions. Gynecol Oncol. 2012;124:164–9. References 1. Menon U, Karpinskyj C, Gentry-Maharaj A. Ovarian cancer prevention and screening. Obstet Gynecol. 2018;131:909–27. 1. Menon U, Karpinskyj C, Gentry-Maharaj A. Ovarian cancer prevention and screening. Obstet Gynecol. 2018;131:909–27. g y 2. Messini I, Doulgeraki T, Chrysanthakis D, et al. Assessing the landscape of ovarian serous borderline tumors. Int J Gynecol Cancer. 2019;29:572–8. y 2. Messini I, Doulgeraki T, Chrysanthakis D, et al. Assessing the landscape of ovarian serous borderline tumors. Int J Gynecol Cancer. 2019;29:572–8. 3. Chui MH, Xing D, Zeppernick F, et al. Clinicopathologic and molecular fea- tures of paired cases of metachronous ovarian serous borderline tumor and subsequent serous carcinoma. Am J Surg Pathol. 2019;43:1462–72. 3. Chui MH, Xing D, Zeppernick F, et al. Clinicopathologic and molecular fea- tures of paired cases of metachronous ovarian serous borderline tumor and subsequent serous carcinoma. Am J Surg Pathol. 2019;43:1462–72. Authors’ contributions 10. Niguez Sevilla I, Machado Linde F, Marín Sánchez MDP, et al. Prognostic importance of atypical endometriosis with architectural hyperplasia versus cytologic atypia in endometriosis associated ovarian cancer. J Gynecol Oncol. 2019. https://​doi.​org/​10.​3802/​jgo.​2019.​30.​e63. Ján Varga developed the original design and performed the initial literature review. Alžbeta Reviczká helped with the patients’ selection and samples har- vesting. Hedviga Háková performed examination by conventional cytogenet- ics. Peter Švajdler covered and performed all the pathological examinations. For the PCR examinations was responsible Miroslava Rabajdová. Ján Varga, Hedviga Háková, Peter Švajdler and Miroslava Rabajdová performed results confrontation. With the patients’ selection, discussion and founding helped Alexander Ostró. Ján Varga prepared a final manuscript. All the authors read and approved the final manuscript. 11. Fukunaga M, Nomura K, Ishikawa E, et al. Ovarian atypical endometriosis: its close association with malignant epithelial tumours. Histopathology. 1997;30:249–55. 12. Ogawa S, Kaku T, Amada S, et al. Ovarian endometriosis associated with ovarian carcinoma: a clinicopathologic and immunohistochemical study. Gynecol Oncol. 2000;77:298–304. 13. Matsuzaki S, Darcha C. Involvement of the Wnt/β-catenin signaling path- way in the cellular and molecular mechanisms of fibrosis in endometrio- sis. PLoS One. 2013. https://​doi.​org/​10.​1371/​journ​al.​pone.​00768​08. Conclusion Endometriosis represents a heterogeneous disease from many aspects. The need for the selection of the patients is appropriate and uniform management is obsolete at pre- sent. In terms of possibility of endometriosis overthrow the cognition of predictive factors is the basis of the issue. Endometriomas due to their close relationship to ovar- ian microenvironment are more susceptible to malignant overthrow. Tubo-ovarian junction plays important role in ovarian carcinogenesis, including those containing endo- metriosis. Specific structural chromosome abnormalities in endometriosis patients predicting its malignant over- throw are not detectable by the conventional cytogenet- ics. By applying the histopathological criteria defining Protein HIF-1 is included in the processes of cell metabolism regulation. Hypoxia leads to decrease in HIF-1α degradation resulting in pro-angiogenic poten- tial as well as potentiation of tumor induced immuno- suppression. This situations were in tissues of different carcinomas and endometriosis confirmed. Whereas in endometriomas an angiogenesis is more prominent in the outer capsule of the cyst comparing to inner part [17]. The inner environment of the tumours is charac- terized by low pH and low oxygen level due to the inad- equate circulation. The fast proliferation of the tumor Table 6  Patients’ characteristics in TIE and TEE Group Peroperative finding No. patients (%) Type of surgery No. patients (%) Residual disease No. patients (%) Unextensive Extensive Optimal Suboptimal NEGAT. POZIT. TIE (n = 26) 20 (76.9%) 6 (23.1%) 25 (96.1%) 1 (3.9%) 22 (84.6%) 4 (15.4%) TEE (n = 34) 14 (41.2%) 20 (58.8%) 22 (64.7%) 12 (35.3%) 22 (47.05%) 18 (52.9%) Page 12 of 13 Page 12 of 13 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research AE a risk group of endometriosis patients with need for more precise observation can be selected. The patients with long-term history of endometriosis, advanced stage and big endometriomas should be under precise observa- tion. EAOC is more common with endometriosis. EOC are usually less aggressive, slowly progressing and often with detectable histological transition from precursor to invasive carcinoma. Increased expression of mentioned genes is most probably related to the disease progression and characterizes early stages of progression. Determina- tion of genes transcription can help to select a risk group of patients, although other studies are needed. Deggendorf, Germany. 3 Department of Laboratory Medicine, subdivision of Medical Genetics L. Pasteur University Hospital, Košice, Slovakia. Conclusion 4 CYTOPA- THOS, s.r.o, Bratislava, Slovakia. 5 Department of Medical and Clinical Biochem- istry, Faculty of Medicine, P. J. Šafárik University, Košice, Slovakia. Author details 1 Angiogenesis. 2010;13:43–58. 33. Carpini JD, Karam AK, Montgomery L. Vascular endothelial growth factor and its relationship to the prognosis and treatment of breast, ovarian, and cervical cancer. Angiogenesis. 2010;13:43–58. 34. Goteri G, Lucarini G, Zizzi A, et al. Proangiogenetic molecules, hypoxia- inducible factor-1alpha and nitric oxide synthase isoforms in ovarian endometriotic cysts. Virchows Arch. 2010;456:703–10. 34. Goteri G, Lucarini G, Zizzi A, et al. Proangiogenetic molecules, hypoxia- inducible factor-1alpha and nitric oxide synthase isoforms in ovarian endometriotic cysts. Virchows Arch. 2010;456:703–10. 34. Goteri G, Lucarini G, Zizzi A, et al. Proangiogenetic molecules, hypoxia- inducible factor-1alpha and nitric oxide synthase isoforms in ovarian endometriotic cysts. Virchows Arch. 2010;456:703–10. Author details 1 20. Hapangama DK, Turner MA, Drury JA, et al. Sustained replication in endo- metrium of women with endometriosis occurs without evoking a DNA damage response. Hum Reprod. 2009;24:687–96. 1 Department of Gynaecology and Obstetrics, Faculty of Medicine, P.J. Šafárik University and L. Pasteur University Hospital, Rastislavova 43 Street, 041 90 Košice, Slovakia. 2 Frauenklinik, DONAUISAR Klinikum Deggendorf, Page 13 of 13 Varga et al. Journal of Ovarian Research (2022) 15:5 Varga et al. Journal of Ovarian Research (2022) 15:5 21. Koerner M, Burckhardt E, Mazzucchelli L. Higher frequency of chromo- somal aberrations in ovarian endometriosis compared to extragonadal endometriosis: a possible link to endometrioid adenocarcinoma. Mod Pathol. 2006;19:1615–23. 22. Dangel A, Medchill MT, Davis G, et al. Cytogenetic studies in endometrio- sis tissue. Cancer Genet Cytogenet. 1994;78:172–4. 23. Tamura M, Fukaya T, Murakami T, et al. Analysis of clonality in human endometriotic cysts based on evaluation of X chromosome inactiva- tion in archival formalin-fixed, paraffin-embedded tissue. Lab Investig. 1998;78:213–8. 24. Kosugi Y, Elias S, Malinak LR, et al. Increased heterogeneity of chro- mosome 17 aneuploidy in endometriosis. Am J Obstet Gynecol. 1999;180:792–7. 25. Bouquet De Jolinière J, Ayoubi JM, Gianaroli L, et al. Endometriosis: a new cellular and molecular genetic approach for understanding the patho- genesis and evolutivity. Front Surg. 2014;1:16. 26. Jacobs IJ, Kohler MF, Wiseman RW, et al. Clonal origin of epithelial ovar- ian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation. J Natl Cancer Inst. 1992;84:1793–8. 27. Sampson J. Endometrial carcinoma of the ovary, arising in endometrial tissue of that organ. Arcg Surg. 1925;10:1–72. 28. Scott RB. Malignant changes in endometriosis. Obstet Gynecol. 1953;2:283–9. 29. Tanase Y, Furukawa N, Kobayashi H, et al. Malignant transformation from endometriosis to atypical endometriosis and finally to endometrioid adenocarcinoma within 10 years. Case Rep Oncol. 2013;6:480–4. 30. Wei JJ, William J, Bulun S. Endometriosis and ovarian cancer: a review of clinical, pathologic, and molecular aspects. Int J Gynecol Pathol. 2011;30:553–68. 31. Stewart CJ, Walsh MD, Budgeon CA, et al. Immunophenotypic analysis of ovarian endometrioid adenocarcinoma: correlation with KRAS mutation and the presence of endometriosis. Pathology. 2013;45:559–66. 32. Geyer JT, López-García MA, Sánchez-Estevez C, et al. Pathogenetic path- ways in ovarian endometrioid adenocarcinoma: a molecular study of 29 cases. Am J Surg Pathol. 2009;33:1157–63. 33. Carpini JD, Karam AK, Montgomery L. 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https://www.frontiersin.org/articles/10.3389/fneur.2018.00760/pdf
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Degraded Synergistic Recruitment of sEMG Oscillations for Cerebral Palsy Infants Crawling
Frontiers in neurology
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ORIGINAL RESEARCH published: 18 September 2018 doi: 10.3389/fneur.2018.00760 Degraded Synergistic Recruitment of sEMG Oscillations for Cerebral Palsy Infants Crawling Zhixian Gao 1 , Lin Chen 1,2 , Qiliang Xiong 1 , Nong Xiao 3 , Wei Jiang 3 , Yuan Liu 3 , Xiaoying Wu 1,4* and Wensheng Hou 1,2,4* 1 Key Laboratory of Biorheological Science and Technology of Ministry of Education, Chongqing University, Chongqing, China, 2 Collaborative Innovation Center for Brain Science, Chongqing University, Chongqing, China, 3 Department of Rehabilitation Center, Children’s Hospital of Chongqing Medical University, Chongqing, China, 4 Chongqing Medical Electronics Engineering Technology Research Center, Chongqing University, Chongqing, China Edited by: Xu Zhang, University of Science and Technology of China, China Reviewed by: Eugene Golanov, Houston Methodist Hospital, United States Rong Song, Sun Yat-sen University, China *Correspondence: Xiaoying Wu wuxiaoying69@163.com Wensheng Hou w.s.hou@cqu.edu.cn Specialty section: This article was submitted to Neurotrauma, a section of the journal Frontiers in Neurology Received: 19 May 2018 Accepted: 22 August 2018 Published: 18 September 2018 Citation: Gao Z, Chen L, Xiong Q, Xiao N, Jiang W, Liu Y, Wu X and Hou W (2018) Degraded Synergistic Recruitment of sEMG Oscillations for Cerebral Palsy Infants Crawling. Front. Neurol. 9:760. doi: 10.3389/fneur.2018.00760 Frontiers in Neurology | www.frontiersin.org Background: Synergistic recruitment of muscular activities is a generally accepted mechanism for motor function control, and motor dysfunction, such as cerebral palsy (CP), destroyed the synergistic electromyography activities of muscle group for limb movement. However, very little is known how motor dysfunction of CP affects the organization of the myoelectric frequency components due to the abnormal motor unit recruiting patterns. Objectives: Exploring whether the myoelectric activity can be represented with synergistic recruitment of surface electromyography (sEMG) frequency components; evaluating the effect of CP motor dysfunction on the synergistic recruitment of sEMG oscillations. Methods: Twelve CP infants and 17 typically developed (TD) infants are recruited for self-paced crawling on hands and knees. sEMG signals have been recorded from bilateral biceps brachii (BB) and triceps brachii (TB) muscles. Multi-scale oscillations are extracted via multivariate empirical mode decomposition (MEMD), and non-negative matrix factorization (NMF) method is employed to obtain synergistic pattern of these sEMG oscillations. The coefficient curve of sEMG oscillation synergies are adopted to quantify the time-varying recruitment of BB and TB myoelectric activity during infants crawling. Results: Three patterns of sEMG oscillation synergies with specific frequency ranges are extracted in BB and TB of CP or TD infants. The contribution of low-frequency oscillation synergy of BB in CP group is significantly less than that in TD group (p < 0.05) during forward swing phase for slow contraction; however, this low-frequency oscillation synergy keep higher level during the backward swing phase crawling. For the myoelectric activities of TB, there is not enough high-frequency oscillation recruitment of sEMG for the fast contraction in propulsive phase of CP infants crawling. Conclusion: Our results reveal that, the myoelectric activities of a muscle can be manifested as sEMG oscillation synergies, and motor dysfunction of CP degrade the 1 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations synergistic recruitment of sEMG oscillations due to the impaired CNS regulation and destroyed MU/muscle fiber. Our preliminary work suggests that time-varying coefficient curve of sEMG oscillation synergies is a potential index to evaluate the abnormal recruitment of electromyography activities affected by CP disorders. Keywords: muscle synergy, cerebral palsy, sEMG oscillations, infants crawling, synergistic recruitment INTRODUCTION sensitive to muscle fatigue than the raw sEMG signal. Moreover, coherence between two sEMG oscillations was used to evaluate the neural control of movement under different conditions, such as low-alcohol and fatigue (13, 14). In addition, studies also reported that coherence can be affected by voluntary force (15). More recent studies employed sEMG oscillation components for abnormal motor functions analysis, and found that the characteristics of sEMG oscillation components correspond to various neuromuscular damages in CP (10, 16). On other hand, sEMG frequency spectrum is correlated the types of recruited MUs, Wakeling et al. have demonstrated that slower and faster MUs in muscles indeed generate low and high sEMG frequency components, respectively (17, 18). Moreover, sEMG oscillations with specific frequency ranges have been successfully used to evaluate the recruitment patterns of corresponding types of MUs in both animal and human muscles during movement (19, 20). It is generally accepted that multiple element synergistic organization is a fundamental strategy for motor control, and the elements can be well-organized by CNS to perform limb movement (21, 22). A variety of methods have been employed to extract multiple oscillations from sEMG (12, 23, 24), and multivariate empirical mode decomposition (MEMD) shows better performance in decomposing multi-channel sEMG signals due to its self-adaptability, fully data-driven and generalized multivariate extension (16, 25). These studies have indicated that the resulted multi-scale oscillations are mode-aligned across channels, and analysis of multiple oscillations is at the same scale. To obtain the synergistic pattern of multiple elements, although component-based algorithms, such as principal component analysis (PCA) (26, 27) and independent component analysis (ICA) (28), have been introduced to extract synergies, nonnegative matrix factorization (NMF) is a better choice (29, 30) due to its non-negative constraint for all the matrices (the raw matrix and the obtained matrices). A few works have demonstrated that NMF can be used to extract muscle synergies from multi-channel sEMG signals in upper limb muscles during motion tasks (31), even to obtain activation patterns of muscletendon units and time-vary coefficient curves from high-density sEMG signals during dynamic motion tasks (32). Inspired by muscle synergy, different types of MUs might also be manifested as specific oscillation synergy patterns. In other words, the synergistic recruitment mechanism should be manifested with coordinated activation of muscle groups and of different types of MUs in individual muscles. Hence, we assumed sEMG frequency components or oscillations might also be recruited with a synergistic pattern for normal motor function, whereas motor dysfunction of CP may affect the organization patterns of oscillation components. To this end, we recorded Cerebral palsy (CP) is a permanent movement disorder caused by brain injury in early childhood with a high prevalence of 2–3 per 1,000 live births (1). The regulation of muscle coordination and motor unit (MU) recruitment are usually affected due to the secondary musculoskeletal morphology alterations and changes in the electrophysiological characteristics of MU followed by cerebral injury (2). Surface electromyography (sEMG) signals regulated by central nervous system (CNS) have been widely used to evaluate MU recruitment patterns and neuromuscular function, and it is widely accepted that motor dysfunction of cerebral palsy destroys the synergistic recruitment of muscular activities (3). Although the impact of CP on synergistic electromyography activities has been observed among the muscle group for limb movement (4), very little is known about how motor dysfunction of CP affects the organization of the myoelectric frequency components due to the abnormal motor unit recruitment patterns. Various features of sEMG signals have been proposed for assessing the abnormal neuromuscular functions in CP patients. The sEMG characteristics from individual muscle, including parameters in time- and frequency-domain, can be used to measure the abnormal overall sEMG activity of CP. Researchers found the changes of magnitude, frequency content and timing from sEMG signals of individuals with CP. The time- and frequency-domain parameters were also utilized to analyze symmetry, cadence, or smoothness of muscle activity for CP patients (5–7). On the other hand, sEMG characters extracted from multiple muscles, such as muscle synergy and co-activation index, can provide the information about relative muscle activation of muscle groups and insights into motor control. Tang et al. (3) reported that CP recruited fewer muscle synergies during upper limb movements with simplified neuromuscular control strategy. Gross et al. (8) found that coactivation index of the rectus femoris/semitendinosus couple was more sensitive to speed, which could be explained by altered motor control. Furthermore, increasing studies suggested that the motor dysfunction caused by CP affected not only the overall sEMG activity but also the sEMG frequency components or oscillations (9, 10). Recent studies have demonstrated that the sEMG spectrum profile or frequency components correlated with physiological status of movement. Von et al. (11) reported that high-frequency components of sEMG from tibialis anterior were activated before heel strike during running, while low-frequency components were dominated after heel strike. Liu et al. (12) declared that the highest frequency components of sEMG were more Frontiers in Neurology | www.frontiersin.org 2 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations the sEMG signals from biceps brachii (BB) and triceps brachii (TB) muscles of the upper limbs during infant crawling, extracted sEMG oscillations and analyzed their synergy patterns to explore the abnormal organization of sEMG oscillations in CP. To specify the crawling phase, 14 markers were attached on the bony landmarks of the bilateral wrist, elbow, shoulder, hip, knee, ankle, the right spine scapula, and the trunk, respectively (Figure 1). The kinematic data of infants were recorded at 100 frames/s by a 3D motion capture system (Raptor-E, Motion analysis corporation, USA) with six high-speed digital cameras. Kinematic data and sEMG data were stored in a desktop computer and a laptop computer respectively synchronized offline. METHOD Participants Under the approval of children’s hospital of Chongqing medical university, 17 infants with typical development (TD, 11.4 ± 1.7 months, 12 males and 5 females) and 12 infants with cerebral palsy (CP, 22.3 ± 5.5 months, 9 males and 3 females) were recruited in this study. Cerebral palsy infants were collected from the department of rehabilitation center in the children’s hospital of Chongqing medical university. The TD infants were recruited from local child health clinics. The inclusion criteria for CP infants included: (1) all CP infants were under the age of 3 years old; (2) crawling continuously on their hands and knees during the test; (3) no other diseases that lead to motor function deficits according to the historical records. The TD infants were born at term with normal birth weight and had no neurological impairment. To make sure all subjects can crawl continuously on hands and knees, the Gross Motor Function Measure (GMFM88) was conducted before experiments. Informed consent forms were obtained from participants’ legal guardians before experiments. Data Analysis As shown in Figure 2, to study the organization of sEMG oscillations during crawling, multivariate empirical mode decomposition (MEMD) was employed to extract the multi-scale myoelectric oscillations, and non-negative matrix factorization (NMF) was used to analyze the patterns of synergistic organization within sEMG oscillations. Then, the recruitment coefficients within each refined crawling phase were evaluated. Pre-processing Firstly, the valid data of more than four consecutive crawling cycles were segmented from raw signals, and sEMG for the BB and TB of both sides and kinematic data were simultaneously recorded (see Figure 3). Then, the valid sEMG signals were processed with a zero-lag high-pass filter (4th order Butterworth filter, 20 Hz). Then, a 50 Hz notch filters were adopted to remove power frequency noise. Experimental Protocol The infants were encouraged to crawl at their own pace from one end of a mat (360 × 120 cm) to the opposite side. Valid crawling cycle sequence requires infants to crawl continuously on hands and knees more than four cycles. Before the experiment, infants crawled on the mat several minutes to warm up. As the major activated muscles of the upper limb during crawling, bilateral BB and TB were selected for sEMG recording. After disposable surface electrodes were attached to the muscle belly and bandaged to reduce motion artifacts (Figure 1), sEMG signals were collected using a sEMG system (ME6000T8, Mega Electronics Ltd, Finland) with a 15–500 Hz bandwidth and a 1,000 Hz sampling rate. Oscillation Extraction With MEMD EMD is a fully data-driven analysis approach which selfadaptively decomposes a non-linear and non-stationary signal into several intrinsic mode functions (34). However, for multichannel signals, applying EMD to each channel could produce a different number of misaligned IMFs. Rehman et al. (35) proposed the MEMD method to extend EMD to multivariate signal decomposition. They treated n-variable time series as ndimensional vectors and employ the spherical coordinate system to project n-dimensional vectors along different directions in (n1)-dimensional space, and the mean value of the envelopes of these n-dimensional projection sequences is obtained as the local mean of multiple time series. After MEMD decomposition, the ndimensional raw signal {v (t)}Tt=1 = {v1 (t) , v2 (t) , · · · vn (t)} can be decomposed into d layers multivariate IMFs hi (t) and residual r(t), which can be described as v (t) = d X hi (t) + r (t) (1) i=1 The IMFs obtained after MEMD analysis have the same number for each channel and orderly align scales across channels (35). Referred literature (16), we firstly concatenated the valid sEMG signal segments from all subject as a 4-channel sEMG FIGURE 1 | Experiment setup of markers and sEMG electrodes. Frontiers in Neurology | www.frontiersin.org 3 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations FIGURE 2 | Block diagram of the proposed oscillation synergy analysis framework. FIGURE 3 | sEMG and kinematic recorded in infant crawling. (A) Diagram of the crawling cycle of left upper limb [Adapted from Patrick et al. (33)]; (B) Schematic of the shoulder joint angle (AS); (C) Refined crawling phases, and the corresponding sEMG signals of bilateral TB and BB (LTB, left TB; LBB, left BB; RTB, right TB; RBB, right BB;) of one crawling cycle for TD (left graph) and CP (right graph). dataset D, which is shown as following formula:   CH11 − CH12 − · · · − CH1j   .. D=  . CHi1 − CHi2 − · · · − CHij the same length as D) were added to the 4-channel sEMG dataset D to composite a 6-channel dataset. Then, the composite dataset was decomposed by MEMD, which yield a total of 23 IMFs with aligned scales across channels, cycles and subjects. Example of raw sEMG signals and their first nine IMFs of BB in a crawling cycle are illustrated in Figure 4, of which the right panel show the corresponding power spectra for raw sEMG and IMFs. The frequency ranges of the first nine IMFs are listed in Table 1, the frequency band ranges of IMFs decrease orderly, and corresponding IMFs for different subjects are scalealigned. (2) where CHij is the ith channel of the valid sEMG signal segments for the jth subject. Here, we recorded four channel sEMG signals, and totally 29 subjects conducted the test; that is to say, i = 1, 2, 3, 4; j = 1, 2, . . . , 29. To reduce the mode mixing in multivariate IMFs (36), 2-channel Gaussian white noise (with Frontiers in Neurology | www.frontiersin.org 4 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations FIGURE 4 | Examples of decomposition results of a raw sEMG signal of BB from one TD subject. (Left panel) sEMG signal and its first nine IMFs, and (right panel) corresponding power spectra of these IMFs. TABLE 1 | Frequency ranges of first nine IMFs determined by 3dB bandwidth. IMF Frequency range (Hz) 1 2 3 4 5 6 7 8 9 284–500 183–352 127–252 81–189 52–116 34–69 23–41 16–24 4–18 Kinematics Data Preprocessing and Crawling Phase Segmentation propulsive phase (PRP) with the minimum of time derivative of AS (wS ). Kinematics data were processed with a zero-lag low-pass filter (4th order Butterworth filter, 4Hz) to remove high-frequency noise. As shown in Figures 3A,B, crawling cycle phases was determined with the z coordinates of the wrist (ZW ) and the shoulder joint angle (the joint angle in sagittal plane) (37). Figure 3C showed the examples of segmentation from two subjects during one crawling cycle. The squared of time derivative of ZW (vZ2 W , velocity squared) was applied to segment swing and stance, and the crawling cycle begins with swing. A threshold of vZ2 W was set at 0.5 (m2 /s2 ) to decide the onset of limb moving and the end moment of a crawling phase (38). To refine the crawling cycle in detail, the swing phase was divided into forward swing phase (FSP) and backward swing phase (BSP) with the maximum of ZW ; and stance was divided into braking phase (BRP) and Frontiers in Neurology | www.frontiersin.org Synergistic Recruitment Analysis of sEMG Oscillations As the frequency of IMF9 is below 20 Hz and out of sEMG frequency range (20–500 Hz), the first eight IMFs are included for oscillation recruitment analysis. Envelopes of the first eight IMFs of each channel sEMG signal were extracted (Hilbert spectrum and median filtering) and segmented into cycles according to kinematics data. The envelopes were re-sampled into 200 points for each cycle. The synergies of sEMG oscillation were extracted with NMF, which decompose a non-negative matrix V into two non-negative matrixes including a base matrix W and corresponding recruitment coefficient matrix C (39). Here, eight envelopes of IMFs resulted from a channel of sEMG within a 5 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations Three Stable Oscillation Synergies With Low-, Medium- and High-Frequency Ranges in Muscles crawling cycle composed the non-negative matrix V. The NMF method can be described as following V m×n = W m×s Cs×n The mean VAF values acquired in the NMF decomposition of the sEMG oscillations are shown in Figure 5. For bilateral BB and TB muscles, the number of oscillation synergies (s) was chosen as 3 according to the selection criteria above. Figure 6 shows the three oscillation synergies composed of the IMFs with different weights for BB and TB in TD and CP group. Each synergy of sEMG oscillation owned a dominant frequency range. The main contributors of synergy1 were IMF1, IMF2, IMF3, and IMF4, and their frequency ranged from 81 to 500 Hz (see Table 1); synergy2 was dominated with IMF4, IMF5 and IMF6, which located the frequencies from 34– 189 Hz. Synergy3 was comprised of oscillations with much lower frequency, IMF6 and IMF7, and the corresponding frequency range was 23–69 Hz. Furthermore, as listed in Table 2, the Pearson’s correlation coefficient of synergy 1 was more than 0.883, which indicated that the composition of IMF1∼IMF8 exhibited high structural similarity for any paired muscles. The composition of IMF1∼IMF8 in synergy 2 or synergy 3 showed high similarity for the BB and TB of CP and/or TD group as well, and corresponding correlation coefficients were more than 0.816 and 0.861, respectively. Altogether, there were three stable oscillation synergies of sEMG for BB and TB both in CP infants or TD infants crawling, and each synergy can be characterized with sEMG oscillation composition (i.e., IMF) of specific frequencies. (3) In this study, the matrix of V m×n represents the envelopes of m IMFs (m = 8), and n is the sample number (n = 200). Each column of W m×s represents an oscillation synergy with m weight factors (1 ≤ s ≤ m) , and s is the number of synergies. Each row of Cs×n represents corresponding recruitment coefficients, which shows how each synergy is modulated over time. To maintain the modulation of information within oscillation synergies, C was normalized to its maximum values of the crawling cycle. The number of oscillation synergies was determined by calculating the variation accounted for (VAF) between the ′ original matrix V and the reconstruction matrix V = WC for each s value (from 1 to 8) (40). VAF is calculated as follow ′ (V−V ) VAF = 1− V2 2 (4) Here, the selection criteria were that the mean of VAFs was larger than 95% and the increment of VAF was <1%. Statistical Analysis The recruitment coefficient values for each oscillation synergy were averaged over 3–4 valid cycles for each subject. For each TD infant or CP infant whose both side limbs were affected, as the symmetric movement for left and right side, the oscillation synergy and recruitment coefficient of sEMG signals recorded from right and left BB and TB muscles have been averaged firstly. In order to compare the recruitment pattern of each oscillation synergy, independent sample t-test was applied on the recruitment coefficients for each phase. The significance level was set to 0.05. All the data were analyzed in SPSS 22.0 statistical software (SPSS Inc., USA). In addition, Pearson’s correlation coefficient (r) between any two oscillation synergies with the same order number was calculated to assess the similarities of oscillation synergy structures. Four kinds of r values were calculated between same within-groups muscles (e.g., BB of TD1 vs. BB of TD2), between different within-groups muscles (e.g., BB of TD1 vs. TB of TD2), between same between-groups muscles (e.g., BB of TD1 vs. BB of CP1) and between different betweengroups muscles (e.g., BB of TD1 vs. TB of CP1). Dynamic Recruitment of Oscillation Synergies for Crawling Movement The recruitment coefficient curves of sEMG oscillation synergies are illustrated in Figure 7. During a crawling cycle, each of those three oscillation synergies was dynamically recruited with time-varied coefficient curves. It can be observed, either for TD or CP infants, that they recruited the synergy 1 in BB with a low level firstly, then the recruitment reached a peak level in the second half crawling phase. Whereas the recruitment strength of synergy 2 and synergy 3 reached a peak quickly at the beginning of crawling cycle (i.e., FSP) and decreased then. For the TB, both TD and CP infants almost synchronously recruited those three synergies, and their activation level reached a peak in the middle phase of crawling (i.e., BSP and BRP). However, the motor dysfunction of CP affected the recruitment of oscillation synergies for crawling movement both in BB and TB. RESULTS The Impact of CP on the Recruitment Pattern of Oscillation Synergies All of the recruited infants in this study can crawl continuously on hands and knees more than 4 valid cycles (TD, 4.9 ± 0.9 cycles; CP, 5.9 ± 1.4 cycles). Their hands-and-knees crawling scores were at least 44 (TD, 50.9 ± 3.3; CP, 63.7 ± 8.5) according to GMFM. Although the crawling motor function scores of CP infants equal or even exceeded TD infants (in this study) after rehabilitation training, their motor development was indeed slower than that of TD infants at the same age. CP infants exhibited abnormal movement behavior and sEMG activities. Frontiers in Neurology | www.frontiersin.org To quantitatively evaluate the impact of CP on the recruitment pattern of oscillation synergies, the amplitude of recruitment coefficient curves was compared in the refined crawling phases of FSP, BSP, BRP, and PRP. As shown in Figure 8, CP recruited significant less synergy3 or low-frequency sEMG oscillations in BB for slow contraction in FSP crawling (Figure 8C, p = 0.023), and less synergy 1 or high-frequency sEMG oscillations in TB for the fast contraction in PRP crawling 6 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations FIGURE 5 | Mean VAF values corresponding to different number of oscillation synergies. FIGURE 6 | Oscillation synergies extracted from 8 sEMG IMFs of BB and TB during one crawling cycle in two groups. (A) BB of TD group; (B) BB of CP group; (C) TB of TD group; (D) TB of CP group. DISCUSSION (Figure 8D, p = 0.007). During the BSP (p = 0.007) and BRP crawling (p = 0.006), CP maintained a high level of synergy3 in BB, which revealed that the sEMG oscillations with low-frequencies cannot be de-recruited effectively (Figure 8C). Furthermore, during CP crawling in FSP (p = 0.002) and BRP (p = 0.047), there was a significantly higher activation level for synergy 2 in BB (Figure 8B), whereas less synergy 2 was recruited in TB for BSP crawling (Figure 8E, p = 0.029). Frontiers in Neurology | www.frontiersin.org To deeply explore the effect of neuromuscular damage of CP on the motor regulation, this study aimed to extract and evaluate the oscillation synergy patterns from sEMG signals with MEMD and NMF during infant crawling. The present work showed that sEMG signals contained stable structures of oscillation synergy, and the motor dysfunction of CP affected the recruitment of oscillation synergies for crawling movement both in BB and TB. 7 September 2018 | Volume 9 | Article 760 0.868 ± 0.018 0.869 ± 0.011 0.861 ± 0.021 0.929 ± 0.010 0.938 ± 0.005 0.893 ± 0.010 0.883 ± 0.061 Crawling requires multiple skeletal muscles to participate in the regulation of limb joint flexion and extension. Muscles play different roles and their contraction patterns also vary with crawling phases. The results showed that the activation pattern of muscles can be expressed as synergistic recruitment of multiscale sEMG oscillations. As shown in Figure 6, the activation of BB or TB muscles during crawling can be represented by the synergistic combination of the oscillation subsets with different frequency ranges, i.e., high-frequency range (synergy1), medium-frequency range (synergy2), and low-frequency range (synergy3). Moreover, as shown in Figure 7, these synergistic sEMG oscillations have been dynamically organized for infant crawling movement. Both for BB and TB, the activation level of high-frequency synergy (C1) keeps to a way of progressively developing pattern, whereas the activation level of mediumand low-frequency synergy (C2 and C3) reach a peak and then descend gradually. These results follow the principle that the recruitment of MUs has been typically graded (41) from slow to fast during dynamic contractions (42) for joint movement, and types of recruited MUs can be manifested as sEMG frequency components (17, 18). In context, the observations in our study suggest that, during the swing and stance of infant crawling, the organization of MUs activation pattern in BB and TB can be represented as synergistic sEMG oscillations, which have been dynamically regulated for different contraction status and crawling phase. Although there is some time delay for BB and TB contraction due to their different role in crawling movement, the coefficient curve for sEMG oscillations with high-, medium-, and low-frequency are alternatively enhanced to a complementary pattern among these synergies, which reveal that different types of MUs have been coordinately recruited for limb movement in crawling. Crawling is a periodic movement of flexion-extension, in which the upper limbs postures alternate with FSP, BSP, BRP, and PRP successively. Meanwhile, BB and TB exhibit alternating coordinate relaxation and contraction. During swing, BB gradually contracts to achieve forward swing of upper limbs in FSP; slow MUs are activated, and the recruitment strength of synergy 2 and synergy 3 with lower-frequency range reach a peak. However, TB presents some time delay in crawling movement and kept extension/relaxation in FSP, and then begin contraction to swing arm backward with fast developing of high-, medium-, and low-frequency oscillations, and medium- and lowfrequency oscillations reached their peak activations. To perform backward swing, BB extends in BSP as slow MUs are de-recruited, and the activation of high-frequency oscillations exceeded the medium- and low-frequency ones. During stance, both BB and TB are activated to response loading period, and fast MUs are dominantly recruited accordingly to absorb the strike shock and quickly stabilize the kinematic behavior of the joint. As shown in Figure 7, the coefficient curve of synergy 1 (C1) maintains a high level, whereas medium- and low-frequency oscillations (C2 and C3) decrease gradually. In other words, more fast MUs for high-frequency oscillations are activated to compensate the 0.816 ± 0.053 0.822 ± 0.021 0.961 ± 0.004 0.848 ± 0.038 0.932 ± 0.010 0.860 ± 0.022 0.897 ± 0.012 0.894 ± 0.015 0.863 ± 0.011 0.927 ± 0.006 0.892 ± 0.011 Synergistic Recruitment of sEMG Oscillations for Crawling Movement 0.883 ± 0.014 TB BB CP Frontiers in Neurology | www.frontiersin.org Data were expressed as mean ± standard error. 0.931 ± 0.017 0.910 ± 0.013 0.929 ± 0.006 0.921 ± 0.015 0.890 ± 0.012 0.907 ± 0.014 0.945 ± 0.003 0.912 ± 0.015 BB TB BB TB BB TB BB 0.896 ± 0.013 TB TB BB TD CP TD CP TD 0.968 ± 0.003 TB BB TD BB CP Synergy3 Synergy2 Synergy1 TABLE 2 | Correlation coefficients between oscillation synergies. 0.877 ± 0.011 Synergistic Recruitment of sEMG Oscillations TB Gao et al. 8 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations FIGURE 7 | Comparison of recruitment coefficient curves for three sEMG oscillation synergies and their performance in every refined phase for TD and CP group during one crawling cycle. (A) BB of TD group; (B) BB of CP group; (C) TB of TD group; (D) TB of CP group. Red (C1), green (C2), and blue (C3) lines represent the recruitment coefficient curves of synergy1, synergy 2, and synergy 3, respectively. FIGURE 8 | The mean recruitment coefficient of three oscillation synergies from BB and TB in two groups. (A) synergy 1 of BB; (B) synergy 2 of BB; (C) synergy 3 of BB; (D) synergy 1 of TB; (E) synergy 2 of TB; (F) synergy 3 of TB. *0.01 < p < 0.05, **0.005 < p < 0.01, ***p < 0.005. Frontiers in Neurology | www.frontiersin.org 9 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations de-recruited slow MUs in stance phase of crawling, which is a mechanism for CNS timely and moderately regulating the recruitment of different types of MUs in muscles for joint movement (42). MUs; another observation showed that spastic diplegic CP loss the ability to fully recruit and optimally activate available motor units because of their central activation failure (39). So, our work reveal that, the synergistic recruitment of MUs can be characterized with the coordinated activation of different types of MU with appropriate proportion, and CP cannot effectively regulate the activation intensity among different MUs. Abnormal Recruitment Pattern of Oscillation Synergies Under Motor Dysfunction of CP Motor dysfunction of cerebral palsy originates from brain injury leading to a series of neurological changes, such as reduced input to the CNS (43) and decreased activity of descending inhibitory system (44). Subsequently, motor dysfunction of CP is also manifested as abnormal myoelectric activities and abnormal movement postures due to the affected cerebral nerve in motor area (2). As illustrated in Figures 6, 7, although three types of synergistic sEMG oscillations with different frequency band are presented during CP infants crawling, there are some changes in recruitment coefficient curves, which suggest that brain injury of cerebral palsy affected the MUs recruitment. Generally, CNS employs a synergistic strategy to organize the multi-element motor system within different scales to simplify motor control (45), and the combination of motor elements with certain weights constructs motor synergy. However, motor dysfunction of CP mainly alters the organization of medium- and low-frequency oscillations of sEMG, especially the intensity proportion between medium- and low-frequency oscillations. Compared with TD, CP infant recruits less low-frequency oscillation (synergy3) during FSP crawling, whereas TB activates less medium-frequency oscillation (synergy2) during BSP crawling. It was reported that the impaired CNS of CP is unable to effectively drive lowthreshold MUs (46–48), our results reveal that the inadequate recruitment of low-threshold MUs induced insufficient activation of low- and medium-frequency oscillations when BB and TB of CP infants perform flexion contraction in FSP and BSP crawling, respectively. In addition to the aforementioned insufficient activation of low- or medium-frequency sEMG oscillations, it also can be observed that motor dysfunction of CP is unable to de-recruit those undesired sEMG oscillations. As shown in Figures 7, 8, for the BB of CP infants, the coefficient curves of synergy3 (C3) and synergy2 (C2) hold on an abnormal level in BRP crawling, and coefficient curve of synergy3 (C3) also keep on an abnormal level in BSP crawling. These results suggest that, during BRP and BSP, CP infants are unable to inhibit their low-frequency and/or medium-frequency sEMG oscillations in the BB muscles. It is accepted that different MU owns different intrinsic contraction properties, and slow MUs are activated for slow contraction and low-intensity activities, while fast MUs are recruited for fast contraction and high-intensity activities (49, 50). Central nervous system selectively recruits desired MUs and de-recruit undesired MUs to produce synergistic contraction pattern, and organizes different types of MUs at a certain activation level can improve the synergistic control for muscular activities and joint movement. For CP infant, motor dysfunction of injured motor nerve or declined activity of descending inhibitory system (31) cause an insufficient organization of different types of Frontiers in Neurology | www.frontiersin.org Synergistic Organization of Muscle Functional Units Exhibited by Oscillation Synergy Patterns The regulation of motor function by the CNS is a synergistic organization of multiple function units, which has been widely confirmed in muscle groups. Variety of methods have been used to evaluate the intensity, spectrum or frequency component of sEMG activity. However, it is still difficult to analyze the organization patterns of different types of MUs. Multiscale oscillation modes in sEMG signals are a combination of MU activation and organization patterns. The results of this study demonstrated that the synergistic organization of different oscillations in sEMG signals can be used to evaluate the organization of MUs. In fact, sEMG frequency components are related to physiological state and have been applied to assess motor function under different conditions (13, 14, 23). However, these studies only provided an overall effect of frequency components in a certain period of time. We decomposed sEMG signals of individual muscles into multiscale oscillations, which is correlated to MU recruitment pattern, and then, utilized NMF to extract oscillation synergies and their recruitment coefficient curves. The time-varying curves represent how CNS regulate oscillation synergies over time, which provided a novel insight to better understand the dynamic process of the CNS organizing different types of MUs. The results of this study showed that three stable multiscale oscillation synergies might be underlying intrinsic structure of EMG activity during crawling. The coefficient curves can further reveal the abnormal MU activation patterns during crawling in CP. Some researchers have found that motor dysfunction of CP manifested as abnormal entropy of multi-scale oscillations of sEMG activities in CP, and a major peripheral cause of these abnormalities is abnormal MU recruitment (10, 16). This study revealed that motor dysfunction of CP also manifested as abnormal recruitment of oscillation synergies. For CP, the motor dysfunction is caused by original brain injury and resulted abnormal descending motor commands. It can be observed that, in CP group, the oscillation synergies with low-frequency range (synergy3) were insufficiently recruited when BB muscles performed slow contraction during swing, on other hand, they insufficiently de-recruited the oscillation synergies with low-frequency range (synergy2 and 3) when BB extended and relaxed in BSP and BRP. These results suggested the recruitment coefficient curves of oscillation synergies can exhibit the time-varying organization of sEMG oscillations modulated by CNS, and the curves also can 10 September 2018 | Volume 9 | Article 760 Gao et al. Synergistic Recruitment of sEMG Oscillations reveal abnormal motor control caused by neuromuscular deficits. in the department of rehabilitation center, children’s hospital of Chongqing medical university. CONCLUSION AUTHOR CONTRIBUTIONS This is the first time to adopt the synergistic mechanism of motor function to characterize the recruitment pattern of sEMG oscillations. The present results reveal that, the myoelectric activities of a muscle can be manifested as sEMG oscillation synergies with different frequency ranges, and motor dysfunction of CP degrade the synergistic recruitment of sEMG oscillations due to the impaired CNS regulation and destroyed MU/muscle fiber. Our preliminary work suggests that, the synergistic organization for motor control can be manifested rather than muscle group, there are oscillation synergies in sEMG signals. Also, the time-varying coefficient curve of sEMG oscillation synergies is a potential index to evaluate the abnormal recruitment of different types of MUs affected by CP disorders. In future work, we would further investigate the sEMG oscillation synergies in other muscles of upper and lower limbs, and the body size of test sample should be enlarged to verify the influence of CP on the synergistic recruitment of sEMG oscillations. ZG analyzed the data and signals. ZG, LC, and WH drafted and revised the work. XW, NX, WJ, and WH interpreted the data. ZG, QX, and YL collected the data. XW and WH proposed the research topic. WH organized the data collection, signal processing, and manuscript draft. FUNDING This work was supported by the National Natural Science Foundation of China (NSFC 31470953, 31771069, 31800824), the Chongqing Science & Technology Program (cstc2016shmszx130060, cstc2015jcyjB0538), and the Fundamental Research Funds for the Central Universities (No. 106112016CDJXZ238826). ACKNOWLEDGMENTS We much appreciated the infants and their parents for participating in this study. We would like to thank the department of rehabilitation center, children’s hospital of Chongqing medical university for recruiting infants and their help in data collection. ETHICS STATEMENT This study is under the approval of children’s hospital of Chongqing medical university, and all the tests are conducted REFERENCES 9. Wakeling J, Delaney R, Dudkiewicz I. A method for quantifying dynamic muscle dysfunction in children and young adults with cerebral palsy. Gait Posture (2007) 25:580–9. doi: 10.1016/j.gaitpost.2006. 06.009 10. Hong T, Zhang X, Ma H, Chen Y, Chen X. 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(2016) 26:36–43. doi: 10.1016/j.jelekin.2015.12.001 31. Tang L, Chen X, Cao S, Wu D, Zhao G, Zhang X. Assessment of upper limb motor dysfunction for children with cerebral palsy based on muscle synergy analysis. Front Hum Neurosci. (2017) 11:130. doi: 10.3389/fnhum.2017.00130 32. Huang C, Chen X, Cao S, Zhang X. Muscle-tendon units localization and activation level analysis based on high-density surface EMG array and NMF algorithm. J Neural Eng. (2016) 13:066001. doi: 10.1088/1741-2560/13/6/066001 33. Patrick SK, Noah JA, Yang JF. Developmental constraints of quadrupedal coordination across crawling styles in human infants. J Neurophysiol. (2012) 107:3050–61. doi: 10.1152/jn.00029.2012 34. Huang NE, Shen Z, Long SR, Wu MC, Shih HH, Zheng Q, et al. Huang, N.E., et al.: The empirical mode decomposition and the hilbert spectrum for nonlinear and non-stationary time series analysis. Proc. R. Soc. Lond A (1998) 454:903–95. 35. Rehman N, Mandic DP. Multivariate empirical mode decomposition. Proc Mathemat Phys Eng Sci. (2010) 466:1291–302. doi: 10.1098/rspa.2009.0502 Frontiers in Neurology | www.frontiersin.org Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2018 Gao, Chen, Xiong, Xiao, Jiang, Liu, Wu and Hou. 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. 12 September 2018 | Volume 9 | Article 760
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PENGEMBANGAN BAHAN AJAR FISIKA SMA BERBASIS INVESTIGASI PADA MATERI FLUIDA DINAMIS UNTUK MENINGKATKAN HASIL BELAJAR SISWA
Jurnal Pendidikan Fisika
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Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Físika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Físika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. PENGEMBANGAN BAHAN AJAR FISIKA SMA BERBASIS INVESTIGASI PADA MATERI FLUIDA DINAMIS UNTUK MENINGKATKAN HASIL BELAJAR SISWA Rizki Maulida dan Mariati Purnama Simanjuntak Prodi Pendidikan Fisikan Pascasarjana Universitas Negeri Medan maulida_qq@yahoo.com Rizki Maulida dan Mariati Purnama Simanjuntak Prodi Pendidikan Fisikan Pascasarjana Universitas Negeri Medan maulida_qq@yahoo.com Abstrak. Penelitian ini bertujuan untuk menganalisis apakah bahan ajar yang dikembangkan dapat meningkatkan respon, aktivitas dan hasil belajar siswa dengan menggunakan pengembangan bahan ajar SMA berbasis investigasi pada materi fluida dinamis berbasis investigasi. Metode yang dilakukan dalam penelitian ini menggunakan penelitian pengembangan Research and Development (R&D). Sampel penelitian ini adalah satu kelas yaitu kelas XI IPA2. Instrumen yang digunakan untuk mengumulkan data ada tiga yaitu dengan menggunakan lembar validasi bahan ajar berupa angket dan kemudian di validkan oleh tim ahli, untuk menilai respon siswa pada bahan ajar instrumen yang digunakan berupa angket yang dibagikan pada setiap pertemuan dan untuk melihat aktivitas siswa menggunakan angket yang dinilai oleh teman sejawat peneliti dalam mengukur aktivitas siswa pada setiap pertemuan serta untuk melihat hasil belajar siswa digunakan penilaian evaluasi berupa 10 pertanyaan dalam bentuk pilihan berganda yang diberikan pada setiap akhir pertemuan. Hasil penelitian diperoleh: Bahan ajar fisika SMA berbasis investigasi yang dikembangkan dapat meningkatakan respon, aktivitas dan hasil belajar siswa pada pertemuan I, II, III yaitu: respon rata-rata pada pertemuan pertama sebesar 76,42. pada pertemuan kedua sebesar 79,77 dan pada pertemuan ketiga 87,36. Aktivitas rata-rata pada pertemuan pertama sebesar 67,01. pada pertemuan kedua sebesar 71,00 dan pada pertemuan ketiga 79,00. Hasil belajar rata-rata pada pertemuan pertama sebesar 40,36. pada pertemuan kedua sebesar 60,71 dan pada pertemuan ketiga 81,07. Kata kunci: hahan ajar, investigasi, hasil belajar Keywords: material teaching, investigation, learning outcomes Keywords: material teaching, investigation, learning outcomes Majid (2009) sumber belajar yang diorganisir melalui suatu rancangan yang dimafaatkan sebagai sumber ajar dapat bermanfaat bagi seorang guru mapun peserta didiknya.Bahan ajar yang sesuai dengan model pembelajaran menjadi hal penting agar pembelajaran dapat bermanfaat dan mencapai tujuannya. Menurut (Wena, 2009) pembelajaran kooperatif adalah pembelajaran kelompok yang memiliki aturan tertentu dan prinsip dasar pada pembelajaran kooperatif ini adalah pembentukan kelompok kecil yang saling mengajar untuk mencapai tujuan bersama. Majid (2009) sumber belajar yang diorganisir melalui suatu rancangan yang dimafaatkan sebagai sumber ajar dapat bermanfaat bagi seorang guru mapun peserta didiknya.Bahan ajar yang sesuai dengan model pembelajaran menjadi hal penting agar pembelajaran dapat bermanfaat dan mencapai tujuannya. Menurut (Wena, 2009) pembelajaran kooperatif adalah pembelajaran kelompok yang memiliki aturan tertentu dan prinsip dasar pada pembelajaran kooperatif ini adalah pembentukan kelompok kecil yang saling mengajar untuk mencapai tujuan bersama. Kata kunci: hahan ajar, investigasi, hasil belajar DEVELOPMENT OF TEACHING MATERIALS PHYSICS BASED INVESTIGATION ON SMA FLUID DYNAMIC MATERIALS TO IMPROVE STUDENT LEARNING OUTCOMES Rizki Maulida dan Mariati Purnama Simanjuntak Physics Education Program, Graduate State University of Medan maulida_qq@yahoo.com Based on the research result obtained: The physics teaching materials of senior high school based on investigation which was developed can improve the response, activity, and learning outcomes of students in meeting I, II, III. The mean response in meeting I was 76.42, 79.77 in meeting II, and 87.36 in meeting III. The mean activity in meeting I was 67.01, 71.00 in meeting II, and 79.00 in meeting III. The mean of learning outcomes in meeting I was 40.36, 60.71 in meeting II, and 81.07 in meeting III. http://jurnal.unimed.ac.id/2012/index.php/jpf Kata kunci: hahan ajar, investigasi, hasil belajar DEVELOPMENT OF TEACHING MATERIALS PHYSICS BASED INVESTIGATION ON SMA FLUID DYNAMIC MATERIALS TO IMPROVE STUDENT LEARNING OUTCOMES Rizki Maulida dan Mariati Purnama Simanjuntak Physics Education Program, Graduate State University of Medan maulida_qq@yahoo.com Rizki Maulida dan Mariati Purnama Simanjuntak Physics Education Program, Graduate State University of Medan maulida_qq@yahoo.com Abstract. The purpose of this study was to analyze whether the teaching materials developed can improve responsiveness, activities, and student learning outcomeusing SMA development of teaching materials based on the material fluid dynamic investigation. The research method was Research and Development (R&D) research. The research sample was randomly Vol. 4 No. 1 Juni 2015 http://jurnal.unimed.ac.id/2012/index.php/jpf 71 Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Físika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 selected by gamble the existing seven classes. The used instrument were three namely using the validation sheet of teaching materials in questioner type and then validated by the expert team, to assess the students’ response in teaching materials so the used instrument was questioner given in each meeting and to find out the students’ activity used questioner assessed by researcher peer in measuring the students’ activity in each meeting and also to find out the students’ learning outcomes used evaluation assessment in ten multiple choice problems given in the end of each meeting. Based on the research result obtained: The physics teaching materials of senior high school based on investigation which was developed can improve the response, activity, and learning outcomes of students in meeting I, II, III. The mean response in meeting I was 76.42, 79.77 in meeting II, and 87.36 in meeting III. The mean activity in meeting I was 67.01, 71.00 in meeting II, and 79.00 in meeting III. The mean of learning outcomes in meeting I was 40.36, 60.71 in meeting II, and 81.07 in meeting III. selected by gamble the existing seven classes. The used instrument were three namely using the validation sheet of teaching materials in questioner type and then validated by the expert team, to assess the students’ response in teaching materials so the used instrument was questioner given in each meeting and to find out the students’ activity used questioner assessed by researcher peer in measuring the students’ activity in each meeting and also to find out the students’ learning outcomes used evaluation assessment in ten multiple choice problems given in the end of each meeting. PENDAHULUAN Tuntutan pada saat ini terhadap kualitas manusia yang sangat besar, menjadi salah satu tanggung jawab bagi para pendidik. Guru merupakan tenaga pendidik yang ikut bertang- gung jawab atas ketercapaian terhadap kualitas manusia yang akan bersaing dimasa depan untuk mewujudkan kehidupan yang lebih baik (Sani, 2014). Metode saintifik pada umumnya melibatkan kegiatan pengamatan atau observasi yang dibutuhkan untuk perumusan hipotesis atau pengumpulan data. Metode saintifik ini yang membutuhkan kegiatan observasi dan pengamatan sebagai bahan untuk pengumpulan data merupakan metode yang mendukung dalam proses pembelajaran fisika. Penggunaan buku sebagai bahan ajar yang saat ini masih menjadi pegangan seorang guru dalam mengajar masih kurang sesuai dengan metode saintifik. Buku yang biasa digunakan oleh guru adalah buku dari penerbit yang mana isi dalam buku tersebut berupa materi dan penugasan dalam bentuk soal.buku yang digunakan guru dari penerbit masih belum selaras dengan model pembelajaran yang saintifik. Bahan ajar adalah seperangkat materi yang disusun secara sistematis tertulis baik maupun tidak sehingga tercipta lingkungan suasana yang memungkinkan siswa untuk belajar (Daryanto, 2014). Dalam pembelajaran di kelas seorang guru juga sebaiknya memiliki bahan ajar yang tepat. Bahan ajar juga menjadi salah satu faktor pendukung terwujudnya pembelajaran yang efisien. Bahan ajar yang sinergis dan berjalan sesuai dengan model pembelajaran yang akan dilakukan diharapkan mampu meningkatkan hasil belajar. Menurut Menurut wawancara yang dilakukan oleh siswa juga masih banyak yang memandang fisika adalah pembelajaran hitung yang rumit, sehingga berakibat pada persepsi siswa yang menganggap fisika adalah pembelajaran yang Vol. 4 No. 1 Juni 2015 72 http://jurnal.unimed.ac.id/2012/index.php/jpf Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Físika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Pengembangan bahan ajar berbasis inves- tigasi ini bertujuan untuk menganalisis pening- katkan respon siswa dan aktivitas belajar siswa serta hasil belajar siswa setelah menggunakan pengembangan bahan ajar berbasis investigasi pada materi fluida dinamis. Manfaat dari hasil penelitian ini dapat memberikan sumbangan pemikiran dalam bentuk bahan ajar yang dapat digunakan guru, sehingga siswa dapat mengem- bangkan aspek kemampuan dasar yang mencakup aspek kognitif siswa dan sebagai bahan informasi bagi peneliti selanjutnya dalam melakukan penelitian lebih lanjut sulit. Hal ini juga menjadi akibat dari penyajian pelajaran fisikan yang masih berpusat pada guru. Pelaksanaan pembelajaran yang terjadi disekolah yaitu guru dalam menyampaikan materi pada saat ini masih lebih dominan menggunakan model pembelajaran langsung, dan berupa penyampaian materi dengan diakhiri penugasan-penugasan. PENDAHULUAN Penggunaan bahan ajar dalam proses pembelajaran juga masih berpusat pada buku paket. Proses pembelajaran yang berpusat pada guru ini juga terjadi di sekolah MAN 1 Medan bahan ajar yang digunakan oleh guru sebagai pegangan menggunakan buku dari penerbit Platinum, dan dalam penerapannya dalam proses pembelajaran masih berupa menjelaskan materi dan membahas soal sebagai penugasan. sulit. Hal ini juga menjadi akibat dari penyajian pelajaran fisikan yang masih berpusat pada guru. Pelaksanaan pembelajaran yang terjadi disekolah yaitu guru dalam menyampaikan materi pada saat ini masih lebih dominan menggunakan model pembelajaran langsung, dan berupa penyampaian materi dengan diakhiri penugasan-penugasan. Penggunaan bahan ajar dalam proses pembelajaran juga masih berpusat pada buku paket. Proses pembelajaran yang berpusat pada guru ini juga terjadi di sekolah MAN 1 Medan bahan ajar yang digunakan oleh guru sebagai pegangan menggunakan buku dari penerbit Platinum, dan dalam penerapannya dalam proses pembelajaran masih berupa menjelaskan materi dan membahas soal sebagai penugasan. METODE PENELITIAN Penelitian ini akan dilaksanakan pada Semester II Kelas XI di MAN 1 Medan yang beralamat Jalan Willem Iskandar no. 7B. Populasi dalam penelitin ini adalah seluruh siswa kelas XI IPA semester 2 pada Tahun 2015 di MAN 1 Medan. Sampel yang ditetapkan dalam penelitian ini adalah satu kelas XIIPA2 dari semua kelas yang ada di tempat penelitian. Bahan ajar yang masih kurang sesuai dengan model pembelajaran yang digunakan ini juga menjadi faktor penentu proses pembela- jaran yang terjadi didalam kelas. didalam kelas Penggunaan bahan ajar yang masih berpusat pada guru ini membuat aktivitas siswa berkurang. Aktivitas siswa yang rendah juga mengakibat- kan kejenuhan siswa dan rendahnya semangat dalam belajar fisika yang mengakibatkan rendahnya hasil belajar kognitif siswa. Peng- gunaan bahan ajar yang menuntut siswa dalam memecahkan masalah melalui konsep, mate- matis dan prinsip fisika akan membuat siswa lebih aktif dan menuntut siswa untuk lebih mengaplikasikan pengetahuannya. Penggunaan bahan ajar dan model pembelajaran yang masih berpusat pada guru ini juga menjadi penyebab rendahnya minat belajr fisika pada siswa, minimnya minat belajar mengakibatkan transfer ilmu menjadi sulit diterima siswa hasil belajar siswa yang rendah menjadi gambaran mengenai kurang maksimal seorang siswa menerima pelajaran.Pada materi tertentu antara lain materi fluida dinamis pada umumnya hasil belajar siswa rendah, sedangkan pada materi tersebut seharusnya banyak hal yang dapat kita kembangkan berdasarkan kehidupan sehari- hari. Kebutuhan akan penggunaan bahan ajar yang sesuai dengan motode saintifik menjadi perhatian penting tersendiri. Penelitian ini dirancang sebagai penelitian pengembangan produk menggunakan metode pengembangan Research and Development (R&D). Metode Research and Development (R&D) ini digunakan untuk menghasilkan produk tertentu, dan menguji keefektifan model tersebut. HASIL PENELITIAN DAN PEMBAHASAN Penelitian ini bertujuan untuk melihat analisis peningkatan respon belajar siswa dan menganalisis aktivitas belajar siswa serta menganalisis hasil belajar siswa. Deskripsi data hasil penelitian ini terdiri dari skor hasil validitas (SV) tim ahli yaitu berupa materi dan media bahan ajar yang dikembangkan, hasil respon siswa (HR) terhadap pengembangan bahan ajar berbasis investigasi, hasil aktivitas (HA) belajar siswa pada saat pembelajaran dengan menggunakan pengembangan bahan ajar berbasis investigasi, dan skor hasil belajar (HB) siswa dengan menggunakan pegembangan bahan ajar berbasis investigasi. Pada tahap pertama pengembangan bahan ajar berbasis Vol. 4 No. 1 Juni 2015 http://jurnal.unimed.ac.id/2012/index.php/jpf 73 Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Físika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. investigasi mendapatkan skor hasil validasi oleh tim ahli tertera pada tabel 1. membuat daftar pustaka. (7) memperjelas bahasa, tulisan dan kosa kata yang digunakan pada bahan ajar. Tabel 1. Hasil Validasi Materi Oleh Tim Ahli No Aspek Persentase 1 Kelayakan Isi 83,85% 2 Kelayakan Penyajian 81,41% 3 Kontekstual 80,56% 4 Kesesuaian dengan Investigasi 86,90% Setelah mendapatkan hasil validasi oleh tim ahli kemudian bahan ajar dilakukan revisi sesuai saran dari tim ahli. Bahan ajar yang sudah dilakukan revisi kemudian dilakukan uji coba terbatas kepada satu kelas yang dipilih pada tempat penelitian. Hasil respon siswa terhadap bahan ajar diperoleh hasil yang tertera pada tabel 3. Adapun aspek penilaian validasi yang dilakukan antara lain: Hasil validasi bahan ajar oleh bahan ajar terdapat empat aspek anataralain aspek kela- yakan dengan jumlah 83,85% dengan kategori valid, aspek kelayakan penyajian dengan jumlah 81,41 dengan kategori valid, aspek kontekstual dengan jumlah 80,56 dengan kategori valid dan aspek kesesuaian dengan investigasi dengan jumlah 86,90% dengan kate- gori sangat valid. 1. Materi Modul: Keluasan Materi, Contoh aplikasi, Latihan-latihan, Bahan Pratikum, Uji Kompetensi 2. Format Modul: Pengetikan, Kalimat, Bahasa yang digunakan, Tampilan bahan ajar 2. Format Modul: Pengetikan, Kalimat, Bahasa yang digunakan, Tampilan bahan ajar Tabel 3. Hasil Respon Siswa Terhadap Bahan Ajar No Pertemuan Persentasi 1 Pertemuan I 77,18% 2 Pertemuan II 81,16% 3 Pertemuan III 88,39% Selain dari validasi pada materi pada bahan ajar juga dilakukan validasi terhadap desain pada bahan ajar. Pada validasi terhadap bahan ajar ini dilakukan oleh tim ahli. Hasil validasi tim ahli terhadap desain bahan ajat tertera pada tabel 2. Hasil respon siswa pada pertemuan I adalah 77,18%. Pada pertemuan UU 81,16 dan pada pertemuan III 88,39%. HASIL PENELITIAN DAN PEMBAHASAN Dari hasil respon yang diperoleh pada setiap pertemuan dapat dilihat bahwa terdapat peningkatan respon siswa yang signifikan pada setiap pertemuan. Hasil dari respon siswa tersebut menjadi dasar untuk melakukan revisi bahan ajar yang sudah digunakan. Untuk lebih jelas peningkatan respon siswa dapat dilihat pada gambar 1. Tabel 2. Hasil Validasi Bahan Ajar Pada j Desain Bahan Ajar Oleh Tim Ahli No Aspek Persentase 1 Ukuran Modul 70,83% 2 Keakuratan Materi 79,17% 3 Desain Isi Modul 88,67% Desain Bahan Ajar Oleh Tim Ahli Hasil validasi oleh tim ahli terhadap bahan ajar terdiri dari tiga aspek yaitu ukuran modul dengan jumlah 70,83% dengan kategori cukup valid, keakuratan materi 79,1 dengan kategori valid dan desain isi modul dengan jumlah 88,68 dengan kategori sangat valid. Gambar 1. Persentase Peningkatan Respon Siswa Pada Setiap Pertemuan http://jurnal.unimed.ac.id/2012/index.php/jpf Terdapat beberapa perbaikan yang dilakukan berdasarkan saran para ahli antara lain: (1) keluasan materi, yaitu menambahkan beberapa aplikasi azas bernoulli pada kehidupan sehari-hari. (2) mengganti rangkaian pipa venturi pada percobaan pipa venturi. (3) memperjelas ganbar dan ilustrasi yang terdapat pada bahan ajar. (4) memperjelas peta konsep. (5) membuat soal dan contoh soal pada setiap submateri. (6) Gambar 1. Persentase Peningkatan Respon Siswa Pada Setiap Pertemuan Vol. 4 No. 1 Juni 2015 http://jurnal.unimed.ac.id/2012/index.php/jpf 74 Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Fisika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 Penilaian hasil belajar dilakukan dengan melakukan evaluasi pada setiap akhir proses pembelajaran. Terdapat tiga hasil evaluasi yang diperoleh pada penelitian ini. Secara ringkas hasil belajar siswa dijelaskan pada tabel 5. Pada hasil respon siswa terdapat beberapa bagian yang dilakukan perbaikan berdasarkan saran dari siswa, antara lain: (1) mengganti contoh soal agar sesuai seperti peristiwa kehi- dupan sehari-hari. (2) menambah pertanyaan agar mendorong rasa ingin tahu. (3) menambah kata-kata motivasi pada bahan ajar. (4) memperjelas kalimat yang terdapat pada bahan ajar. (5) merubah tampilan bahan ajar menjadi lebih menarik Tabel 5. Hasil belajar siswa No Pertemuan Rata-rata 1 Pertemuan I 40,36 2 Pertemuan II 60,71 3 Pertemuan III 81,07 Pada penelitian ini selain respon siswa terhadap bahan ajar, juga dilihat aktivitas dan hasil belajar siswa. Secara ringkas hasil aktivitas belajar siswa dengan menggunakan pengembangan bahan ajar berbasis investigasi terlihat pada tabel 4 Hasil belajar siswa pada penelitian ini terlihat bahwa pada pertemuan I rerata hasil belajar siswa adalah 40,36. HASIL PENELITIAN DAN PEMBAHASAN Selanjutkan pada pertemuan II hasil belajar siswa adalah 60,71 serta pada pertemuan III hasil belajar siswa adalah 81,07. Penjelasan pada tabel 5 terlihat jelas bahwa terdapat peningkatan hasil belajar siswa yang signifikan antara hasil belajar siswa pada pertemuan I, II dan III. aktivitas belajar siswa dengan menggunakan pengembangan bahan ajar berbasis investigasi terlihat pada tabel 4 Tabel 4. Hasil aktivitas siswa dengan menggunakan pengembangan bahan ajar berbasis investigasi No Aspek Penilaian Pertem uan I Pertemu an II Pertemu an III 1 Visual 77,3% 80,9% 85,7% 2 Oral 76,2% 77,3% 82,1% 3 Listening 76,2% 80,9% 82,1% 4 Writing 78,5% 82,1% 83,3% 5 Motor 78,5% 79,7% 83,3% 6 Mental 64,2% 73,8% 79,9% 7 Emotional 77,3% 79,7% 80,9% Rata-rata 76,19% 79,25% 85,71% Rerata hasil aktivitas belajar siswa pada pertemuan I, II dan III terlihat pada gambar 2. Gambar 2. Persentase nilai aktivitas belajar siswa pada setiap pertemuan Tabel 4. Hasil aktivitas siswa dengan menggunakan pengembangan bahan ajar berbasis investigasi berbasis investigasi No Aspek Penilaian Pertem uan I Pertemu an II Pertemu an III 1 Visual 77,3% 80,9% 85,7% 2 Oral 76,2% 77,3% 82,1% 3 Listening 76,2% 80,9% 82,1% 4 Writing 78,5% 82,1% 83,3% 5 Motor 78,5% 79,7% 83,3% 6 Mental 64,2% 73,8% 79,9% 7 Emotional 77,3% 79,7% 80,9% Rata-rata 76,19% 79,25% 85,71% Rerata hasil aktivitas belajar siswa pada Penelitian tentang pengembangan ini juga pernah dilakukan oleh beberapa peneliti sebelumnya yaitu Parmin (2012) hasil dari penelitiannya adalah 72% siswa menyatakan modul sangat diperlukan dan 68% mahasiswa yang dijadikan sasaran mendapatkan nilai A dan Budiyasa (2013) hasil penelitiannya adalah bahan ajar online efektif meningkatkan prestasi belajar dalam pembelajaran IPA tingkat SMA kelas delapan semester dua. Astawan (2013) hasil penelitiannya adalah tes hasil belajar siswa sebelum menggunakan modul dan sesudah menggunakan modul dengan menggu- nakan modul meningkat. Satria (2014) hasil penelitiannya adalah siswa mengalami kenaikan nilai sebesar 32,30% setelah menggunakan bahan ajar yang telah dikembangkan. p , p g Gambar 2. Persentase nilai aktivitas belajar siswa pada setiap pertemuan Gambar 2. Persentase nilai aktivitas belajar siswa pada setiap pertemuan KESIMPULAN Berdasarkan hasil analisis data temuan dan pembahasan selama pembelajaran dengan menggunakan pengembangan bahan ajar berbasis investigasi diperoleh beberapa kesimpulan yang berupa jawaban dari pertanyaan-pertanyaan yang diajukan dalam rumusan masalah. Kesimpulan- kesimpulan tersebut sebagai berikut (1) Pembe- Gambar 2. Persentase nilai aktivitas belajar siswa pada setiap pertemuan Vol. 4 No. 1 Juni 2015 Vol. 4 No. 1 Juni 2015 Vol. 4 No. 1 Juni 2015 http://jurnal.unimed.ac.id/2012/index.php/jpf 75 Maulida, R. dan Simanjuntak, M.P.: Pengembangan Bahan Ajar Fisika SMA Berbasis Investigasi Pada Materi Fluida Dinamis Untuk Meningkatkan Hasil Relajar Siswa. Jurnal Pendidikan Fisika p-ISSN 2252-732X e-ISSN 2301-7651 basis yang berbeda pada pengembangan bahan ajar yang akan dilakukan basis yang berbeda pada pengembangan bahan ajar yang akan dilakukan basis yang berbeda pada pengembangan bahan ajar yang akan dilakukan lajaran dengan menggunakan pengembangan bahan ajar berbasis investigasi terdapat pening- katakan respon siswa pada pertemuan I, II, III yaitu respon rata-rata pada pertemuan pertama sebesar 76,42, pada pertemuan kedua sebesar 79,77, dan pada pertemuan ketiga 87,36%. (2) Pembelajaran dengan menggunakan pengem- bangan bahan ajar berbasis investigasi terdapat peningkatakan aktivitas siswa pada pertemuan I, II, III yaitu aktivitas rata-rata pada pertemuan pertama sebesar 67,01%, pada pertemuan kedua sebesar 71,00%, dan pada pertemuan ketiga 79,00%. (3) Pembelajaran dengan menggunakan pengembangan bahan ajar berbasis investigasi terdapat peningkatakan hasil belajar siswa pada pertemuan I, II, III yaitu aktivitas rata-rata pada pertemuan pertama sebesar 40,36, pada pertemuan kedua sebesar 60,71, dan pada pertemuan ketiga 81,07. DAFTAR PUSTAKA DAFTAR PUSTAKA Astawan. 2013. Pengembangan Modul Berbasis Model Pembelajaran Kooperatif Tipe Group Investigation Pada Mata Pelajaran Server Jaringan Di Smk Ti Bali Global Sarjana. E-Journal Program Pasca- sarjana Universitas Pendidikan Ganesha. Vol 3-2013. Budiyasa, I.M. 2013. Pengembangan bahan ajarassessment alternatif online mata pelajaran IPA tingkat SMP kelas Delapan dengan model Dick and Carey. E-Journal Program Pascasarjana Universitas Pendidikan Ganesha. Vol 3-2013 Daryanto. 2014. Pengembangan perangkat Pembelajaran. Yogyakarta: Gava Media. p g Berdasarkan hasil pembahasan dan simpulan, saran yang dapat dikemukakan adalah (1) Bagi guru yang ingin menerapkan pengembangan bahan ajar berbasis investigasi dalam kegiatan belajar mengajar hendaknya melakukan pembagian kelompok dengan kombi- nasi kemampuan siswa yang bervariasi untuk membantu mengatasi terbatasnya ketersediaan waktu dalam pembimbingan pada fase-fase praktik. (2) Bagi guru yang ingin menerapkan pengembangan bahan ajar berbasis investigasi sebaiknya melakukan analisis dan kesimpulan lembar kerja siswa dengan menunjuk siswa selain menunggu kesediaan siswa untuk menghindari dominasi oleh siswa tertentu dalam kelompok dan menghilangkan adanya waktu menunggu. (3) Bagi peneliti selanjutnya yang ingin mengembangkan bahan ajar fisika dalam pembeljaran hendaknya menggunakan Majid, A. 2009. Mengembangkan Standar Kompetensi Guru. Bandung: Remaja Rosdakarya. Parmin. 2012. Pengembangan Modul Mata Kuliah Strategi Belajar Mengajar IPA Berbasis Hasil Penelitian Pembelajaran. Jurnal Pendidikan IPA Indonesia. Vol 1(2012)8-15. Satria, B. 2014. Pengembangan bahan ajar berbasis model problem based learning pada pokok bahasan pencemaran lingkungan untuk meningkatkan hasil belajar siswa kelas X SMA Negeri Grunjungan Bondowoso. Universitas Jember. Sani, R.A. 2014. Pembelajaran Saintifik. Jakarta: PT Bumi Aksara. Wena, M. 2009. Strategi dan Inovasi Pembelajaran Kontemporer. Jakarta: Bumi Aksara. http://jurnal.unimed.ac.id/2012/index.php/jpf Vol. 4 No. 1 Juni 2015 76
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Decentralization matters - Differently organized mental health services relationship to staff competence and treatment practice: the VELO study
International journal of mental health systems
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Published: 18 May 2009 International Journal of Mental Health Systems 2009, 3:9 doi:10.1186/1752-4458-3-9 tional Journal of Mental Health Systems 2009, 3:9 doi:10.1186/ is article is available from: http://www.ijmhs.com/content/3/1/9 This article is available from: http://www.ijmhs.com/content/3/1 © 2009 Bjorbekkmo et al; licensee BioMed Central Ltd. © 2009 Bjorbekkmo 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. Decentralization matters – Differently organized mental health services relationship to staff competence and treatment practice: the VELO study Svein Bjorbekkmo*1, Lars H Myklebust2, Reidun Olstad2,3, Stian Molvik4, Asle Nymann5 and Knut Sørgaard3,6 y Svein Bjorbekkmo*1, Lars H Myklebust2, Reidun Olstad2,3, S Asle Nymann5 and Knut Sørgaard3,6 Address: 1Nordland Hospital Trust Vesteraalen, DPS, N-8450 Stokmarknes, Norway, 2Psychiatric Research Center of North Norway, University Hospital of North Norway, Postboks 6124, N-9291 Tromsø, Norway, 3University of Tromsø, Institute of Clinical Medicine, Dept. of Clinical Psychiatry, N-9291 Tromsø, Norway, 4Nordland Hospital Trust Bodø, Psychiatric Division, Kløveraasveien 1, N-8092 Bodø, Norway, 5Nordland Hospital Trust Lofoten, DPS, N-8372 Gravdal, Norway and 6Psychiatric Research Center of North Norway, Nordland Hospital Trust, Kløveraasveien 1, N-8092 Bodø, Norway Email: Svein Bjorbekkmo* - svein.bjorbekkmo@nordlandssykehuset.no; Lars H Myklebust - Lars.Henrik.Ryther.Myklebust@unn.no; Reidun Olstad - Reidun.olstad@unn.no; Stian Molvik - Stian.Molvik@Nordlandssykehuset.no; Asle Nymann - asle.nymann@unn.no; Knut Sørgaard - knut.sorgaard@nordlandssykehuset.no * Corresponding author Received: 26 February 2009 Accepted: 18 May 2009 Published: 18 May 2009 International Journal of Mental Health Systems BioMed Central BioMed Central Open Access Background is basic professional education and relevant postgraduate education. Second are clinical skills, developed in a pro- fessional setting through encounter with patients and from supervision, and third, there are professional ethics in the sense of a reflective relation to knowledge and to one's own professional behaviour. Roth and Fonagy [9] conclude in a study of psychotherapy that good outcome of psychotherapy is more strongly related to competence in terms of skills and knowledge than to length of experi- ence. g During the last decades, the developmental trend in psy- chiatric services in western countries has been away from large psychiatric hospitals to more locally based psychiat- ric services [1]. The community based services have become the cornerstone of the mental health treatment systems, and the role of the central hospitals has changed towards delivering more time limited back-up service in periods of crises for the patients and services for patients with special needs [2]. To describe and characterize psychiatric services has been called "a difficult and dangerous task" [10]. It may be "dif- ficult" because we do not have good enough theoretical concepts and methods, and "dangerous" because it describes treatment practice instead of just counting beds or consultations. It has therefore been necessary to develop instruments and methods that can be used to describe and differentiate between mental health practices and programs in a valid, reliable and time saving/eco- nomical manner [11-13]. Such instruments are necessary tools for service planners and clinical leaders in their efforts to evaluate and develop the services. Different scales for description of organizational culture, climate and treatment philosophy at different mental health serv- ices have been developed [14-19]. Hargreaves and Jerrell [20] at the University of California introduced in 1991 the Community Program Philosophy Scale (CPPS), later renamed Community Program Practice Scale [21]. The scale intends to be an instrument for the staff to describe treatment philosophy and treatment practice at their psy- chiatric outpatient service. The scale has been translated to Norwegian by Ruud [22] and augmented with 40 items adapted to inpatient staff. This development may be seen partly as a consequence of disillusionment concerning large psychiatric hospitals' suitability for modern treatment of psychiatric illnesses, and partly as related to a general socio-political trend favouring more locally based solutions [3]. Background Central in this development is the idea that it is better for the patient to stay in and receive treatment in their community, and that this will ease access to the services, lead to better coopera- tion between service levels and reduce stigma. This developmental trend has also characterised the devel- opment of psychiatric services in Norway during the last three decades [4]. The number of patients in central psy- chiatric institutions has been reduced, and specialized outpatient and inpatient services are transferred to Com- munity Mental Health Centres (CMHC), often integrated in local general hospitals. In addition, community based mental health services provided by the municipalities have been strengthened. The role of the CMHC is described by the Norwegian Directorate of Health and Social Welfare [5,6] as a specialist health service collabo- rating with the municipal organisations and backed by specialized hospital services. Initially there were no strong directives from the Norwegian health authorities as to how the decentralized specialist services (CMHC) should be organized. This led to a variety of organizational mod- els, often expressing the treatment philosophy of local professionals. Variations in the organizational structure and treatment philosophy of Norwegian CMHCs are not uncommon. In Northern Norway, two of the CMHC units developed along very different lines with regards to organization, staffing, clinical services, use of and access to inpatient treatment [23]. In these two organizations, although serv- ing close to equal sized populations in demographically similar areas, the staffing ratio is about 3:1. The ratio for use of psychiatric beds are equal in the two areas, about 1 bed pr 1000 inhabitants [24]. But whereas one of the cen- tres has no psychiatric inpatient services and uses beds in the county's central psychiatric hospital, the other has established 3 small decentralized inpatient units in their catchment area and uses beds at the Central Mental Hos- pital to a very limited extent and only for very selected purposes such as compulsory treatment and severe eating disorders. Thus, in one of the centres the inpatient services are integrated in a comprehensive community mental health centre that works in accordance with community mental health principles, whereas the other uses the inpa- tient services in a traditional and far-off mental hospital. Mental health care is almost exclusively based on human resources (human technology), and seldom, as may be the case in other areas of health care, on instrumental technology. Abstract Background: The VELO study is a comparative study of two Community Mental Health Centres (CMHC) in Northern Norway. The CMHCs are organized differently: one has no local inpatient unit, the other has three. Both CMHCs use the Central Mental Hospital situated rather far away for compulsory and other admissions, but one uses mainly local beds while the other uses only central hospital beds. In this part of the study the ward staffs level of competence and treatment philosophy in the CMHCs bed units are compared to Central Mental Hospital units. Differences may influence health service given, resulting in different treatment for similar patients from the two CMHCs. Methods: 167 ward staff at Vesterålen CMHCs bed units and the Nordland Central Mental Hospital bed units answered two questionnaires on clinical practice: one with questions about education, work experience and clinical orientation; the other with questions about the philosophy and practice at the unit. An extended version of Community Program Philosophy Scale (CPPS) was used. Data were analyzed with descriptive statistics, non-parametric test and logistic regression. Results: We found significant differences in several aspects of competence and treatment philosophy between local bed units and central bed units. CMHC staff are younger, have shorter work experience and a more generalised postgraduate education. CMHC emphasises family therapy and cooperation with GP, while Hospital staff emphasise diagnostic assessment, medication, long term treatment and handling aggression. Conclusion: The implications of the differences found, and the possibility that these differences influence the treatment mode for patients with similar psychiatric problems from the two catchment areas, are discussed. Page 1 of 10 (page number not for citation purposes) Page 1 of 10 (page number not for citation purposes) http://www.ijmhs.com/content/3/1/9 International Journal of Mental Health Systems 2009, 3:9 International Journal of Mental Health Systems 2009, 3:9 Page 2 of 10 (page number not for citation purposes) Background This is the case for diagnostic processes as well as for therapy. It is a central challenge in the mental health field to make sure there is staff with optimal educa- tion and experience, and that the organizational climate and the motivation of the personnel facilitates the condi- tions for the best outcome of the treatment. Human resources are not static, but will deteriorate if there is not a continual process of maintaining and refreshing professional knowledge and skills. According to Thornicroft and Tansella [7], there is a striking lack of research in this field. Rosenvinge [8] describes three cen- tral elements of clinical competence. First knowledge, that Page 2 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 distance to Bodø from the region is about 1/2 – 1 hour by plane or 4–5 hours by car/ambulance. distance to Bodø from the region is about 1/2 – 1 hour by plane or 4–5 hours by car/ambulance. In this article, we will focus on the relationship between differently organized treatment systems and the philoso- phy of the treatment staff as measured by CPPS. Two groups of inpatient staff are studied: (i) those employed at the CMHC with integrated and decentralised inpatient units, and (ii) the staff at the wards of the county Central Mental Hospital (Nordland Hospital) that primarily serves the CMHC units that operate without access to local inpatient facilities. To our knowledge no similar compar- isons have been done before, and the study has an explor- ative character. We expected to find some systematic differences in program philosophy and practice between the two groups of inpatient staff, reflecting the different context of their clinical work. We also wanted to examine differences in the level of experience and competence between the ward staff in local and central inpatient units. Three wards at the Central Mental Hospital participated in the study: (i) An acute ward with 10 beds, 33 staff includ- ing 6 psychologists and psychiatrists. The ward is used mainly for short term stay for acutely ill patients. (ii) A general inpatient ward with 24 beds, 52 staff including 13 psychologists and psychiatrists. The ward treats patients with mood disorders, post-traumatic disorders, eating dis- orders and personality problems. Stays are usually for three months, occasionally longer. (iii) A rehabilitation ward with 37 beds. The context of the study Vesterålen CMHS has a catchment area of 30500 inhabit- ants, and consists of an outpatient clinic and an inpatient ward consisting of three small units with 8, 6 and 6 beds. The three inpatient units are decentralized in three differ- ent municipalities. The inpatient units are multi-purpose, open, short stay psychiatric units, mainly used for shorter crises and relief stays, but occasionally for longer stays for several months. The inpatient staff work together with outpatient staff in the patients' home environment, and doctors and psychologists at the outpatient clinic also have the responsibility for treatment in the inpatient units. The full-time equivalent staff is 77. Background The number of staff is 117 including 11 psychologists and psychiatrists. The ward serves patients with serious mental disorders (mainly psychosis) on a long term (up to one year) basis. In this article the term CMHC-units refers to the decentral- ised units in Vesterålen, whereas hospital wards refers to the Central Mental Hospital's acute, general and rehabili- tation units in Bodø. (see Table 1) *ranges from 1,3 to 4,6 (4,6 in a security ward) Design A A cross-sectional design was used. Leaders, clinicians and ward staff at the Vesterålen CMHS, and the acute, general and rehabilitation units at the psychiatric division at Nor- dland Hospital in Bodø, were informed about the study and asked to participate. It was emphasised that participa- tion was voluntary and that all data were confidential. After accepting and signing an agreement form, they were delivered the two forms. 252 employees answered the questionnaires. To examine our main hypothesis, that there is a connection between organisational context and treatment philosophy, we chose the ward staff at the inpa- tient units in Vesterålen CMHC and the three units at the psychiatric department of Nordland Hospital in Bodø. All compulsory and some acute hospitalizations from the region are remitted to the psychiatric hospital in Bodø. Some patients are also referred to the Central Mental Hos- pital for long term treatment and for rehabilitation. The Table 1: Organizational outline of Nordland Hospital, psychiatric division. Vesterålen CMHC Nordland Hospital acute Nordland Hospital general Nordland Hospital rehab. Units Outpatient clinic 1 - - - Daycare centres - - - - Bed units 3 1 2 7 Number of beds 20 10 24 37 Staff at inpatient units Number of ward staff 57 27 39 106 Ward personnel number per bed 2,85 2,75 1,62 2,95* Treatment staff rate (doctor/psychologist a.o) per bed 0,2 0,6 0,5 0,3 Total equivalent full- time staff 77 33 52 117 *ranges from 1,3 to 4,6 (4,6 in a security ward) Table 1: Organizational outline of Nordland Hospital, psychiatric division. http://www.ijmhs.com/content/3/1/9 International Journal of Mental Health Systems 2009, 3:9 167 ward staff answered the questionnaires. The response rate was 94% in CMHC and 71% in Hospital units. women is higher at the CMHC wards and the Hospital general ward compared to Hospital acute and rehabilita- tion wards. The staff at the CMHC is also younger. Mean years of practice in the mental health field is higher at all hospital units. Here we also find the highest proportion of registered nurses among the staff. The proportion of staff with postgraduate education is generally high, and there are many specialists within own profession. Competence in cognitive psychology is more common at the hospital units, while the CMHC staff have more generalized post- graduate education aimed at treating and caring for per- sons with severe mental illness. Data analysis Statistical computations were performed with SPSS statis- tical package version 16.0. Descriptive statistics were used to describe the sample in terms of personal characteristics, education and work experience. Parametric (t-test), non-parametric tests (Mann-Whitney U, Chi-square) and logistic regression were used for studying between-group differences. Results In a logistic regression analysis, scales with univariate p-val- ues < .20 were included in the analyses as potential explana- tory variables. These variables were innovation, substance abuse emphasis, medication, long term emphasis, handle aggression, diagnostic emphasis, contact with GP and family therapy. Then a backward stepwise analysis with CMHC staff and Central hospital staff as the dependent variables (table 5) was performed. The table shows that hospital staff con- Instruments To study competence a modified version of a question- naire ("Assessment of competence in the health services") developed by Ruud [22] was used. This form includes questions about age, sex, basic and postgraduate educa- tion, amount of work experiences, competence, clinical orientation and task priorities at the work place. The Community Program Philosophy Scale (CPPS) meas- ures practice and treatment philosophy at the working place. An extended Norwegian version of CPPS was used. The first 80 items in 20 sub-scales are identical with Jer- rells and Hargreaves original scale [20]. In addition 40 items in 10 sub-scales developed by Ruud [22] are included. These items are specially designed for inpatient units, and have sub-scales for sheltering, handling aggres- sion, amount of care, etc. Ward staff were asked to describe climate, philosophy and practice at their unit by responding to the 120 statements on a five point Likert scale (1 = strongly disagree, 5 = strongly agree). State- ments are grouped into 30 sub-scales with 4 statements in each. Five of the sub-scales are about internal climate while the rest of the sub-scales describe treatment practice. We chose to use the extended Norwegian version (NeCPPS) because it has scales for inpatient practice, and offers an opportunity to compare our results to interna- tional studies as well as research from Norwegian mental health services. The psychometric qualities of the Norwe- gian version are equal to the American version as meas- ured by Cronbach's alpha [22]. Therapeutic Profile CPPS ratings show that all units rate help with socio-eco- nomic rights, out of office orientation, teamwork, user participation and medication emphasis high, as well as the climate scales involvement and staff support high or very high. Psychotherapy is generally rated in the low to medium range. Univariate analyses (T-test/Mann-Whitney) were per- formed on all the the CPPS sub-scales. A number of signif- icant differences between the CMHC wards and the Central Mental Hospital wards were found as shown in table 3 and 4. On the ward level, the main differences were between the CMHC units and the hospital acute ward (significant dif- ferences on 15 of 30 sub-scales) and between the CMHC units and the hospital rehabilitation wards (18 of 30 sub- scales). The differences were most evident when the CMHCs were compared to the rehabilitation units. The most substantial differences were found on scales like emphasis on help with housing, work rehabilitation, medication, evaluation, working through, and above all on long term treatment. Hospital rehabilitation ward all ranked these higher than the CMHC. The hospital acute ward and rehabilitation units were similar in that they put significantly more importance on medication and han- dling of aggression than the CMHC and the hospital gen- eral unit. Most similarities were found between the CMHC and the hospital general ward (significant differ- ences on 11 of 30 scales). The Regional Ethics Committee and The Norwegian Social Science Data Services approved the study. Design A The number per bed of doctors/psychiatrists and psychologist is also higher at the hospital units. Competence and Experience Table 2 shows data from the competence questionnaire and socio-demographic data for ward staff at Vesterålen CMHC and the three psychiatric units at Nordland Hospital. Comparing the ward staff in the CMHC to the ward staff at the three hospital units, we find that the percentage of Page 4 of 10 (page number not for citation purposes) Page 4 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 Table 2: Ward staff competence and experience at CMHC and hospital bed units Vesterålen CMHC Nordland Hospital acute Nordland Hospital general Nordland Hospital rehab Response rate (%) 94 80 64 72 Sex (%) Women 83,3 65,0 95,2 *61,1 Age (%) < 30 13,0 5,0 0 4,2 30–39 24,1 30,0 28,6 26,4 40–49 40,7 30,0 28,6 31,9 50–59 20,4 35,0 33,3 27,8 > 60 1,9 0 9,5 4,2 Education (%) Nurses 29,6 60,0 71,4 41,7 Social educator 9,3 0 0 0 Social workers 3,7 10,0 4,8 11,1 Teachers 9,2 0 0 8,3 Other university college education 3,7 0 0 9,0 Total university college education: 55,5 **70,0 **76,2 **70,1 Assistant nurses 18,5 15,0 4,8 19,4 Others 18,5 10,0 9,5 5,6 Assistents 5,6 5,0 4,8 2,8 Total without univ. college education: 42,6 30 19,1 27,8 Postgraduate education (%) Specialist within own profession 42,0 55,0 65,0 41,0 Supervisory competence within own profession 1,9 5,0 9,5 9,7 Competence in cognitive therapy 11,0 20,0 23,8 27,9 Psychosocial rehabilitation of psychoses competence program 18,5 0 4,6 8,0 Work experience Total (mean years) 19,6 20,3 23,2 20,9 Total (range) (1–40) (2–42) (9–36) (2–46) In psychiatric care (mean years) 6,1 *10,1 **14,3 **13,7 At present work place (mean years) 5,3 **10,2 8,6 **10,6 * significantly different from Vesterålen CMHC p < 0,05 ** significantly different from Vesterålen CMHC p < 0,005 Table 2: Ward staff competence and experience at CMHC and hospital bed units * significantly different from Vesterålen CMHC p < 0,05 ** significantly different from Vesterålen CMHC p < 0,005 * significantly different from Vesterålen CMHC p < 0,05 ** significantly different from Vesterålen CMHC p < 0,005 with work in mental health services and (b) on their rank- ing of the wards' treatment orientation. Competence and Experience On competence the main differences were that the ward staff at the hospi- tal generally has longer experience from working in psy- chiatric care, have longer and more medically oriented education and more specialized post graduate education. ceptualized their work more in terms of long term treatment, handling aggression, emphasis on evaluation/diagnostics and emphasis on medication, whereas their CMHC-inpa- tient colleagues emphasised use of family therapy and, to a somewhat lesser extent, contact with GPs. ceptualized their work more in terms of long term treatment, handling aggression, emphasis on evaluation/diagnostics and emphasis on medication, whereas their CMHC-inpa- tient colleagues emphasised use of family therapy and, to a somewhat lesser extent, contact with GPs. Discussion The univariate analyses showed that on treatment philos- ophy, all units strongly emphasised help with socio-eco- nomic rights, out of office orientation, teamwork, user participation and medication. Psychotherapy was gener- ally given a low priority. In this study of competence and treatment philosophy or treatment profile we have compared the staff at three inpatient wards in a community mental health centre to the staff at the inpatient wards in a central mental hospital with regard to (a) demography, education, experience Page 5 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 Table 3: CPPS subcale scores; means and standard deviations. Significant differences in ward staff ratings between CMHC inpatient units and hospital units. Variable Vesterålen CMHC Nordland Hospital acute Nordland Hospital general Nordland Hospital rehab Total Climate scales 1. Innovation 3,4 (0,5) 3,0 (0,4)* 3,5 (0,5) 3,3 (0,6) 3,3 (0,6) 2. Involvement 4,4 (0,5) 4,3 (0,4) 4,5 (0,4) 4,1 (0,5)* 4,3 (0,5) 3. Clarity 3,3 (0,7) 3,5 (0,5) 3,8 (0,4)* 3,7 (0,6)** 3,6 (0,6) 4. Mutual staff support 4,1 (0,5) 4,2 (0,4) 4,2 (0,4) 4,1 (0,5) 4,1 (0,5) 5. Supervisory support 3,7 (0,5) 3,7 (0,7) 4,0 (0,5) 3,6 (0,6) 3,7 (0,5) Practice scales 6. Out-of office orientation 4,0 (0,5) 3,9 (0,6) 3,8 (0,5) 4,0 (0,6) 4,0 (0,6) 7. Team vs individual 4,0 (0,6) 4,2 (0,4)* 4,2 (0,4) 4,2 (0,6)* 4,1 (0,5) 8. Help with housing 3,6 (0,6) 3,6 (0,6) 2,6 (0,6)** 4,1 (0,6)** 3,7 (0,8) 9. Severe mental disorders 3,9 (0,5) 4,0 (0,5) 3,5 (0,4)** 3,9 (0,5) 3,9 (0,5) 10. Involve family 3,5 (0,7) 3,0 (0,8)* 3,4 (0,4) 3,5 (0,8) 3,4 (0,7) 11. Substance abuse emphasis 3,6 (0,6) 3,9 (0,5) 3,4 (0,5) 3,8 (0,8) 3,7 (0,7) 12 Help with socio- econom rights 4,2 (0,5) 4,3 (0,4) 4,3 (0,4) 4,5 (0,4)* 4,3 (0,4) 13 Emergency accessability 3,3 (0,7) 3,4 (0,5) 3,5 (0,6) 3,4 (0,7) 3,4 (0,7) 14 Follow up emphasis 3,6 (0,6) 3,6 (0,5) 3,3 (0,6)* 4,2 (0,6)** 3,8 (0,6) 15 Cooperation with externals 3,6 (0,5) 3,3 (0,4) 3,4 (0,6) 3,6 (0,6) 3,5 (0,6) 16 Users perspective 3,9 (0,5) 3,7 (0,5) 4,1 (0,5) 3,9 (0,6) 3,9 (0,6) 17 Work rehabilitation 3,5 (0,4) 3,0 (0,6)** 3,2 (0,6) 3,8 (0,5)** 3,5 (0,6) 18 Psychoterapy emphasis 2,8 (0,5) 3,0 (0,5) 3,0 (0,4) 2,5 (0,7)** 2,7 (0,6) 19 Medication emphasis 3,6 (0,5) 4,1 (0,3)** 3,6 (0,5) 4,0 (0,7)** 3,8 (0,6) 20. Discussion Long term emphasis 2,9 (0,6) 2,2 (0,4)** 2,3 (0,7)** 4,0 (0,5)** 3,2 (0,9) 21. Handle aggression 2,9 (0,6) 4,3 (0,4)** 2,4 (0,7)* 3,5 (1,0)** 3,2 (0,9) 22 Evaluation, diagnostic empha 3,3 (0,7) 2,8 (0,7)* 4,1 (0,6)** 3,7 (0,6)** 3,5 (0,7) 23. Support emphasis 3,6 (0,5) 4,1 (0,5)** 3,2 (0,6)** 3,9 (0,6)* 3,8 (0,6) 24. Sheltering 3,5 (0,5) 4,3 (0,4)** 2,5 (0,6)** 3,4 (0,8) 3,4 (0,8) 25. Contact GP 3,5 (0,6) 3,1 (0,5)* 3,4 (0,4) 3,2 (0,7)* 3,3 (0,6) 26. Responsibility for untreated 2,8 (0,5) 3,3 (0,6)** 2,4 (0,5)** 2,3 (0,5)** 2,6 (0,6) 27. Working through emphasis 3,3 (0,5) 2,9 (0,6)** 3,3 (0,6) 3,6 (0,6)** 3,4 (0,6) 28. Family therapy emphasis 3,5 (0,6) 2,6 (0,6)** 3,3 (0,4) 3,2 (0,7)* 3,2 (0,7) 29. Group therapy emphasis 3,6 (0,6) 3,1 (0,6)** 4,4 (0,6)** 3,4 (0,8) 3,5 (0,8) 30. Skill training emphasis 3,3 (0,5) 2,8 (0,4)** 3,6 (0,6) 3,6 (0,6)* 3,4 (0,6) * significantly different from Vesterålen CMHC p < 0,05 Mann Whitney U Test ** significantly different from Vesterålen CMHC p < 0,005 Mann Whitney U Test Range emphasis: very small 1 – 1,8, small 1,9 – 2,6, moderate 2,7 – 3,4, large 3,5 – 4,2, very large 4,3 – 5,0 Table 3: CPPS subcale scores; means and standard deviations. Significant differences in ward staff ratings between CMHC inpatient units and hospital units. Table 3: CPPS subcale scores; means and standard deviations. Significant differences in ward staff rat units and hospital units. ans and standard deviations. Significant differences in ward staff ratings between CMHC inpatient * significantly different from Vesterålen CMHC p < 0,05 Mann Whitney U Test ** significantly different from Vesterålen CMHC p < 0,005 Mann Whitney U Test Range emphasis: very small 1 – 1,8, small 1,9 – 2,6, moderate 2,7 – 3,4, large 3,5 – 4,2, very large 4,3 – 5 ward staff, but it is a paradox that specialised treatment services seem to downplay the role of specific therapeutic approaches. The proportion of hospital ward staff with training in cognitive therapy was sizeable. A related ques- tion is the almost uniform importance attributed to med- ication in treatment units covering a rather heterogeneous patient population. ward staff, but it is a paradox that specialised treatment services seem to downplay the role of specific therapeutic approaches. The proportion of hospital ward staff with training in cognitive therapy was sizeable. Discussion A related ques- tion is the almost uniform importance attributed to med- ication in treatment units covering a rather heterogeneous patient population. One may wonder why, with the exception of medication, treatment approaches were not prioritized. All these treat- ment units are expected to give highly specialised services, but there seemed to be an informal agreement that organ- isational and practical approaches were more important than psychotherapy, group therapy and family therapy. It is noteworthy that psychotherapy, next to responsibility for the untreated, was given the lowest average rating of all the CPPS items. Neither was assessment (diagnosis, eval- uation) considered especially important. The explanation for this may be that the participants in the study were On the ward level we found, as might be expected, the largest differences in treatment philosophy between the hospital acute unit and the CMHC. Even though CMCH Page 6 of 10 (page number not for citation purposes) Page 6 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 Table 4: Scores on CPPS subcales; Univariate analyses: means and standard deviation. Variable Vesterålen CMHC Nordland Hospital units Total Asymp sig. 2 tailed Mann-Whitney Climate scales 1. Innovation 3,4 (0,5) 3,3 (0,6) 3,3 (0,6) .196 2. Involvement 4,4 (0,5) 4,2 (0,5) 4,3 (0,5) .053 3. Clarity 3,3 (0,7) 3,7 (0,6)* 3,6 (0,6) .002** 4. Mutual staff support 4,1 (0,5) 4,1 (0,5) 4,1 (0,5) .797 5. Supervisory support 3,7 (0,5) 3,6 (0,6) 3,7 (0,5) .578 Practice scales 6. Out-of office orientation 4,0 (0,5) 3,9 (0,6) 4,0 (0,6) .350 7. Team vs individual 4,0 (0,6) 4,2 (0,5)** 4,1 (0,5) .003** 8. Help with housing 3,6 (0,6) 3,7 (0,8) 3,7 (0,8) .180 9. Severe mental disorders 3,9 (0,5) 3,8 (0,5) 3,9 (0,5) .504 10. Involve family 3,5 (0,7) 3,4 (0,8) 3,4 (0,7) .281 11. Substance abuse emphasis 3,6 (0,6) 3,7 (0,7) 3,7 (0,7) .165 12 Help with socio-econom rights 4,2 (0,5) 4,4 (0,4)* 4,3 (0,4) .046* 13 Emergency accessability 3,3 (0,7) 3,4 (0,7) 3,4 (0,7) .630 14 Follow up emphasis 3,6 (0,6) 3,9 (0,6)* 3,8 (0,6) .014* 15 Cooperation with externals 3,6 (0,5) 3,5 (0,6) 3,5 (0,6) .696 16 Users perspective 3,9 (0,5) 3,9 (0,6) 3,9 (0,6) .596 17 Work rehabilitation 3,5 (0,4) 3,5 (0,6) 3,5 (0,6) .348 18 Psychoterapy emphasis 2,8 (0,5) 2,7 (0,7) 2,7 (0,6) .171 19 Medication emphasis 3,6 (0,5) 4,0 (0,6)** 3,8 (0,6) .000** 20. Discussion Long term emphasis 2,9 (0,6) 3,3 (1,0)** 3,2 (0,9) .002** 21. Handle aggression 2,9 (0,6) 3,4 (1,0)** 3,2 (0,9) .000** 22 Evaluation, diagnostic empha 3,3 (0,7) 3,6 (0,8)** 3,5 (0,7) .002** 23. Support emphasis 3,6 (0,5) 3,8 (0,6)** 3,7 (0,6) .039* 24. Sheltering 3,5 (0,5) 3,4 (0,9) 3,4 (0,8) .559 25. Contact GP 3,5 (0,6)* 3,2 (0,6) 3,3 (0,6) .029* 26. Responsibility for untreated 2,8 (0,5)** 2,5 (0,7) 2,6 (0,6) .000** 27. Working through emphasis 3,3 (0,5) 3,4 (0,6) 3,4 (0,6) .151 28. Family therapy emphasis 3,5 (0,6)** 3,1 (0,7) 3,2 (0,7) .000** 29. Group therapy emphasis 3,6 (0,6) 3,5 (0,9) 3,5 (0,8) .577 30. Skill training emphasis 3,3 (0,5) 3,4 (0,6) 3,4 (0,6) .512 Significant differences in ward staff ratings between CMHC inpatient units and hospital units. * significantly higher p < 0,05 Mann Whitney U Test ** significantly higher p < 0,005 Mann Whitney U Test Range emphasis: very small 1 – 1,8, small 1,9 – 2,6, moderate 2,7 – 3,4, large 3,5 – 4,2, very large 4,3 – 5.0 Table 4: Scores on CPPS subcales; Univariate analyses: means and standard deviation. Vesterålen CMHC Nordland Hospital units Total Asymp sig. 2 tailed Mann-Whitney Vesterålen CMHC Nordland Hospital units Total Asymp sig. 2 tailed Mann-Whitney need for compulsory detention, but in need for inpatient treatment. In one system they are hospitalized locally, in the other in central hospital. As expected, we found that the treatment philosophy surrounding the beds in Vest- erålen was comparable to the treatment principles in the hospital general psychiatric unit. But the hospital rehabil- itation units were more similar to the acute unit, putting wards admit patients for shorter crisis stay, in contrast to hospital acute ward, they do not use coercive measures and do not receive patients for compulsory detention and treatment. On the other hand we would expect the hospi- tal general unit and rehabilitation unit to fill functions more similar to the local CMHC units, because these units probably receive similar patient groups, patients not in Table 5: Logistic regression with CMHC-inpatients staff vs Hospital staff as dependent variable (N = 1 Logistic regression with CMHC-inpatients staff vs Hospital staff as dependent variable (N = 161) Table 5: Logistic regression with CMHC-inpatients staff vs Hospital staff as dependent variable (N = 161) Variable B S.E. Exp (B) 95% C.I for EXP (B) 19. Medication emphasis .785 .381 2.193 1.038 4.632 20. Significant differences in ward staff ratings between CMHC inpatient units and hospital units. g y g p y Range emphasis: very small 1 – 1,8, small 1,9 – 2,6, moderate 2,7 – 3,4, large 3,5 – 4,2, very large 4,3 – 5.0 Discussion These differences in treatment orientation might also reflect a conscious strat- egy to recruit professionals with different educational background and experience to CMHC-team than what is usual in traditional mental hospital teams. There is also a difference in postgraduate education between the CMHC and hospital units. CMHC emphasises generalized reha- bilitation programs while hospital emphasises cogntive therapy. This may reflect an intended strategy or a differ- ence in treatment orientation that is related to organiza- tional frames or differences in professional background. When we compare, the CPPS reference values from USA [21] show more emphasis on basic care, social support and medication in the USA values. This difference may be connected to dissimilarities between the Norwegian pub- lic health service and the American health service, and in differences in social legislation. In Norway, the most important single factor behind the deinstitutionalisation was the disability pension which, in addition to other social welfare laws, made living outside mental hospital possible for seriously mentally ill persons [3]. Higher pop- ulation density in USA makes it more natural to develop more specialized programs for different groups of patients. In more scattered populated areas in Norway, it is necessary to give a public general psychiatric service. Alternative private health services, especially for the less severe psychiatric problems, are supposed to be better available in USA, while it is almost non-existent in the regions in Norway that we study [4]. If reported treatment philosophy in our study influences and reflects actual treatment practice, this means that patients referred to the Central Mental Hospital units to some extent receive different treatment compared to the CMHC treated patients. We have mentioned that the acute ward at the hospital is used for some of the acutely ill patients from the CMHC region, and to some extent the differences in program profile may be functional. On the other hand, it may seem that the more family- and GP-ori- ented treatment is more functional for patients in need of long term follow up. This is in accordance with what is found in earlier studies [1]. Discussion Long term emphasis .577 .251 1.780 1.089 2.909 21. Handle aggression .492 .242 1.636 1.018 2.629 22 Evaluation, diagnostic empha 1.209 .338 3.349 1.726 6.498 25. Contact GP -.725 .385 .484 .228 1.030 28. Family therapy emphasis -1.275 .419 .279 .123 .634 International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 significantly more weight on medication, on handling aggression and on support than the CMHC and the Hos- pital general unit. The general impression was that the CMHC staff were most similar to the hospital general unit staff both with regard to competence and treatment orien- tation. We also expected many similarities in competence and treatment philosophy between the CMHC wards and the hospital rehabilitation ward, because we thought patients in need for rehabilitation programs would be a relatively homogenious group. These expectations were not confirmed. The largest differences were found between the CMHC wards and hospital rehabilitation unit. Significant differences were found on treatment ori- entation scales like psychotherapy, medication and family therapy, and on cooperation with other parts of the health service (follow up, contact with GP), on threshold for intake, and above all, on long term treatment. present study accept acutely ill patients. The hospital pro- file becomes more evident when we compare it with the attitudes of the CMHC staff and their orientation towards family treatment and collaboration with GPs. In spite of considerable overlap in professional attitudes and philos- ophy the distinctness of the different profiles seems to be real, even though the mechanism behind them is not clear. It has been argued that a decentralised mental health organization might have problems with recruitment of personnel with relevant competence and maintenance of a high professional standard [7,25]. To some extent we find support for this view. This might to some degree be balanced with an advantage in geographical closeness to municipality services and the patient's family and social network, giving better conditions for continuity of care than a more far away central hospital system might give. The differences in emphasis on family therapy, contact with GP, medication and long term treatment might reflect that the CMHC wards have a more resource and social network oriented practice looking for solutions in cooperation with the patient's local support system, while the hospital system focuses more on reduction of symp- toms and strengthening of the patient's function through therapeutic efforts within the ward. Strength and limitations Our material is based on a relatively small number of ward staff from several different units. But the response rate is high, and some differences concerning competence and treatment profiles are very distinct. Doctors and psy- chologists are not included in our analyses. This is due to their somewhat different roles in relation to CMHC bed units and hospital units. In hospital units their job is fully connected to treatment of inpatients, while in the CMHC their job is partly connected to inpatients and partly to outpatients. It would not be possible to distinguish to what extent their ratings had their source in outpatient or inpatient experience. Preliminary analyses of data includ- ing doctors, psychologists and other treatment personnel indicate that including them had minimal impact on the results. It is not possible to draw conclusions about qual- ity of treatment on the basis of our data on competence and treatment orientation and there are always differences between what people say they do and what they actually do [17]. However, this affects both the hospital and the CMHC staff to the same degree. Closer knowledge of groups of patients receiving different treatment in the units would strengthen our knowledge about aspects of the differences. Data from other parts of the VELO study will contribute to this. A central aim for health authorities is to give the popula- tion equal access to health services regardless of popula- tion density and geographical distances [6]. Our results may indicate that an unintended consequence of differ- ences in organization models is that even if the aim of equal access to services is reached, the services given may be different. But comprehensive information is required on the characteristics of the patients that receive inpatient services in the two regions before firm conclusions can be made. CPPS is used in projects in USA and Norway. It seems to be a useful and economical instrument for comparing psychi- atric services and for evaluating the services over time. Fur- ther research with the scale and analyses of its psychometric strength based on accessible Norwegian data is needed. To our knowledge this is the first international publication of a study using inpatient ward staffs CPPS ratings. It is also an aim to give a coordinated health service to patients with the most severe mental illness close to their local network. Discussion When CMHC and hospital staff were used as a dependent variable in the logistic regression analysis, it became apparent that the main differences between the two groups were that the hospital staff conceptualized their work more in terms of long term treatment, handling aggression, emphasis on evaluation/diagnostics and emphasis on medication, whereas their CMHC-inpatient colleagues emphasised use of family therapy and, to a somewhat lesser extent, contact with GPs. In spite of many univariate similarities, there seemed to be a distinct hospital professional profile characterized by a more diag- nostically and medically oriented approach directed at patients with aggression and long term problems. The importance of aggression may be caused by some of these wards often receiving patients who are dangerous to oth- ers or to themselves, more often acutely ill and disorgan- ized. But many hospital inpatients do not suffer from such problems, and only one of the hospital wards in the What determines treatment practice in the mental health field? [3]. Is it mainly socio-political trends, the structure or history of organizations, geography, the patient popu- lation or the staff's age, professional background and experience? Treatment programs are to a large extent Page 8 of 10 (page number not for citation purposes) Page 8 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 http://www.ijmhs.com/content/3/1/9 different in respect of inpatient specialized service from the two catchment areas. adjusted to different clinical types of patients. This is sup- posed to be a central determinant. But our data indicates that differences in staff competence and education, and organizational characteristics may influence treatment philosophy and consequently practice. On the basis of our results, we have created a hypothesis of the relation- ship of these variables (figure 1). If results from the present study can be generalized, there seems to be a ten- dency that hospitals give what could be called a more medically oriented treatment over longer time with emphasis on evaluation and coping with aggression and less emphasis on cooperation with municipal health and social services. Lack of tradition and geographical close- ness to patient's network may give newer organizations like CMHC with more flexible organizational frames and younger staff with educations and training that implies other treatment philosophies an advantage over presuma- bly more slow-to-change large hospital organizations. When it comes to treatment of severely ill patients this hypothesis must be tested further. Conclusion 7. Thornicroft G, Tansella M: The Mental Health Matrix. A manual to improve services Cambridge University Press; 1999. • How psychiatric services are organised may influ- ence the formulation of the treatment philosophy. In spite of considerable overlap between the CMHC and the hospital staff with regard to attitudes and priori- ties, there appeared a distinct but not radical differ- ence between the units' treatment profiles. • How psychiatric services are organised may influ- ence the formulation of the treatment philosophy. In spite of considerable overlap between the CMHC and the hospital staff with regard to attitudes and priori- ties, there appeared a distinct but not radical differ- ence between the units' treatment profiles. p g y 8. Rosenvinge JH, Larsen E, Skaarderud F, Thune-Larsen KB: The per- ception and development of clinical competence. Journal of the Norwegian Psychological Assosiation 2004, 41(9):706-712. g y g ( ) 9. Roth A, Fonagy P: What Works for Whom? A Critical Review of Psycho- therapy Research New York: Guilford Press; 2005. py 10. Bickman L: The most dangerous and difficult question in men- tal health service research. Mental Health Services Research 2000, 2:71-72. 11. Johnson S, Salvador-Carulla L, the EPCAT group: Description and classification of mental health services: a European perspec- tive. European Psychiatry 1998, 13(7):333-341. • The hospital staff to a larger degree conceptualized their work in terms of long term treatment with emphasis on handling aggression, evaluation and medication, whereas their CMHC-inpatient colleagues emphasised family therapy and contact with GPs. p y y ( ) 12. Bryant DM, Bickman L: Methodology for Evaluating Mental Health Case Management. Evaluation and Program Planning 1996, 19(2):121-129. ( ) 13. Lloyd-Evans B, Johnson S, Slade M: Assessing the content of men- tal health services: a review of measures. Social Psychiatry and Psychiatric Epidemiology 2007, 42:673-682. 14. Aarons GA, Sawitzky AC: Organizational Climate Partially Mediates the Effect of Culture on Work Attitudes and Staff Turnover in Mental Health Services. Administration and Policy in Mental Health Service Research 2006, 33(3):289-301. • Somewhat surprisingly, on treatment philosophy all units rated help with socio-economic rights, out of office orientation, teamwork, user participation and medication as most important. Psychotherapy was given a low priority, with other therapeutic modalities (e.g. group therapy, skill training) in between. ( ) 15. Moos RH, Finney JW, Ouimette PC, Suchinsky RT: A comparative evaluation of substance abuse treatment: I. Authors' contributions All authors participated in the design of the study. SB, KS, RO and LM wrote the manuscript. SB, KS and RO per- formed the statistical analysis. All authors read and approved the final manuscript. 21. Hargreaves WA, Jerrell JM, Lawless SM, Unick J: Doing the difficult and dangerous: The community program practice scale. Administration and Policy in Mental Health and Mental Health Service Research 2007, 34:138-149. 22. Ruud T, Breimoen M: Practical advice for the use of some eval- utation methods. Evaluation of Psychiatric Health Services, Norwegian Directorate for Health and Social Affairs. Oslo 1996. Conclusion Treatment ori- entation, amount of care, and 1-year outcomes. Alcoholism: Clinical and Experimental Research 1999, 23:529-536. p 16. Glisson C, Landsverk J, Schoenwald S, Kelleher K, Hoagwood KE, Mayberg S, Green P: Assessing the Organizational Social Con- text (OSC) of Mental Health Services: Implications for Research and Practice. Administration and Policy in Mental Health Service Research 2008, 35:98-113. Our study shows that organizing community mental health specialist services with or without access to locally situated beds makes a difference that might influence the mental health services given to patients and their families, and the support given to the municipital health service in relation to patients with similar psychiatric disturbances. 17. Rossberg JI, Friis S: Patients' and Staff's Perceptions of the Psy- chiatric Ward Environment. Psychiatric Services 2004, 55(7):798-803. 18. Cohen-Mansfield J, Parpura-Gill A: Practice style in the nursing home: dimensions for assessment and quality improvement. International Journal of Geriatric Psychiatry 2007, 23:376-386. 19. 19. Burt MR, Duke AE, Hargreaves WA: The Program Environmen- tal Scale: Assessing Client Perseption of Community-Based Programs for Severely Mentally Ill. American Journal of Commu- nity Psychology 1998, 26(6):853-878. The authors declare that they have no competing interests. The authors declare that they have no competing interests. y y gy ( ) 20. Jerrell JM, Hargreaves WA: The Operating Philosophy of Com- munity Programs. In Working Papers Series 18 Berkeley (CA): Insti- tute of Mental Health Service Research; 1991. Acknowledgements 23. Nymann A, Eisemann MR, Olstad R: Selection and use of need indexes for mental health services in the catchment areas of two Community Mental Health Centres in the county of Nordland, Norway University of Tromsø Norway; 2006. The study was initiated and funded by the Northern Norway Regional Health Authorities. Health Authorities. 24. 24. Myklebust LH, Bjorbekkmo S, Nymann A, Molvik S, Sørgaard K, Olstad R: The utilization pattern of psychiatric beds in two dif- ferently organized community mental health service sys- tems in northern Norway. The VELO-project. Social Psychiatry and Psychiatric Epidemiology in press. Strength and limitations Our study may indicate that patients with long term rehabilitation needs receive treatment that is Page 9 of 10 Possible factors influencing outcome in mental health services Figure 1 Possible factors influencing outcome in mental health services. Patient outcome Treatment practice Treatment philosophy Patient factors - Age - Diagnoses - Functional level Staff factors Organizational - Age factors - Gender - Ideology - Formal education - History - Years of practice - Architecture - Geography Staff factors - Age - Gender - Formal education - Years of practice Patient factors - Age - Diagnoses - Functional level Organizational factors - Ideology - History - Architecture - Geography Treatment practice Treatment philosophy Patient outcome Possible factors influencing outcome in mental health services Figure 1 Possible factors influencing outcome in mental health services. Possible factors influencing outcome in mental health services Figure 1 Possible factors influencing outcome in mental health services. Page 9 of 10 (page number not for citation purposes) International Journal of Mental Health Systems 2009, 3:9 6. Norwegian Directorate for Health and Social Affairs: Community Men- tal Health Centres – with the eye at the municipalities and backed by spe- cialized hospital services. Oslo 2006. References 1. Killaspy H: From the asylum to community care:learning from experience. British Medical Bulletin 2006, 79 and 80:245-258. 1. Killaspy H: From the asylum to community care:learning from experience. British Medical Bulletin 2006, 79 and 80:245-258. p 2. Thornicroft G, Tansella M: Balancing community-based and hospital-based mental health care. World Psychiatry 2002, 1(2):84-90. 25. y p gy p 25. Bjertnaes OA, Garratt A, Ruud T: Family Physicians' Experiences With Community Mental Health Centers: A Multilevel Anal- ysis. Psychiatric Services 2008, 59(8):864-870. ( ) 3. Soergaard KW: From institutional treatment to community health care. Reasons behind the reforms of mental health care. Journal of the Norwegian Psychological Association 2002, 39:796-805. 4. Kolstad A, Hjort H: Mental Health Services in Norway. In Men- tal Health Systems Compared: Great Britain, Norway, Canada, and the United States Edited by: Olson RP. Springfield, Illinois: Charles C Tho- mas Publisher; 2006. 4. Kolstad A, Hjort H: Mental Health Services in Norway. In Men- tal Health Systems Compared: Great Britain, Norway, Canada, and the United States Edited by: Olson RP. Springfield, Illinois: Charles C Tho- mas Publisher; 2006. 5. Norwegian Ministry of Health and Care Services: Mental Health Serv- ices in Norway. Prevention, treatment, care. Mental Health 1999–2008. Oslo 2004. 6. Norwegian Directorate for Health and Social Affairs: Community Men- tal Health Centres – with the eye at the municipalities and backed by spe- cialized hospital services. Oslo 2006. 6. Norwegian Directorate for Health and Social Affairs: Community Men- tal Health Centres – with the eye at the municipalities and backed by spe- cialized hospital services. Oslo 2006. Page 10 of 10 (page number not for citation purposes)
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Pressure ulcers: risk, predisposing factors and hospital prognosis in patients older than 65 years
Revista virtual de la Sociedad Paraguaya de Medicina Interna
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ERROR: type should be string, got "https://doi.org/10.18004/rvspmi/2312-3893/2021.08.02.23\n\nARTÍCULO ORIGINAL\nÚlceras por presión: riesgo, factores predisponentes y pronóstico\nhospitalario en pacientes mayores de 65 años\nPressure ulcers: risk, predisposing factors and hospital prognosis in\npatients older than 65 years\n1,2\n\nMiguel Morales Ojeda\n3,2\nMarisa Ileana Gómez\n4\nIsmael Morales Ojeda\n5\nBárbara Cerda Aedo\n2\nMiguel Ángel Meriño\n1\n\nUniversidad Adventista del Plata. Villa Libertador San Martín. Entre Ríos, Argentina.\n\n2\n\nUniversidad Adventista de Chile. Núcleo de Investigación en Salud. Chillán, Chile.\n\n3\n\nUniversidad Adventista del Plata. Facultad de Ciencias de la Salud. Entre Ríos, Argentina.\n\n4\n\nUniversidad Adventista de Chile. Carrera de enfermería. Dirección de Investigación. Chillán, Chile.\n\n5\n\nUniversidad Adventista de Chile. Dirección de Investigación. Chillán, Chile.\n\nRESUMEN\nIntroducción: las úlceras por presión (UPP) influyen drásticamente en la estancia hospitalaria\nproduciendo malestar y dolor a los pacientes.\nObjetivo: determinar el riesgo de desarrollar UPP y evaluar conjuntamente nutrición / alimentación y\ngrado de independencia en pacientes mayores de 65 años.\nMétodo: diseño descriptivo y transversal. Se evaluó a 444 pacientes con y sin UPP mediante la Escala\nde Braden, el Mini Nutritional Assessment Short y Prisma 7, internados en el Hospital Adventista\nSilvestre, Rio de Janeiro, Brasil, entre 2018 y 2019.\nResultados: respecto de la Escala de Braden el grupo con mayor representación es el Riesgo\nModerado de sufrir UPP (37,6%). La estratificación en grupo del Prima 7 muestra 2 grupos principales,\nel mayor (51,8%) que corresponde a Prisma positivo (>3), seguido por el grupo de Prisma negativo\n(35,1%). Según la escala Mini Nutritional Assessment Short 41,9% de los pacientes internados tiene\nriesgo de desnutrición y 34% se halla con desnutrición.\n\nArtículo recibido: 7 abril 2021\n\nArtículo aceptado: 26 mayo 2021\n\nAutor correspondiente:\nDr. Ismael Morales Ojeda\nCorreo electrónico: ismaelmoralesojeda@gmail.com\nEste es un artículo publicado en acceso abierto bajo una Licencia Creative Commons CC-BY 4.0\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n23\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nConclusión: el estudio relaciona las UPP con una serie factores como la malnutrición, la inmovilidad y\npérdida de la independencia. La escala de Braden demostró ser un buen predictor de UPP. Se evidenció\nque las UPP son un problema con gran prevalencia en el hospital, que el acompañamiento\nmultidisciplinar para el cuidado y tratamiento de estas lesiones es fundamental.\nPalabras claves: úlcera por presión, causalidad, anciano\n\nABSTRACT\nIntroduction: Pressure ulcers (PUs) drastically influence the hospital stay, causing discomfort and\npain to patients.\nObjective: To determine the risk of developing PUs and jointly evaluate nutrition / diet and degree of\nindependence in patients over 65 years of age.\nMethod: Descriptive and cross-sectional design. Four hundred and forty-four patients with and\nwithout PUs, admitted to the Silvestre Adventist Hospital, Rio de Janeiro, Brazil between 2018 and\n2019, were evaluated using the Braden Scale, the Mini Nutritional Assessment Short Form and the\nPrisma 7.\nResults: Regarding the Braden Scale, the group with the highest representation was the Moderate\nRisk of suffering Pus (37.6%). The group stratification of Prima 7 showed two main groups, the largest\n(51.8%) corresponding to positive Prism (> 3), followed by the negative Prism group (35.1%).\nAccording to the Mini Nutritional Assessment Short scale, 41.9% of hospitalized patients were at risk\nof malnutrition and 34% were malnourished.\nConclusion: The study related PUs to a series of factors such as malnutrition, immobility and loss of\nindependence. The Braden scale proved to be a good predictor of UPP. It was evidenced that PUs are a\nhighly prevalent problem in the hospital, and that multidisciplinary support for the care and treatment\nof these injuries is essential.\nKeywords: pressure ulcer, causality, aged\n\nINTRODUCCIÓN\nLas lesiones de la piel conocidas como úlceras por presión (UPP) comprenden a áreas\nlocalizadas de isquemia con posterior necrosis de los tejidos, las que son ocasionadas por la\ncompresión prolongada, cizallamiento o fricción de los tejidos blandos entre prominencias óseas y la\n(1)\nsuperficie externa .\nLa aparición de UPP en pacientes hospitalizados constituye un problema importante para la\nsalud y puede causar molestias físicas al paciente, aumentar los costos de tratamiento por concepto\nde cuidados intensivos, hospitalización prolongada, aumento del riesgo de desarrollar complicaciones\nadicionales que pueden incluir incluso la necesidad de cirugía correctiva y el riesgo en el aumento de la\n(2,3)\ntasa de mortalidad\n. La etiología de la UPP es multifactorial y depende de factores de riesgo\nintrínsecos y extrínsecos.\nLa determinación del nivel riesgo para el paciente en el desarrollo de la UPP es la primera\nmedida adoptada para evitar estas lesiones. Debe ser realizada a la admisión del paciente y por lo\nmenos cada 48 horas, o cuando exista algún cambio de sus condiciones de salud (4,5). Esto debe\nhacerse especialmente en pacientes en estado crítico o en enfermos que poseen un gran número de\nfactores de riesgo.\n24\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nEn la literatura se han descrito e implementado un gran número de estrategias por parte de\ninvestigadores y profesionales de la salud, en un intento de establecer normas de asistencia\n(6,7)\nenfocados a la prevención de las UPP y estimular mejoras en la calidad de atención\n. Entre estas\nestrategias se destaca el uso de la escala de Braden como instrumento predictor de riesgo y el\ndesarrollo de protocolos específicos para pacientes con problemáticas particulares. La escala de\nBraden es la herramienta de evaluación del riesgo más ampliamente probada y usada, la cual tiene\nespecificidad y sensibilidad para la población hospitalaria. Se presenta como un instrumento eficaz\npara ayudar en la toma de decisiones de las medidas preventivas a adoptarse en conformidad con el\n(8,9)\nriesgo para cada paciente\n. Debe tenerse en cuenta que, con el uso de esta escala, es posible\nevaluar al individuo hospitalizado en aspectos fundamentales como: estado nutricional, nivel de\nmovilidad, percepción sensorial, fricción, cizallamiento, humedad y grado de actividad física. Por ello,\nel objetivo principal de esta investigación fue determinar el riesgo de desarrollar UPP versus la\naparición efectiva de las mismas y evaluar la existencia de correlación con las variables nutrición, vía\nde alimentación, grado de independencia social, y de esta forma determinar su relación frente a la\nresolución hospitalaria en los pacientes internados, mayores de 65 años, del Hospital Adventista\nSilvestre, en la ciudad de Rio de Janeiro, Brasil.\n\nMATERIALES Y MÉTODOS\nSe aplicó un diseño descriptivo, transversal. La población estuvo formada por 444 pacientes\nmayores de 65 años que fueron internados en el Hospital Adventista Silvestre, en la ciudad de Rio de\nJaneiro, Brasil, entre los años 2018-2019. Para la selección se emplearon los siguientes criterios:\nestar hospitalizado, ser mayor de 65 años, responder por sí mismos o con la ayuda de un familiar los\ntópicos a ser evaluados en los instrumentos aplicados que así lo requieran, y poseer registro e historia\nclínica en los sistemas de la institución. Se realizó el estudio considerando la resolución de la\ninternación de los pacientes.\nSe aplicaron la Escala de Braden, el Mini Nutritional Assessment Short (MNA short) y Prisma 7.\n(10)\nLa escala de Branden validada\nofrece seis parámetros para evaluación por sus subescalas: 1percepción sensorial; 2- humedad; 3- actividad; 4- movilidad; 5- la nutrición; 6- fricción de dominio y\nde cizallamiento. Cada subescala tiene una puntuación que varía entre 1 y 4, con la excepción de\nfricción de dominio y de cizallamiento. La suma total va entre valores de 6 a 23 puntos(11). Una\npuntuación igual o menor de 16 indica que el paciente adulto está en riesgo para el desarrollo de UPP.\nEl MNA short validado(12) consta de un cuestionario de 7 preguntas que incluye además la posibilidad\nde utilizar el perímetro de la pantorrilla cuando no es posible obtener el IMC del paciente. Posee una\npuntuación máxima de 14 puntos. Una puntuación entre 12-14 indica que el paciente tiene un buen\n(13)\nestado nutricional. Una puntuación entre 8-11 identifica a los pacientes en riesgo nutricional . El\nPrisma 7 es un instrumento validado(14)que consta de varios temas: edad mayor a 85 años, si el\nentrevistado es del sexo masculino, si posee problemas de salud que limiten sus actividades físicas, si\nnecesita ayuda regularmente, si posee algún problema de salud que lo obligue a quedarse en su casa,\nsi en caso de necesidad puede contar con la ayuda de alguien que esté cerca suyo, si necesita\n(15)\nregularmente del uso de bastón, andador o silla de ruedas para moverse\n. Los criterios se\nobjetivaron con las respuestas sencillas de “sí “o “no”. Según el número de respuestas positivas “sí”\nque el entrevistado haya dado, se clasificó en 0-2 puntos (pacientes prisma negativo), 3 puntos\n(pacientes borderline) y 4-7 puntos (pacientes prisma positivo). Para análisis, los adultos mayores\nclasificados como borderline y prisma positivo fueron agrupados en una sola categoría. Para el análisis\nde los resultados se utilizó el programa SPSS, presentándose los resultados con estadística\ndescriptiva, tablas de frecuencia / porcentaje junto con medias y desviación estándar. Para la\nestadística analítica aplicada a los instrumentos ya mencionados se determinó la normalidad de las\nvariables con la prueba de Kolmogorov-Smirnov y posteriormente se utilizó la prueba no paramétrica\ncorrespondiente, en este caso las pruebas U de Mann Whitney y correlación de Spearman. Ambas\npruebas fueron realizadas con un nivel de significancia de p ≤ 0,05.\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n25\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nEl proyecto fue aprobado por el Comité de Ética de la Universidad Adventista del Plata,\nArgentina. Se realizó un procedimiento de consentimiento informado previo a la recolección de datos.\n\nRESULTADOS\nLas características de la UPP se describen en la tabla 1.\nTabla 1. Descripción de las características de las úlceras por presión (UPP) (n 109)\n\nPacientes con UPP\nAmbiente del evento\n\nLugar principal de UPP\n\nÚnicas o múltiples\n\nGrado de UPP principal\n\nCriterios\n\nFrecuencia Porcentaje\n\nAntes de internación/casa\nInternación\nTerapia intensiva\nNo definido\nSacra y/o interglútea\nCalcáneo\nTrocánter\nMaléolo\nOtros lugares\n\n51\n26\n31\n1\n91\n8\n3\n4\n3\n\n46,8\n23,9\n28,4\n0,9\n83,5\n7,3\n2,8\n3,7\n2,8\n\nÚnica UPP\nMúltiples incluyendo\nregión sacra\nMúltiples sin incluir región\nsacra\n\n83\n23\n\n76,1\n21,1\n\n3\n\n2,8\n\nI\nII\nIII\nIV\nSin clasificar/necrosis/sin\nregistro\n\n26\n52\n18\n7\n6\n\n23,9\n47,7\n16,5\n6,4\n5,5\n\nLos resultados de asociar los resultados de las escalas se detallan en la tabla 2.\n\n26\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nTabla 2. Tabla de contingencia respecto de la clasificación según los instrumentos Prisma 7, MNA\nShort y Braden según presencia de úlceras por presión (UPP)\n\nPrisma 7 clasificación\nPrisma negativo (< 3)\nPrisma borderline (= 3)\nPrisma positivo (> 3)\nTotal\n\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\n\nMNA Short clasificación\nEstado nutricional\nnormal (12 a 14)\nEn riesgo de\ndesnutrición (8 a 11)\nDesnutrido\n(0 a 7)\nTotal\n\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\n\nEscala de Braden\nSin riesgo\n(21 a 23)\nBajo riesgo\n(18 a 20)\nRiesgo Moderado\n(12 a 17)\nRiesgo Alto\n(06 a 11)\nTotal\n\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\nRecuento\n% dentro de UPP\n\nUPP\nNo\nSi\n143\n13\n42,7%\n11,9%\n47\n11\n14,0%\n10,1%\n145\n85\n43,3%\n78,0%\n335\n109\n100,0% 100,0%\nUPP\nNo\nSi\n101\n6\n30,1%\n5,5%\n151\n35\n45,1%\n32,1%\n83\n68\n24,8%\n62,4%\n335\n109\n100,0% 100,0%\nUPP\nNo\nSi\n93\n3\n27,8%\n2,8%\n88\n9\n26,3%\n8,3%\n125\n42\n37,3%\n38,5%\n29\n55\n8,7%\n50,5%\n335\n109\n100,0% 100,0%\n\nTotal\n156\n35,1%\n58\n13,1%\n230\n51,8%\n444\n100,0%\nTotal\n107\n24,1%\n186\n41,9%\n151\n34,0%\n444\n100,0%\nTotal\n96\n21,6%\n97\n21,8%\n167\n37,6%\n84\n18,9%\n444\n100,0%\n\nLa prueba U de Mann Whitney demostró diferencias en la escala de Braden entre el grupo “con\nUPP” y el grupo “sin UPP” (p=0,000). La prueba U de Mann Whitney demostró diferencias en la escala\nPrisma 7 entre el grupo “con UPP” y el grupo “sin UPP” (p=0,000). La prueba U de Mann Whitney\ndemostró diferencias en la Escala MNA Short entre el grupo “con UPP” y el grupo “sin UPP” (p=0,000)\n(figuras 1, 2 y 3)\nHaciendo un análisis de las medianas con la prueba de correlación de Spearman, se demostró\nque la variable edad se correlacionó con: a) El número de días de internación (p = 0,004 r = 0,136), b)\nLa escala de Braden (p = 0,000 r = -0,398), c) La escala Prisma 7 (p = 0,000 r = 0,553), d) MNA Short\n(p = 0,000 r = -0,313)\nLa variable número de días de internación se correlacionó con: a) La escala de Braden (p =\n0,000 r = -0,292), b) La escala Prisma 7 (p = 0,001 r = 0,163) c) MNA Short (p = 0,000 r = -0,261) La\nEscala de Branden se correlacionó con: a) La escala Prisma 7 (p = 0,000 r = -0,691), b) MNA Short (p\n= 0,000 r = -0,612). La Escala Prisma 7 se correlacionó con: MNA Short (p = 0,000 r = -0,559).\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n27\n\n\fBraden\n\nÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nUPP\n\nPrisma 7\n\nFigura 1. Gráfico de caja de la escala de Braden frente a los grupos con y sin úlceras por presión (UPP)\n\nUPP\n\nFigura 2. Gráfico de caja de la escala Prisma 7 frente a los grupos con y sin úlceras por presión (UPP)\n\n28\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n\fMNA Short\n\nÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nUPP\n\nFigura 3. Gráfico de caja de la escala MNA Short frente a los grupos con y sin úlceras por presión\n(UPP)\nEn la tabla 3 se observa la descripción de las características de las úlceras por presión, respecto\na lo observado en los pacientes (n=109).\nTabla 3. Descripción de las características de las úlceras por presión (n 109)\n\nPacientes con\nUPP\nAmbiente del\nevento\n\nCaracterísticas\n\nFrecuencia Porcentaje\n\nAntes de internación/casa\nInternación\nCTI/UCO\nNo definido\n\n51\n26\n31\n1\n\n46,8\n23,9\n28,4\n0,9\n\nLugar principal de\nUPP\n\nSacra y/o interglútea\nCalcáneo\nTrocánter\nMaléolo\nOtros lugares\n\n91\n8\n3\n4\n3\n\n83,5\n7,3\n2,8\n3,7\n2,8\n\nÚnicas o múltiples\n\nÚnica UPP\nMúltiples incluyendo región\nsacra\nMúltiples sin incluir región\nsacra\n\n83\n23\n3\n\n76,1\n21,1\n2,8\n\nGrado de UPP\nprincipal\n\nI\nII\nIII\nIV\nSin clasificación/necrosis/sin\nregistro\n\n26\n52\n18\n7\n6\n\n23,9\n47,7\n16,5\n6,4\n5,5\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n29\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nAl analizar la resolución de los pacientes en el momento del cierre de los datos, estos revelan\nque 88,7% fueron dados de alta o continuaban internados (estos últimos corresponden solo a 6\ncasos). Por otra parte, 50 pacientes, que equivale al 11,3%, fueron a óbito. De 50 pacientes que\nfueron a óbito, 36 de ellos tuvieron o tenían UPP, dando un 72% de incidencia de UPP en este grupo,\nfrente a un 18,5% de incidencia de los pacientes que recibieron el alta.\n\nDISCUSIÓN\nLas localizaciones más frecuentes de las UPP fueron en la zona sacra, comparando con otros\n(16,17)\ntrabajos podemos también ver la predominancia de este tipo de UPP\n. Según se indica, la\ndesnutrición, además de aumentar el riesgo de UPP, dificulta la curación por reducción de nutrientes\ndisponibles para el reparo y mantención tecidual, resultando en la pérdida del efecto amortiguador del\n(18-20)\ntejido adiposo, menor resistencia cutánea, debilidad en general, movilidad reducida y edema\n.\n(19)\nCuando existe pérdida de la masa magra e inmovilidad aumenta el riesgo de UPP en un 74% .\nA partir de la escala de Braden se observa que la población se ubica en su mayoría bajo la\ncategoría de riesgo moderado, lo cual es similar a lo presentado en el estudio hecho por Borghardt, en\n(21)\nel cual la mayoría de la población también se encontraba dentro de este grupo .\nEn la misma escala, de los pacientes con UPP el promedio de 12,75 (riesgo moderado) y dentro\ndel grupo sin UPP el promedio es de 17,4 (riesgo moderado y bajo riesgo). Los grupos con y sin UPP\nson distintos entre sí respecto a la escala de Braden según la prueba U de Mann Whitney (p<0,00). Al\nanalizar la distribución de categorías dentro de los 109 pacientes con UPP, encontramos que 50,5%\nque se ubica dentro de la categoría de riesgo alto, similar a lo expuesto por Borghardt en el mismo\nestudio mencionado anteriormente donde el 59% de la población con UPP se encontraba también\ndentro de la categoría de riesgo alto(21). A su vez dentro de este mismo grupo viene precedido por\n38,5% dentro de la categoría de riesgo moderado, que junto con la categoría anterior representan\n89% dentro de los pacientes que desarrollaron UPP. Al comparar con el grupo sin UPP tenemos 37,3%\nque se ubicó dentro del grupo de riego moderado, seguido de 27,8% dentro de la categoría sin riesgo,\nevidenciando una tendencia a puntajes mayores, lo que significa que poseen menos riesgo de\ndesarrollar UPP.\nRespecto de la sobrevivencia, estudios muestran que existe un riesgo aumentado de\nmortalidad, que puede ser 4 a 6 veces mayor en pacientes con UPP(20,22). En un estudio similar(18), el\ndesarrollo de UPP también se asoció significativamente a mayores índices de mortalidad (50,0%\ncontra 8,6% p<0,001), que es semejante a la relación encontrada en el presente estudio.\nEn cuanto a las limitaciones en esta investigación, se destaca que la presencia de UPP,\nmalnutrición, inmovilidad y pérdida de la independencia son manifestaciones de origen multifactorial,\npor lo cual el presente estudio no establece la causalidad de las mismas, sino que evidencia un\nproblema social que tiene su origen en la falta de recursos o experiencias de las familias o\ncomunidades. Como fortaleza en este estudio se registró la problemática del adulto mayor en este\nhospital. Se recomienda implementar en los centros de atención de salud, un seguimiento estadístico\ny metodológico que permita intervenir tempranamente ante la aparición de los primeros síntomas. Se\nenfatiza en la capacitación y educación a los cuidadores de adultos mayores de la comunidad.\n\nCONCLUSIÓN\nEl presente estudio señala la relación entre los indicadores de la escala de Braden, Prisma 7 y\nMNA, demostrando la relación entre estos en pacientes mayores de 65 años. La escala de Braden\ndemostró ser un buen predictor de UPP, sobre todo considerando que estas últimas son un problema\nfrecuente en el hospital. Es necesario prestar atención sanitaria que incluya acompañamiento\nmultidisciplinar, cuidado y tratamiento constante a las lesiones por presión.\n30\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nConflictos de interés\nLos autores, declaran no tener conflictos de intereses respecto a la investigación realizada.\nContribución de los autores\nMiguel Morales\u0001\na, b, c, d,e,g\nMarisa Ileana Gómez \u0001\na,b,c,d,e,f,g,\nIsmael Morales\u0001\nc, d, e, f, h,i\nBárbara Cerda\u0001\nd, e, i,j,k (adecuación a las normas de revista)\nMiguel Ángel Meriño\u0001\nd,e,f,h,i\na\u0001\nb\u0001\nc\u0001\nd\u0001\ne\u0001\nf\u0001\ng\u0001\nh\u0001\ni\u0001\nj\u0001\nk\u0001\n\nConcepción y diseño del trabajo\nRecolección/obtención de resultados\nAnálisis e interpretación de datos\nRedacción del manuscrito\nRevisión crítica del manuscrito\nAprobación de su versión final\nAporte de pacientes o material de estudio\nObtención de financiamiento\nAsesoría estadística\nAsesoría técnica o administrativa\nOtras contribuciones (definir)\n\nFinanciación\nAutofinanciado\n\nREFERENCIAS BIBLIOGRÁFICAS\n1. Cavalcanti EO, Kamada I. Lesão por pressão relacionada a dispositivo médico em adultos:\nRevisão integrativa. Texto Contexto Enferm [Internet].2020[citado 30 Abr 2020]; 29: e20180371.\nDisponible en:\n\nhttps://www.scielo.br/j/tce/a/srh9bf5wLRDLGLDw6W8CbQg/abstract/?lang=pt\nhttps://doi.org/10.1590/1980-265X-TCE-2018-0371\n2.\u0001 Ponchio Pachá HH, Lamana Faria JI, de Olivera KA, Beccaria LM. Pressure ulcer in intensive care\nunits: a case-control study. Rev Bras Enferm [Internet]. 2018 [citado 30 Abr 2020]; 71(6):3027-34.\nDisponible en: https://www.scielo.br/pdf/reben/v71n6/0034-7167-reben-71-06-3027.pdf.\nhttps://doi.org/10.1590/0034-7167-2017-0950\n3.\u0001 Fernandes Soares C, Schülter Buss Heidemann IT. Promoção da saúde e prevenção da lesão\npor pressão: expectativas do enfermeiro da atenção primária. Texto Contexto Enferm [Internet].\n2018 [citado 30 Abr 2020]; 27(2): e1630016. Disponible en:\nhttp://www.scielo.br/j/tce/a/6zsFqCkRtG75SMQhrcJxdSw/?format=pdf&lang=pt.\nhttps://doi.org/10.1590/0104070720180001630016\n4.\u0001 Guerrero Miralles M. Úlceras por presión: un problema potencial en los servicios de urgencias\ncolapsados. Gerokomos [Internet]. 2008 [citado 30 Abr 2020]; 19(2):99-106. Disponible en:\nhttp://scielo.isciii.es/pdf/geroko/v19n2/99rincon.pdf\n5.\u0001 López-Casanova P, Verdú-Soriano J, Berenguer-Pérez M, Soldevilla-Agreda J. Prevención de las\núlceras por presión y los cambios de postura. Revisión integrativa de la literatura. Gerokomos\n[Internet]. 2018 [citado 30 Abr 2020]; 29(2):92-9. Disponible en:\nhttp://scielo.isciii.es/pdf/geroko/v29n2/1134-928X-geroko-29-02-00092.pdf\n6.\u0001 Garza Hernández R, Meléndez Méndez MC, Fang Huerta MA, González Salinas JF, CastañedaHidalgo H, Argumendo Pérez NE. Conocimiento, actitud y barreras en enfermeras hacia las medidas\nde prevención de úlceras por presión. Cienc enferm [Internet]. 2017 [citado 30 Abr 2020]; 23(3):47Rev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n31\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\n58. Disponible en: https://scielo.conicyt.cl/pdf/cienf/v23n3/0717-9553-cienf-23-03-00047.pdf.\nhttp://dx.doi.org/10.4067/S0717-95532017000300047\n7.\u0001 Knoch Mendonça P, Dias Rolan M, Pereira Frota O, Schiaveto de Souza A. Prevenção de lesão\npor pressão: ações prescritas por enfermeiros de centros de terapia intensiva. Texto Contexto\nEnferm [Internet]. 2018 [citado 30 Abr 2020]; 27(4):1-10. Disponible en:\nhttp://www.scielo.br/j/tce/a/Z9CwyVqcD8MJqtqhy8gYjMG/?format=pdf&lang=pt.\nhttps://doi.org/10.1590/0104-07072018004610017\n8.\u0001 Caniupán J, Rivas E, Bustos L. Capacidad diagnóstica de las escalas Braden Q y Norton para\núlceras por presión en pacientes pediátricos de unidades críticas. Enfermería (Montevideo)\n[Internet]. 2018 [citado 30 Abr 2020]; 7(1):39-47. Disponible en:\nhttp://www.scielo.edu.uy/pdf/ech/v7n1/en_2393-6606-ech-7-01-17.pdf.\nhttp://dx.doi.org/10.22235/ech.v7i1.1539\n9.\u0001 Milena Roa Díaz Z, Parra DI, Camargo-Figuera F. Validación e índices de calidad de las escalas de\nBraden y Norton. Gerokomos [Internet]. 2017 [citado 30 Abr 2020]; 28(4):200-4. Disponible en:\nhttp://scielo.isciii.es/pdf/geroko/v28n4/1134-928X-geroko-28-04-00200.pdf.\n10. Faria Serpa L, Conceição de Gouveia Santos VL, Gonçalves Faustino Campanili TC, Queiroz M.\nValidez predictiva de la escala de Braden para el riesgo de desarrollo de úlceras por presión, en\npacientes críticos. Rev. Latino-Am. Enfermagem [Internet]. 2011 [citado 20 May 2021]; 19(1): 5057. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S010411692011000100008&lng=en. https://doi.org/10.1590/S0104-11692011000100008\n11. Albuquerque de Sousa C, dos Santos I, Dopico da Silva L. Aplicando recomendações da Escala\nde Braden e prevenindo úlceras por pressão - evidências do cuidar em enfermagem. Rev. bras.\nenferm. [Internet]. 2006 [citado 20 May 2021]; 59(3): 279-84. Disponible en:\nhttp://www.scielo.br/j/reben/a/Ct5FpP6n8FY7hgVWwthBVdP/?lang=pt&format=pdf.\nhttps://doi.org/10.1590/S0034-71672006000300006\n12. Fernandes Cabral J, Cândido da Silva AM, Echenique Mattos I, Queiroz Neves Á de, Lima Luz\nL, Bittencourt Ferreira D, et al. Vulnerabilidade e fatores associados em idosos atendidos pela\nestratégia saúde da família. Ciênc. saúde coletiva [Internet]. 2019 [citado 20 May 2021]; 24\n(9): 3227-236. Disponible en:\nhttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S141381232019000903227&lng=en.\nhttps://doi.org/10.1590/1413-81232018249.22962017\n13. Soysal P, Veronese N, Arik F, Kalan U, Smith L, Isik AT. Mini nutritional assessment scale-short\nform can be useful for frailty screening in older adults. Clin Interv Aging /Internet/. 2019 Apr 17/\ncited 2021 May 20/; 14:693-99. Available from:\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475097/pdf/cia-14-693.pdf.\ndoi: 10.2147/CIA.S196770\n14. Flores Saenger AL, Pereira Caldas C, Raîche M, Branco da Motta L. Identifying the loss of functional\nindependence of older people residing in the community: Validation of the PRISMA-7 instrument in\nBrazil. Arch Gerontol Geriatr. 2018; 74: 62-7. doi: 10.1016/j.archger.2017.09.008\n15. Hoffmann S, Wiben A, Kruse M, Jacobsen KK, Lembeck MA, Holm EA. Predictive validity of\nPRISMA-7 as a screening instrument for frailty in a hospital setting. BMJ Open / Internet/. 2020\nOct 28 /cited 20 May 2021/; 10(10): e038768. Available from:\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594371/pdf/bmjopen-2020-038768.pdf.\ndoi: 10.1136/bmjopen-2020-038768\n16.\u0001 Mitchell A. Adult pressure area care: Preventing pressure ulcers. Br J Nurs [Internet]. 2018\n[cited 2020 Abr 30]; 27(18):1050-2. Available from:\nhttps://www.magonlinelibrary.com/doi/epdf/10.12968/bjon.2018.27.18.1050.\nhttps://doi.org/10.12968/bjon.2018.27.18.1050\n17.\u0001 Briggs M, Collinson M, Wilson L, Rivers C, McGinnis E, Dealey C, et al. The prevalence of pain at\npressure areas and pressure ulcers in hospitalised patients. BMC Nurs [Internet]. 2013 [cited 2020\nAbr 30]; 12(1):19. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765382/.\ndoi: 10.1186/1472-6955-12-19\n18.\u0001 Bielecki M, Bielecki P, Żebrowski P, Misiak B, Lewko J. Operative treatment of pressure ulcers\n32\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n\fÚlceras por presión: riesgo, factores predisponentes y pronóstico hospitalario en pacientes mayores de 65 años\n\nen: https://www.umb.edu.pl/photo/pliki/progress-file/current_issue/progres_8.2/doi/105111_bielecki.pdf. DOI: 10.5604/01.3001.0012.8329\n19.\u0001 Meijers JMM, Schols JMGA, Jakson PA, Langer G, Clark M, Halfens RJ. Differences in nutritional\ncare in pressure ulcer patients whether or not using nutritional guidelines. Nutrition [Internet]. 2008\n[cited 2020 Abr 30]; 24(2):127-32. Available from:\nhttps://www.ncbi.nlm.nih.gov/pubmed/18061405. doi: 10.1016/j.nut.2007.10.010\n20.\u0001 Ho Ch, Jiang J, Eastwood CA, Wong H, Weaver B, Quan H. Validation of two case definitions to\nidentify pressure ulcers using hospital administrative data. BMJ Open [Internet] 2017 [cited 2020 Abr\n30]; 7(8) : e016438. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629722. doi:\n10.1136/bmjopen-2017-016438\n21.\u0001 Tomazini Borghardt A, Nascimiento do Prado T, Silveira Bicudo SD, Silveira de Castro D, de\nOliveira Bringuente ME. Pressure ulcers in critically ill patients: Incidence and associated factors.\nRev Bras Enferm [Internet]. 2016 [cited 2020 Abr 30]; 69(3):431-8. Available from:\nhttp://www.scielo.br/j/reben/a/9fxyf6GssK6fpN643Fh8H7J/?format=pdf&lang=en.\nhttps://doi.org/10.1590/0034-7167.2016690307i\n22.\u0001 Costa MP, Sturtz G, Pereira da Costa FP, Castro Ferreira M, Barros Filho TEP. Epidemiologia e\ntratamento das úlceras de pressão: Experiência de 77 casos. Acta Ortop bras [Internet]. 2005\n[citado 30 Abr 2020]; 13(3):124-33. Disponible en:\nhttp://www.scielo.br/j/aob/a/wC3d7VBNCgfBHnPBcyvjGSM/?format=pdf&lang=pt\nhttps://doi.org/10.1590/S1413-7852200 5000300005\n\nRev. virtual Soc. Parag. Med. Int. setiembre 2021; 8 (2):23-33\n\n33\n\n\f"
https://openalex.org/W19593112
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CANCER: Childhood Leukemia and Proximity to Nuclear Power Plants
Environmental health perspectives
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public-domain
4,371
Consumer Protection Act Sees Uneven Launch Some of those provisions have been addressed by the CPSC, but Dwyer thinks there are others. The biggest area of concern remains the expense and logistics of testing, he says. For instance, handheld X-ray fluores­ cence (XRF) machines provide an affordable method for determining the presence of lead in products but may not provide sensitive enough measures of lead content to meet CPSC requirements when the stay is lifted next February. In the case of lead testing, Dwyer says, “Our advice to our members is to use XRF testing as a marker. If it’s com­ ing up with lead content, make sure you do your due diligence and if necessary perform the necessary wet chemistry testing to verify compliance with the lead-level limits.” The Consumer Product Safety Improve­ ment Act (CPSIA), passed by Congress in 2008 in response to widespread recalls of lead-containing toys manufactured in China, imposed new testing and documen­ tation requirements upon manufacturers of juvenile products and gave the Consumer Product Safety Commission (CPSC) new enforcement powers to ensure compliance. Six months after the new law was scheduled to go into effect, however, it is still the sub­ ject of controversy and confusion. Another requirement, which did go into effect August 14, calls for manufacturers and importers to include tracking labels on all chil­ dren’s products manufactured after that date. Tracking labels must provide information about when and where a product was made, which will help officials locate specific chil­ dren’s products in the event of a safety recall. The rule also originally required that manu­ facturers include specific information such as batch and lot numbers, but small manufac­ turers objected to this provision because they lack the production volume to justify the sophisticated batching systems employed by larger manufacturers. In response, the CPSC allowed small manufacturers to comply solely by maintaining adequate records of the com­ ponents they use. The new law set maximum permissible levels of 600 ppm lead (used to inexpensively increase durability, weight, and paint bright­ ness) and 0.1% of three specified phthalates (plastic softeners) for products intended for children aged 12 years and younger. A more stringent lead level was phased in 14 August 2009, when the permissible level dropped to 300 ppm; in two years it will drop again, to 100 ppm. Manufacturers were to ensure their products do not exceed those levels prior to release into the marketplace. Consumer Protection Act Sees Uneven Launch p Rachel Weintraub, director of product safety and senior counsel at the Consumer Federation of America, is an advocate for lead-free children’s products and a strong CPSIA, but agrees that the stay on testing was a good idea. “There was confusion,” she says. “The CPSC was not as clear as they could have been.” During a 10 September 2009 hearing by the House Subcommittee on Commerce, Trade, and Consumer Protection, newly appointed CPSC chair­ woman Inez Tenenbaum pledged to “con­ tinue to solicit feedback from all involved par­ ties, and work to implement commonsense rules that are squarely focused on maximizing product safety and reducing administrative burdens.” Despite the confusion surrounding CPSIA, manufacturing interests and children’s health advocates alike agree it is an essential But the testing requirements met strong opposition from manufacturers, who argued that the timeline and content of the rules Jewelry may consist of a lead core coated with another material such as nickel. It may also be decorated with lead-containing enamel or assembled with lead solder. Weintraub points out that CPSIA marks a profound change for the CPSC because that agency had never had pre-market jurisdiction before. She contends the CPSC needs time to work out regulatory details of the law. “When the whole regulatory structure of an industry changes, there are going to be growing pains,” she says. “But once we see how it’s working, I think it will show that our products have changed for the better.” Jewelry may consist of a lead core coated with another material such as nickel. It may also be decorated with lead-containing enamel or assembled with lead solder. Jewelry may consist of a lead core coated with another material such as nickel. It may also be decorated with lead-containing enamel or assembled with lead solder. In the meantime, public health officials recommend that parents wash their children’s hands several times a day in case they have handled lead-containing products. They also suggest parents prevent children from put­ ting painted, metal, or plastic items in their mouths unless the parent is certain the item is safe. law, albeit a work in progress. “We supported the legislation from day one, as long as it didn’t put small operators out of business,” says Michael Dwyer, executive director of the Juvenile Products Manufacturers Association. Laws, regulations, and policy have to comply with some of these provi­ sions that are just unrealistic, that is a serious concern to us.” 2009 Federal Register that wood, natural fibers, and gemstones joined selected metals and alloys as materials that are exempt from testing. In addition, the CPSC clarified the conditions that must be present—such as security against leaching—in order for a lead- containing component part to be deemed “inaccessible” during “normal and reasonably foreseeable use and abuse of the product by a child.” Inaccessible component parts con­ tained inside a product, which typically are not touched or mouthed by a child, also are on the list of testing exemptions. Boston freelance writer Richard Dahl has contributed to EHP since 1995. He also writes periodically for the Massachusetts Institute of Technology. Forum News | Forum Forum News | Forum CANCER Childhood Leukemia and Proximity to Nuclear Power Plants The German KiKK (Kinderkrebs in der Umgebung von Kernkraftwerken) study, a case–control study described by Peter Kaatsch and colleagues in the 15 February 2008 issue of the International Journal of Cancer, found that children under age 5 years living within 5 km of an NPP were at more than double the normal risk of developing leukemia. Although similar links have been reported by several other authors, a number of ecological studies suggest that children living near NPPs are at no greater risk than other children. The KiKK study pinpointed the distance of individual case homes from each of the 16 German NPPs and was therefore better able to classify exposure than ecological studies, which use approximate distances to classify exposure. Nussbaum argues that ecological studies tend to average out the significant risk–distance association, especially when the number of cases is small. Others believe this stance is premature, given that the KiKK researchers were unable to adjust for any potential confounders besides sex and age—leaving the possibility that some factor besides radiation caused the children’s disease. Currently, however, ionizing radiation is the only established environmental risk factor for childhood leukemia, according to a review by Martin Belson and colleagues published in the January 2007 issue of EHP. At the very least, Nussbaum argued in the July–September 2009 issue of the International Journal of Occupational and Environmental Health, the KiKK study points out the need for a critical reexamination of the fundamental assumptions and models underlying current radiation safety standards and regulations. “Moreover,” says Bithell, “we are looking at a very small effect in terms of the actual numbers of sick children involved, and the statistical tools used have not always had the necessary power to allow conclusions to be drawn. Add all this to the fact that we do not actually know [all] the causes of leukemia, and you can see that it becomes difficult to firmly establish a link between it and NPPs.” According to industry records and the presently accepted canon of radiobiology as defined by national and international radiation regulatory bodies, children living near NPPs are exposed to doses of radiation orders of magnitude below those thought to cause leukemia. CANCER Childhood Leukemia and Proximity to Nuclear Power Plants wrote, “Since the deter­ mination of distance using the central point of the community was much less exact than using individual residential addresses, as in the case–control study, a correspondingly less clear measure of effect was to be xpected. In this respect the two approaches re not contradictory.” Co-author Claudia pix, deputy director of the German Childhood Cancer Registry at the Univ­ rsity of Mainz, explains, “We wished to demonstrate the basic agreement of the esults obtained by both approaches.” h l d h h Gundremmingen Nuclear Power Plant Günzburg, Bavaria of low-dose health effects from inhaled or ingested radioactive fallout at large distances from the Chernobyl nuclear disaster. Since the first report of increased childhood leukemia rates around Britain’s Sellafield nuclear power plant (NPP) in 1983, contro­ versy has surrounded the possible link between the disease and proximity to nuclear reactors. Twenty-five years later the debate rages on, with different studies yielding seemingly con­ tradictory findings. A public sensitized to the dangers of nuclear power might well ask the question: why aren’t we sure by now? It is challenging—but not impossible—to estimate the effective dose of ionizing radiation from an NPP to which a child may have been exposed over the years, says Joseph Mangano, executive director of the nonprofit Radiation and Public Health Project. Measurements of in-body levels of radioactivity are critical to resolve this issue, he says. Perhaps the most feasible way to take such measurements is to test for bone-seeking isotopes in baby teeth. Some researchers conclude that the consistency between the KiKK study findings and comparable ecological studies proves the real controversy is no longer about the validity of the leukemia–distance association. “Rather,” says Nussbaum, “it involves both the mechanism of the disease initiation and the public health implications of the confirmed leukemia clusters near NPPs.” “The many studies that have been performed are difficult to compare because of differences in their methodology,” explains John Bithell, honorary visiting fellow at the Childhood Cancer Research Group, University of Oxford. These differences include the age groups studied, the geographical areas considered, and potential confounding factors such as socioeconomic status. Adrian Burton is a biologist living in Spain who also writes regularly for The Lancet Oncology, The Lancet Neurology, and Frontiers in Ecology and the Environment. Consumer Protection Act Sees Uneven Launch “For bigger companies, it might still be a burden, but they have the resources at their disposal [for testing and tracking]. But when you’ve got mom-and-pop companies that imposed an unrealistic burden on small and large producers alike. Days before the require­ ments were to go into effect, the CPSC issued a one-year stay of enforcement until 10 February 2010 to provide agency staff time to develop guidance on when and how testing is to be done, and on what materials. Such guidance has begun to emerge. The CPSC announced in the 7 August CPSC A 436 volume 117 | number 10 | October 2009  •  Environmental Health Perspectives Gundremmingen Nuclear Power Plant Günzburg, Bavaria Sweeteners Persist in Waterways Artificial sweeteners are widespread in European sewage treatment plant effluent, waterways, groundwater, and even drinking water, a growing body of research demonstrates. One of the latest studies, pub­ lished in the July 2009 issue of Analytical and Bioanalytical Chemistry, presents data on four common sweeteners found in German water and demonstrates the persistence of these additives. Two, acesulfame and sucralose, were remarkably resistant to treatment by conventional sewage treatment plants as well as by a more advanced soil aquifer treatment plant, report environmental engineer Marco Scheurer and colleagues from the Water Technology Center in Karlsruhe. All the sources interviewed for this article agree there’s little risk that acesulfame and sucralose in drinking water will cause human health problems. The implications for the aquatic environment are less clear, however. Because these sweeteners have been classified as safe for human consumption, they have undergone virtually no envi­ ronmental testing. Yet the remarkable persistence of acesulfame and sucralose gives some experts pause, given that environmental concen­ trations will likely rise over time with continued consumption. y p Henrik Kylin, an environmental chemist at the Norwegian Institute for Air Research and a coauthor of a study on sucralose presented at the Society of Environmental Toxicology and Chemistry Europe 17th Annual Meeting in 2007, points out that sucralose mimics sucrose, a structurally similar molecule involved in biological functions from the regulation of genes related to photosynthesis to feeding cues in zooplankton. “If those [functions] are affected, you may end up with serious ecosystem effects,” he says. In samples taken from four German rivers, concentrations of ace­ sulfame exceeded 2 μg/L, whereas concentrations of sucralose, cycla­ mate, and saccharin were an order of magnitude lower. Coauthor Frank T. Lange, an analytical chemist, notes that a person would have to drink 2–3 L of water with sweetener concentrations similar to those of the German rivers every day for years before they would con­ sume the amount contained in a single sweetener tablet (the concen­ trations detected in water were well below human taste thresholds). Three other sweeteners—aspartame, neotame, and neohesperidin dihydrochalcone—were not detected in any of the samples. y p y y Kylin is also concerned by the finding, reported in the October 2006 issue of Plant, Cell & Environment, that sucralose at least partially inhibited sucrose transport in sugarcane. Rebecca Kessler, based in Providence, Rhode Island, is a senior editor at Natural History and writes about science and the environment for various publications. She is a member of the National Association of Science Writers and the Society of Environmental Journalists. Sweeteners Persist in Waterways “There are very many vascular plants in the aquatic ecosystem,” he says, “and if they are [similarly] affected, it would affect very many other organisms.” The findings echo those of four recent studies documenting artificial sweeteners in sewage treatment plant effluent and waterways throughout Europe. Preliminary results from two separate studies— one unpublished and one accepted 11 September 2009 for publica­ tion in Marine Chemistry—also show sucralose in Arizona wastewater treatment plant effluent and several downstream rivers, as well as in coastal and Gulf Stream waters off the southeastern United States. Rosa Krajmalnik-Brown, an assistant professor of environmental biotechnology at Arizona State University, has been working for more than two years to identify a microorganism that can degrade sucralose and hasn’t found one yet—although she says she’s not giv­ ing up. Still, there may be a bright side to sweeteners’ persistence: Scheurer and other researchers have proposed using them as markers for detecting wastewater spills—a welcome finding for scientists who have long sought a failsafe marker. Acesulfame and sucralose, which is sold in the United States as Splenda®, have proven to be the most commonly found and resilient sugar substitutes. They are added to a wide variety of foods, beverages, pharmaceuticals, and toiletries, and they pass through the human body virtually unchanged. Cyclamate and saccharin are much less persistent. School Averts EMF-Related Closure The debate over the safety of electromagnetic fields (EMFs) nearly closed a New Jersey school this fall until the state’s biggest electric utility and the Sussex County Board of Education reached an eleventh-hour settlement. The board had planned to close the K–6 Fredon Township School 1 October 2009 because the existing high-voltage power line crossing the school’s playground had been found to Fredon Township School playground concentrations inside vehicles increased twofold for every cigarette smoked, and while opening the windows reduced smoke somewhat, it did not eliminate exposure within the vehicle. Breysse et al. state the levels are unacceptable for nonsmoking passengers, especially children, who are at increased risk for secondhand smoke– related health problems.. The Beat e Beat | by Erin E. Dooley School Averts EMF-Related Closure The debate over the safety of electromagnetic fields (EMFs) nearly closed a New Jersey school this fall until the state’s biggest electric utility and the Sussex County Board of Education reached an eleventh-hour settlement. The board had planned to close the K–6 Fredon Township School 1 October 2009 because the existing high-voltage power line crossing the school’s playground had been found to Fredon Township School playground by Erin E. Dooley Vehicles Concentrate Nicotine A study by Patrick Breysse et al. published online 25 August 2009 ahead of print in Tobacco Control found that vehicle passengers riding with smokers may be exposed to nicotine levels 40–50% higher than those found in restaurants and bars that permit smoking. Nicotine Left to right: Aaron Amat/Shutterstock; Aristide Economopoulos/The Star-Ledger CANCER Childhood Leukemia and Proximity to Nuclear Power Plants However, Rudi Nussbaum, a professor emeritus of physics and environmental sciences at Portland State University, says evidence of extreme radiation sensitivity of embryos and fetuses has been largely ignored in this canon, as have reports y g In a time when many governments are exploring alternatives to fossil fuel– based energy, nuclear power also remains controversial because of unresolved questions about the safe storage of radioactive waste and the potential for radioactive contamination stemming from accidents or terrorist attacks. In any area where science, politics, and powerful commercial interests meet, it is critical to focus on the science, says Mangano. “Studies of childhood cancer and leukemia from exposure to nuclear reactor emissions have been clouded by political factors,” he says. “A challenge to objective, dispassionate science is to overcome this and help policy makers make the right decisions in this emotive area.” Alfred Körblein, a retired physicist formerly with the Munich Environmental Institute, a German nongovernmental organization, makes a similar observation regarding a recent re-analysis of the KiKK data published in volume 105, issue 42 (2008) of Deutsches Ärtzeblatt International, which used approximate distances to estimate exposures. “This ecological analysis of the same data yielded only a nonsignificant 41% increase [in leukemia incidence] in the 0- to 5-km zone compared to the 119% increase in the superior case–control analysis,” he says. “But that’s what you’d expect when using the weaker ecological approach.” Suggested Reading Baker PG and Hoel DG. Eur J Cancer Care 2007;16:355–363 Bithell JF et al. Radiat Prot Dosimetry 2008;132:191–197 Busby C et al. Med Confl Surviv 2009;25:20–40 COMARE 10th report. J Radiol Prot 2005;25:335–336 Evrard AS et al. Br J Cancer 2006;94:1342–1347 Fairlie I. Med Confl Surviv 2009;25:197–220 Hoffmann W et al. Environ Health Perspect 2007;115:947–952 Kaatsch P et al. Int J Cancer 2008;122:721–726 Kaatsch P et al. Dtsch Ärztebl Int 2008;105:725–732 Laurier D et al. Radiat Prot Dosimetry 2008; 132[2]:182–190 Mangano J and Sherman JD. Eur J Cancer Care 2008;17:416–418 Nussbaum RH. Int J Occup Environ Health 2009;15:318–323 In fact, in their 2008 ecological analysis Kaatsch and colleagues A 437 Environmental Health Perspectives  •  volume 117 | number 10 | October 2009 Forum Cyclamate has been banned in the United States since 1970 as a pos­ sible human carcinogen, but the Food and Drug Administration is considering reapproval. And although saccharin has been found to cause cancer in rats, it is considered safe for human consumption. volume 117 | number 10 | October 2009  •  Environmental Health Perspectives Next Course in Organic Debate With the Northern Hemisphere’s peaking summer produce crop came a new iteration of the question of whether organic food is worth the extra expense. According to a review commissioned by the U.K. Food Standards Agency and published 29 July 2009 ahead of print in the September 2009 American Journal of Clinical Nutrition, organically grown food is no more nutritious than conventionally grown food. But organic proponents question the findings and also note that the health benefits of organic agriculture can go beyond nutrition. Another criticism: the study did not require organic fields to have been used as such for a minimum number of years. “We know from a large body of research that the biological benefits of organic farm­ ing mostly come from improvements in soil quality,” says Benbrook. Moreover, long-term studies of organic and conventional tomatoes by food chemist Alyson Mitchell at the University of California, Davis, have demonstrated that soil organic matter takes at least five years to reach optimal levels. And national standards defining organic produc­ tion practices were not established until 2002. The review authors identified more than 52,000 studies dating back to 1958 that compared organic and conventional foods. Of these, 55 studies were deemed of sufficient quality. None of the studies predate 1990—an important point, says first author Alan D. Dangour, a senior lecturer at the London School of Hygiene & Tropical Medicine, because one chief criticism of the review has been the fact that agriculture has changed markedly over the past 60 years. Mitchell cautions against drawing sweeping conclusions from com­ parison studies of organic versus conventional. She notes that organic practices vary immensely, with some modern industrial-scale organic farms using methods more similar to those of conventional farms, such as growing just a single crop (monoculture). Of 11 parameters examined, organic crops had significantly higher phosphorus content and titratable acidity (which is not a nutrient but a food processing metric) whereas conventional crops had higher nitrogen content. Differences in levels of vitamin C, phenolic compounds, mag­ nesium, calcium, potassium, zinc, total soluble solids (mostly sugar), and copper were insignificant. But Benbrook says sustainably conducted organic farming offers benefits beyond nutrition, including improved health of pollinators and cleaner waterways resulting from minimal pesticide use, significant carbon sequestration as soil builds up through organic cultivation, and potential shrinkage of oceanic dead zones due to reduced nitrogen fertilizer pollu­ tion. Take-Home Dust Boosts Car Seat Lead Levels the August 2009 issue of HortScience finds they can also release these chemicals. The authors found these four types of houseplants released 12–23 VOCs, and although the researchers did not quantify potential exposures, they did note that emissions were higher during the day than at night. The authors attributed the VOCs to pesticides used in nurseries, microorganisms living in the growing medium, and offgassing of plastic planters. Several studies have established that lead- exposed workers can carry lead-contaminated dust off the jobsite on their clothing, shoes, and tools. In the 21 August 2009 MMWR Tina Bernier et al. report the first known cases of childhood lead poisoning attributed to take- home lead dust deposited onto car safety seats. No contamination was found in the six children’s homes, leading the researchers to examine family vehicles and car seats, where high lead levels were found. Although previous studies have recommended monitoring blood lead levels among children of lead-exposed workers, no standards exist for levels of lead dust contamination in vehicles or on child car safety seats. David C. Holzman writes from Lexington and Wellfleet, Massachusetts, on science, medicine, energy, economics, and cars. He has written for EHP since 1996. Next Course in Organic Debate Melissa Perry, an associate professor of occupational epidemiology at the Harvard School of Public Health, also says children on organic diets have shown significantly lower levels of pesticide metabolites in their urine than children on conventional diets. As to the dangers thereof, Perry says the risk assessments conducted so far by the Environmental Protection Agency are limited because they do not routinely account for cumulative and potentially synergistic effects of multiple pesticides. The results contradict a review published as the report New Evidence Confirms the Nutritional Superiority of Plant-Based Organic Foods in March 2008 by The Organic Center, a nonprofit food research outfit. This review found that total phenolics, vitamin E, vitamin C, quercetin, and total antioxidant capacity of organics exceeded that of conventionally grown produce—in the case of total antioxidant capac­ ity, by 80%. Conventional products had higher levels of potassium, phosphorous, and total protein, all basic constituents of conventional fertilizers. Nutrition scientist Denis Lairon reported similar findings in a review published online 8 July 2009 ahead of print in Agronomy for Sustainable Development. emit EMF levels more than 8 times the WHO- recommended maximum of 3 milligauss. Under the settlement, Public Service Electric & Gas Co. agreed to pay $95,000 to relocate playgrounds located under its lines. There is limited evidence that EMFs from power lines may be a risk factor for childhood leukemia. diet and nutrition diet and nutrition Charles Benbrook, The Organic Center’s chief scientist and coauthor of the New Evidence report, criticizes the U.K. review for not requiring the individual studies to have used the same cultivars on organic and conven­ tional plots. Differences in plant varieties—for instance, between hybrid and heirloom varieties of tomatoes—can result in dramatic differences in nutrient content, he says. “We went with what was available,” responds Dangour. “In general I recall most studies compared the same cultivars.” Colorimetric “Nose” Alerts Chemical Handlers A 439 In a report published online 13 September 2009 in Nature Chemistry, Kenneth Suslick and colleagues present a postage stamp–sized electronic sensor capable of quickly and inexpensively detecting toxic chemicals and their concentrations through color visualization. The pattern created by color changes in the disposable 36-dye sensor array identifies both the toxicant and its concentration. The sensor can detect more chemicals than previous methods and produces most results within 2 minutes. The researchers have developed a handheld version similar to a card-scanning device that uses LED illumination and an ordinary camera. USDA Maps Local Food Production USDA Maps Local Food Production Until recently, low fuel prices meant the U.S. food system could rely on food imported from other countries, but rising transport costs and regional food shortages and crop failures are among several factors encouraging a closer look at local food production. Wayne Honeycutt and colleagues at the Agricultural Research Service are now mapping data from Maine to Virginia on weather, soil, land use, and water availability to model potential crop production and determine local food production capacity. They say expanding opportunities for local food production could stimulate rural development and offset the risk of food shortages by diversifying and increasing local production in other areas. Left to right: Aaron Amat/Shutterstock; Aristide Economo School Averts EMF-Related Closure A 438 volume 117 | number 10 | October 2009  •  Environmental Health Perspectives Forum Forum Environmental Health Perspectives  •  volume 117 | number 10 | October 2009 Indoor Greenery Releases VOCs Indoor Greenery Releases VOCs Peace lilies, snake plants, weeping figs (ficus trees), and areca palms are just a few of the houseplants that have been shown to remove volatile organic compounds (VOCs) from indoor air, but a new study by Dong Sik Yang et al. in A 439 Environmental Health Perspectives  •  volume 117 | number 10 | October 2009
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Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength
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Clark, A. P., Howard, K. L., Woods, A. T., Penton-Voak, I. S., & Neumann, C. (2018). Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength. PLoS ONE, 13(1), Article e0190393. https://doi.org/10.1371/journal.pone.0190393 Clark, A. P., Howard, K. L., Woods, A. T., Penton-Voak, I. S., & Neumann, C. (2018). Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength. PLoS ONE, 13(1), Article e0190393. https://doi.org/10.1371/journal.pone.0190393 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1371/journal.pone.0190393 Link to publication record on the Bristol Research Port PDF-document Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1371/journal.pone.0190393 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via PLOS at https://doi.org/10.1371/journal.pone.0190393 . Please refer to any applicable terms of use of the publisher. Clark, A. P., Howard, K. L., Woods, A. T., Penton-Voak, I. S., & Neumann, C. (2018). Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength. PLoS ONE, 13(1), Article e0190393. https://doi.org/10.1371/journal.pone.0190393 Clark, A. P., Howard, K. L., Woods, A. T., Penton-Voak, I. S., & Neumann, C. (2018). Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength. PLoS ONE, 13(1), Article e0190393. https://doi.org/10.1371/journal.pone.0190393 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/ RESEARCH ARTICLE Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength Andrew P. Clark1*, Kate L. Howard2, Andy T. Woods3,4, Ian S. Penton-Voak2, Christof Neumann5 1 Brunel University London, Uxbridge, United Kingdom, 2 University of Bristol, Bristol, United Kingdom, 3 Xperiment, Lausanne, Switzerland, 4 University of Oxford, Oxford, United Kingdom, 5 Universite´ de Neuchaˆtel, Neuchaˆtel, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * andrew.clark@brunel.ac.uk * andrew.clark@brunel.ac.uk * andrew.clark@brunel.ac.uk Abstract We introduce “EloChoice”, a package for R which uses Elo rating to assess pairwise com- parisons between stimuli in order to measure perceived stimulus characteristics. To demon- strate the package and compare results from forced choice pairwise comparisons to those from more standard single stimulus rating tasks using Likert (or Likert-type) items, we inves- tigated perceptions of physical strength from images of male bodies. The stimulus set com- prised images of 82 men standing on a raised platform with minimal clothing. Strength- related anthropometrics and grip strength measurements were available for each man in the set. UK laboratory participants (Study 1) and US online participants (Study 2) viewed all images in both a Likert rating task, to collect mean Likert scores, and a pairwise compari- son task, to calculate Elo, mean Elo (mElo), and Bradley-Terry scores. Within both studies, Likert, Elo and Bradley-Terry scores were closely correlated to mElo scores (all rs > 0.95), and all measures were correlated with stimulus grip strength (all rs > 0.38) and body size (all rs > 0.59). However, mElo scores were less variable than Elo scores and were hundreds of times quicker to compute than Bradley-Terry scores. Responses in pairwise comparison trials were 2/3 quicker than in Likert tasks, indicating that participants found pairwise com- parisons to be easier. In addition, mElo scores generated from a data set with half the partic- ipants randomly excluded produced very comparable results to those produced with Likert scores from the full participant set, indicating that researchers require fewer participants when using pairwise comparisons. Editor: Iratxe Puebla, Public Library of Science, UNITED KINGDOM Received: June 15, 2016 Accepted: December 14, 2017 Published: January 2, 2018 Copyright: © 2018 Clark 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 from this study is shared in a public repository (figshare: https://figshare.com/articles/Why_rate_when_you_ could_compare_Elo_strength_data/4902977). Funding: Leverhulme Trust (www.leverhulme.ac. uk) provided a Research Grant [F/00 182/CJ (2011-2013)] to ISP-V and APC. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, and did not provide support in the form of salaries. The specific roles of these authors OPEN ACCESS Citation: Clark AP, Howard KL, Woods AT, Penton- Voak IS, Neumann C (2018) Why rate when you could compare? Using the “EloChoice” package to assess pairwise comparisons of perceived physical strength. PLoS ONE 13(1): e0190393. https://doi. org/10.1371/journal.pone.0190393 Editor: Iratxe Puebla, Public Library of Science, UNITED KINGDOM Editor: Iratxe Puebla, Public Library of Science, UNITED KINGDOM More generally, using a rating scale can increase cognitive demand as it requires conscientious raters to monitor their responses to each stimuli and retrospectively compare them to responses from previous perceptive states, whereas paired comparisons imposes no such constraint [9]. One alternative to Likert rating is to show stimuli in pairs and ask participants to choose which one better expresses some characteristic, for example, which of two faces is more attrac- tive. The results of these pairwise comparisons can be used to quantify perceptions of a stimu- lus according to a mathematical model that calculates its probability of being judged to be more or less expressive than a given set of alternatives. This method was first developed in the context of psychological research by Thurstone [10], but developed further by Bradley & Terry [11]. A class of mathematically-related models are now often known as Bradley-Terry models [12–13], and include a variety of sophisticated extensions and approaches to model-fitting [9, 14–16]. Also related are models that were independently derived to rank chess players—the earliest by Zermelo [17] and the most famous by Elo [18], which in turn has led to more com- plex derivatives such as Glicko [19] and TrueSkill™[20]. There are a number of advantages associated with pairwise comparisons. They are less com- plicated for participants: there are only two choices for each decision, the choices do not require an understanding of number lines, and there is no need to track responses from previous deci- sions. Besides this, each decision made provides information about two stimuli, therefore representing a more efficient use of participant time. In previous contrasts, results from Likert ratings correlate strongly with pairwise comparison methods [21–22]. Nevertheless, pairwise comparisons are much less used, perhaps because many Bradley-Terry models, although simple by the standards of mathematicians, are comparatively complex and computationally intensive. However, this is no longer a serious constraint as the hardware and software capabilities to over- come it are readily available. Elo rating, in particular, is a relatively simple approach that offers conceptual accessibility, widespread familiarity and use [23–27], and recent programming implementations [28–29]. The purpose of this paper is to demonstrate the application of Elo rat- ing to pairwise comparisons of stimuli, and introduce “EloChoice”, an R package optimized for this use and designed to be accessible even to those with limited experience of the free, and increasingly popular, R programming environment [30]. Why rate when you could compare? attractiveness (“characteristic”) of an image depicting a face (i.e. “stimulus”) along a gradient between not attractive at all and highly attractive that could be scored along integers between 1 and 7. This is often referred to as a Likert scale [1–3] but this is technically inaccurate as Likert himself used “scale” to refer to a group of multiple such items [4]. Discrete Visual Analogue Scale is suggested as a general alternative [5], but we will simply refer to the practice as “Likert rating”. When inter-rater agreement is high, and average responses are calculated from a rea- sonable sample of raters, Likert rating tends to produce repeatable results that convey useful information about the stimuli [6]. This represents a mathematically straight-forward process that is easy for researchers with even minimal technical ability to complete using very basic and readily available hardware and software tools. are articulated in the ‘author contributions’ section. Xperiment, a commercial company founded by ATW, had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Neither Xperiment nor ATW were paid to perform the study - he provided his services and the platform that he created for free. The specific role of this author is articulated in the ‘author contributions’ section. are articulated in the ‘author contributions’ section. Xperiment, a commercial company founded by ATW, had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Neither Xperiment nor ATW were paid to perform the study - he provided his services and the platform that he created for free. The specific role of this author is articulated in the ‘author contributions’ section. Competing interests: One of our authors, ATW, is the founder of Xperiment, but neither he nor the company were paid in relation to this study. This affiliation did not interfere with the full and objective presentation of our results, nor alter our adherence to PLOS ONE policies on sharing data and materials. We declare no further competing interests. From an individual participants’ perspective, however, the rating experience could be bet- ter. The scale itself can be a challenging concept for users with a weak grasp of number lines; for instance, small children [7], or members of some traditional, pre-literate cultures [8]. The package including its source code is available from https://cran.r-project.org/web/packages/EloChoice/index.html and includes a detailed manual (https://cran.r-project.org/web/packages/EloChoice/vignettes/tutorial.pdf). Likert-type rating vs pairwise comparisons When a researcher wants to quantify some perceived characteristic for stimuli within a set, one way to achieve this is to ask participants to rate each stimulus along an integer scale, anchored on either end with bivalent labels. For example, a rater could be asked to evaluate the When a researcher wants to quantify some perceived characteristic for stimuli within a set, one way to achieve this is to ask participants to rate each stimulus along an integer scale, y p p g g anchored on either end with bivalent labels. For example, a rater could be asked to evaluate the PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 1 / 16 The following paragraphs outline the Elo rating process in brief, but further details and more in-depth discussion are readily available [18, 23, 31, 29]. Elo rating The following paragraphs outline the Elo rating process in brief, but further details and more in-depth discussion are readily available [18, 23, 31, 29]. 2 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Why rate when you could compare? Elo rating was developed to quantify and track over time skill distribution amongst chess players using information available from their previous matches. After a match between two players, points are exchanged depending on both players’ prior probability of winning the match. Each player enters with a score based on their match records, and the disparity between scores is used to predict the probability of outcomes, according to the following formulas, which assume a logistical distribution [18]: E1 ¼ 1 1 þ 10ðS2S1Þ=400 ð1Þ E2 ¼ 1 1 þ 10ðS1S2Þ=400 ð2Þ ð1Þ ð2Þ where E1 and E2 are the estimated probabilities of a win for players 1 and 2 respectively, and S1 and S2 are their current scores. Players with equal scores are expected to be equally likely to win. After each new match, more information is available and is used to update each player’s scores, according to the following formulas: S0 1 ¼ S1 þ kðO1 E1Þ ð3Þ S0 2 ¼ S2 þ kðO2 E2Þ ð4Þ ð3Þ S0 2 ¼ S2 þ kðO2 E2Þ ð4Þ ð4Þ where O1 and O2 are the actual outcomes for players 1 and 2 respectively (win = 1, loss = 0), S’1 and S’2 are their new scores, and k is a constant representing maximum point exchange. The exact value of k affects how quickly scores change and their eventual range, but, given a suffi- cient number of matches, will have a limited effect on player rankings [29, 31–32]. A key feature of the system is that the less expected the win, the more scores change (both for winner and loser). An unsurprising victory over a far weaker opponent will not result in a large score change, but an upset win will. Therefore, the highest scores require wins over most other opponents, including those with above-average ability. Elo rating can be readily adapted to quantify the distribution of perceived attributes for a set of stimuli on an interval scale, allowing simple side-by-side comparisons while taking into account disparity between stimuli. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Elo rating Each pairwise comparison can be seen as a contest between two stimuli, with the one chosen as a better exemplar of some attribute being the winner. All stimuli begin with the same starting score, but diverge as successive comparisons take place. Because the number of points exchanged in any comparison is symmetrical (the winner’s gains equal the loser’s losses), the mean score will always equal the starting score, so long as the number of stimuli in the set remains stable. When stimuli are paired randomly, the scores should quickly converge to a stable ranking, assuming sufficient variation among stimuli and a reasonable degree of shared perceptions among rating participants. As with chess players, there is no requirement that all combinations of stimuli are observed, and, indeed, this is one of the system’s chief advantages [33]. There are at least two problems associated with the Elo rating system in the context of this application, but both are readily surmountable. The first problem arises from the fact that final scores can be influenced by the sequence order of contests, because the same contest outcomes will produce different results depending on the order in which they are observed. This is appropriate when the targets being scored are entities that may become weaker or stronger with time (such as players gaining or losing skill), but psychological stimuli are typically static in this regard and hence the order of events is not capturing any useful information. As there is nothing special about the sequence of contests presented to participants, any variation in PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 3 / 16 Why rate when you could compare? final scores introduced by sequence variation is undesirable noise. To counter this problem, we propose a simple fix; randomly shuffle the original sequence presented to participants mul- tiple times to create multiple virtual sequences, and average Elo scores for each stimuli from the different sequences to generate mean Elo scores that are free from sequence noise. final scores introduced by sequence variation is undesirable noise. To counter this problem, we propose a simple fix; randomly shuffle the original sequence presented to participants mul- tiple times to create multiple virtual sequences, and average Elo scores for each stimuli from the different sequences to generate mean Elo scores that are free from sequence noise. Elo rating A second problem is that there is currently no commonly accepted way to measure consis- tency across paired comparison trials. It is often desirable to measure the consistency of ratings for stimuli across multiple raters (to gauge the extent to which raters share perceptions about targets), and in the case of Likert rating this is most appropriately achieved by calculating the intraclass correlation coefficient (ICC) [34–35]. To provide a conceptually (but not directly) comparable value, our solution is to propose a novel measure, the Elo consistency index, which tracks how often outcomes deviate from predictions based on previous judgments (that is, those trials in which the stimulus with the lower Elo score was chosen–in other words, an upset or reversal). Because the preceding Elo score is generated by the judgements made in previous comparisons, violations of expected outcomes can be seen as inconsistency between judgements. This inconsistency can occur both between and within raters. The index will be biased towards tracking one form of inconsistency or the other depending on the ratio of raters to the times each rater makes a judgement on each stimulus. In the case that each rater makes only one judgement on each stimulus, the index will be entirely tracking between-rater consis- tency (because each preceding Elo score was generated by judgements made by previous raters only). The index can be calculated for any sequence of trials (across any number of raters) and is defined by the following formula: R ¼ 1 PN i¼1 ui N ð5Þ ð5Þ where u is a vector of 1’s and 0’s, in which a 1 indicates that the expected outcome was violated and a 0 indicates that it was not, and N is the total number of trials for which an expectation existed (that is, for all trials in which the preceding Elo score difference was not 0). The result- ing index value will vary between 0 and 1. Index values approach 1 as fewer expectation viola- tions (or upsets/reversals) are observed, and approach 0 as more are observed. An index value of 0.5 indicates random choice, but values of less than 0.5 are technically possible. Index values for each sequence generated can be averaged to produce a mean consistency index score to correspond with mean Elo scores. Methods Target stimuli. Stimuli consisted of digital images (399 x 710 pixels) of 82 men (mean age = 21.4, sd = 2.5, min = 18, max = 30) depicted standing on a raised platform and facing the camera. The men are wearing only a pair of black boxer briefs. Their heads are digitally blurred, making positive identification from face alone impossible. Images were all captured from the same camera, mounted on a tripod at the same height, location and orientation. Anthropometric measures were collected from each man on the day he was image was cap- tured. The men who posed for the images gave written informed consent beforehand, and were reimbursed for their time (£40 GBP). Approval for their participation was given by the Faculty of Science Human Research Ethics Committee, at the University of Bristol. Stimuli measures. We used hand grip strength as an assay of overall physical strength, as it easy to collect and previous research has shown it to be highly correlated with other mea- sures, such as chest and shoulder strength [36]. Each man who posed for stimulus images was instructed to squeeze a hand dynamometer as hard as he could using one hand. Trials were completed for both the right hand and the left hand separately. For each man, the average of these two squeezes (measured in kg) was taken and used as the grip strength score associated with the stimulus image (mean = 39.5, sd = 7.1). Body size has also been found to correlate with upper-body strength in males [36]. We used six anthropometric measures to construct a proxy of body size: height, weight, shoulder circumference, chest circumference, bicep circumference and forearm circumference. We per- formed a principal components analysis on these measures and used scores for the first princi- pal component (explaining 72.4% of total variance) as our body size score (PC1 of body size; mean = 0, sd = 2.1). Grip strength and PC1 of body size were moderately correlated (r = 0.46). Rating participants. 56 participants (28 male, 28 female; mean age = 19.9, sd = 1.98) took part in the rating study. These participants were students recruited from the University of Bristol either by opportunity sampling (word of mouth) or for partial course credit in the undergraduate Psychology programme. Participants gave written informed consent before completing any task. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Elo rating A weighted version of the Elo consistency index can be calculated by taking into account the score disparity observed in expectation violations, according to the following formula: R0 ¼ 1 XN i¼1 uiwi P w ð6Þ ð6Þ where u is the same vector described above and w is the absolute difference in preceding Elo scores between members of a trial. This can be understood as the proportion of all points exchanged in a sequence that were correctly predicted by preceding Elo scores. The weighted consistency index will again vary between 0 and 1, and larger expectation violations (that is, where the difference in preceding Elo scores is greater) will negatively impact values of R more. Generally the weighted consistency index value is expected to be greater than the unweighted value, as the magnitude of difference between Elo scores should negatively predict probability of an expectation violation. To demonstrate Elo rating in action and to compare it both to Likert rating and to a basic Bradley-Terry model, we use the example of perceived physical strength from stimuli com- posed of images of human male bodies. Previous research using Likert-type ratings have shown that participants judging strength from photographs of men’s bodies display high inter- PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 4 / 16 Why rate when you could compare? rater agreement, and mean ratings correlate robustly with men’s actual measured strength and strength-related body measurements [36]. We use both laboratory (Study 1) and online partic- ipants (Study 2) to demonstrate that the Elo-rating method is appropriate in both contexts. Why rate when you could compare? right. Again, response time was recorded. 41 responses were required to complete each block, and 82 responses were required to complete the task. right. Again, response time was recorded. 41 responses were required to complete each block, and 82 responses were required to complete the task. Rating measures. For each stimulus image, Likert responses from all rating participants were averaged to generate a mean Likert rating score. To generate Elo scores for each stimulus image, all paired comparison trials were arranged in their original sequence, ordered first by rating participant number and then by trial number. Beginning Elo scores were set to 0, and k was arbitrarily set to 100, following precedent [29]. Using Formulas 1–4, updates to Elo scores were calculated for each trial sequentially—each trial involved updating scores for two images. Final Elo scores were the scores for each image after the calculation performed for the last trial within the sequence. Elo scores were calculated for each image using the original sequence, as described above, and then for 99 randomized trial sequences. Final Elo scores from each of the 100 sequences were averaged together to generate mean Elo (mElo) scores for each image. For comparative purposes, an additional set of mElo scores was generated by averaging Elo scores across 1000 sequences (the original sequence plus 999 randomized sequences). These are referred to as mElo(M) scores in the subsequent analyses. Scores were collected by extracting worth parameters from a Bradley-Terry model fit using the paired comparison data. As these scores are highly skewed, they are log-transformed for the purposes of correlations. Statistical software. All analyses were conducted in R version 3.2.1 [30]. Principal components analysis was conducted using the “FactoMineR” package [37]. El d El d th El i t i di l l t d i th “El Ch i ” Elo and mElo scores and the Elo consistency indices were calculated using the “EloChoice” package [38]. Bradley-Terry models were fit and worth parameters extracted using the “psychotools” package [39]. Comparisons between overlapping and non-overlapping correlations from dependent groups were conducted using the “cocor” package [40] and Williams’s t [41]. Intraclass correlation coefficients were calculated using “irr” package [42]. Comparisons between means from dependent groups were conducted using the “perfect-t- test” script [43]. Methods Approval for their participation was given by the Faculty of Science Human Research Ethics Committee, at the University of Bristol. Rating procedure. Participants viewed each image three times, once within a Likert rating task, and twice within a paired comparison task. The order of these tasks was counterbalanced. The tasks were presented on a laboratory computer using e-Prime software. Each participant completed the tasks in private, and no time requirement was specified. For the Likert rating task, images were presented sequentially in random order. Each image was presented for 2 seconds before text appeared, reading, “How strong is this man?” accom- panied by a 7-point scale anchored by “very weak” at point 1 and “very strong” at point 7. Par- ticipants used the keyboard to indicate a response, and time to make a response was recorded. 82 responses were required to complete the task. The paired comparison task was presented in two blocks. In each block, each image was randomly paired with a different image and each pair was presented in random order. The paired images were presented side by side. Each pair was presented for 2 seconds before text appeared, reading, “Which man is stronger?”. Participants used the keyboard to indicate a response by pressing ‘z’ to choose the image on the left, and ‘m’ to choose the image on the PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 5 / 16 Results All analyses shown incorporate responses from male and female rating participants together, as we were not interested in sex differences for this project. Descriptive statistics of rating measures. Table 1 displays descriptive statistics for Likert ratings of perceived strength, and Elo scores (from the original sequence), mElo scores, mElo (M) scores, and Bradley-Terry scores from perceived strength comparisons for the sample of 82 stimulus images. Table 1. Descriptive statistics for mean Likert ratings, Elo scores, mElo scores (using 100 iterations), mElo(M) scores (using 1000 iterations) and Bradley-Terry scores for the 82 stimulus images. mean standard deviation median minimum maximum Likert 3.97 0.90 3.89 2.14 5.91 Elo 0 289.5 -11 -727 755 mElo 0 292.8 -29.6 -686.2 628.3 mElo(M) 0 292.2 -33.2 -672.7 635.0 Bradley-Terry 0.012 0.024 0.0028 0.000085 0.15 atings, Elo scores, mElo scores (using 100 iterations), mElo(M) scores (using 1000 iterations) and Bradley-Terry Table 1. Descriptive statistics for mean Likert ratings, Elo scores, mElo scores (using 100 iterations), mElo(M) scores (us scores for the 82 stimulus images. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 6 / 16 Why rate when you could compare? Table 2. Correlations between mean Likert ratings, Elo scores, mElo scores (100 iterations), mElo(M) scores (1000 iterations), and log-transformed Bradley-Terry scores for the 82 stimulus images. Likert Elo mElo mElo(M) Likert - Elo 0.91 - mElo 0.96 0.95 - mElo(M) 0.96 0.95 1 - Bradley-Terry 0.96 0.95 1 1 https://doi.org/10.1371/journal.pone.0190393.t002 Table 2. Correlations between mean Likert ratings, Elo scores, mElo scores (100 iterations), mElo(M) scores (1000 iterations), and log-transformed Bradley-Terry scores for the 82 stimulus images. Correlations between rating measures and stimuli measures. All rating measures were strongly correlated with each other (see Table 2), particularly in the cases of mElo, mElo(M) and Bradley-Terry scores (log-transformed), which were almost perfect correlated (all rs > 0.999). All ratings measures were correlated with stimuli measures (grip strength and PC1 of body size) to a similar extent (see Table 3), although these relationships were weakest for mean Likert ratings. The very similar results obtained by mElo and mElo(M) suggest that there is little to be gained by generating mElo scores from greater than 100 sequences. Comparing Elo and mElo scores. The 100 sequences used to calculate the mElo scores each produced their own set of Elo scores. Results Considered separately, each set significantly corre- lated with grip strength, but the strength of these correlations varied (0.35 < all rs < 0.49). This was also the case for correlations with PC1 of body size (0.56 < all rs < 0.68). These 100 sets of Elo scores were all strongly correlated with each other, but again with considerable vari- ation (0.86 < all rs < 0.95). Fig 1 shows Elo scores from the original sequence and mElo scores for individual stimulus images on the same plot for comparison. Comparing Likert ratings and mElo scores from reduced data sets. To show how the relationships between measures of perceived strength and stimulus variables is affected by the number of raters, Likert ratings of perceived strength and mElo scores from perceived strength comparisons were recalculated using just the first half of rating participants (n = 28; 14 males and 14 females), and then again using just the first quarter of rating participants (n = 14; 7 males and 7 females). Correlation coefficients (rs) for these relationships using the full, half, and quarter participant sets are show in Table 4. For the Likert ratings, correlations from the halved set were significantly smaller than those from the full set (grip strength: t = -2.100, df = 79, p = 0.039; PC1 of body size: t = -2.412, df = 79, p = 0.018), and correlations from the quartered set were significantly smaller than those from the halved set (grip strength: t = -3.616, df = 79, p < 0.001; PC1 of body size: t = -3.203, df = 79, p = 0.004). For the mElo scores, correlations from the halved set were significantly smaller than those from the full set (grip strength: t = -2.144, df = 79, p = 0.035; PC1 of body size: t = -4.524, df = 79, p < 0.001), but correlations from the quartered set were not significantly smaller than those from the halved set. Table 3. Correlations between stimuli measures (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores (100 itera- tions), mElo(M) scores (1000 iterations), and log-transformed Bradley-Terry scores) for the 82 stimulus images. Likert Elo mElo mElo(M) Bradley-Terry grip strength 0.4 0.43 0.44 0.44 0.45 PC1 of body size 0.6 0.68 0.65 0.65 0.65 https://doi.org/10.1371/journal.pone.0190393.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 7 / 16 Table 3. Results Correlations between stimuli measures (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores (100 itera- tions), mElo(M) scores (1000 iterations), and log-transformed Bradley-Terry scores) for the 82 stimulus images. (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores (100 itera- g-transformed Bradley-Terry scores) for the 82 stimulus images. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 7 / 16 7 / 16 Why rate when you could compare? Fig 1. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo score. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. https://doi org/10 1371/journal pone 0190393 g001 Fig 1. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo score. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. Fig 1. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo score. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. https://doi org/10 1371/journal pone 0190393 g001 Fig 1. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo score. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. https://doi.org/10.1371/journal.pone.0190393.g001 Consistency of Likert ratings and mElo scores. To measure inter-rater consistency of Likert ratings, an average score intraclass correlation coefficient (model: two-way; type: consis- tency) was calculated [34]. The result (ICC = 0.984, 95% CI: 0.979 < ICC < 0.989) indicates very high agreement between raters about the perceived strength of targets. To measure inter-trial consistency of perceived strength comparisons used to calculate mElo scores, we used the novel consistency indices described (formulas 5 and 6). Results The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g002 Fig 2. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–56, in increments of 1). The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g002 https://doi.org/10.1371/journal.pone.0190393.g002 raters. This could also be used as an indication of the minimal number of raters required to produce relatively stable rankings of mElo scores. Comparing decision times for Likert ratings and paired comparison tasks. Mean deci- sion times (in milliseconds) for Likert ratings (mean = 995.6, sd = 392.6) were compared to mean decision times (in milliseconds) for paired comparisons (mean = 635.8, sd = 233.7) using a paired t-test. The result indicates a significant difference between the two (t55 = 7.65, p < 0.001), and a large effect size (Hedges’ g = 1.1, 95% CI: 0.76 < g < 1.46). Fig 3 shows mean decision times for Likert ratings and paired comparisons. Comparing computation times for mElo and Bradley-Terry scores. Both mElo and Bradley-Terry models involve more computation than either Likert or Elo. To compare the time it takes to compute scores via these methods, we timed each procedure on the same lap- top computer (2.2 GHz processor speed, 8 GB RAM), using R’s system.time function. The elapsed time for computing mElo scores for the entire data set was 3 seconds. The elapsed time for computing Bradley-Terry scores for the entire data set was 1017 seconds. Study 2—Online rating The results of Study 1 suggest that mElo scores correspond well with Likert ratings for tasks completed within the lab, but with the advantages offered by web-based data collection (speed, cost, diversity of participants) it is important to check whether comparable results are obtained for tasks completed online. Previous research indicates that online performance is generally good compared to performance in labs [44] and that attention to instructions is actually better [45]. Results The mean unweighted consistency index was 0.766, while the mean weighted consistency index was 0.864. It is important to note that these values are not directly comparable with ICC values. The unweighted value indicates that 76.6% of trial outcomes were concordant with the predic- tions of preceding Elo scores, and the weighted value indicates that the direction of 86.4% of all points exchanged was predicted by preceding Elo scores. Both values indicate high consis- tency of judgements between trials. How many raters are needed to establish stable consistency indices?. Values for the consistency indices are expected to become more accurate as the number of raters increases. This is because the number of trials with an expected result will increase, effectively increasing the sample size from which to estimate consistency. To demonstrate how many raters are needed before these estimates become stable, the weighted consistency index was re-calculated multiple times starting with data from just the first rater (according to the original sequence), then adding the second rater, then the third rater, and so on until all 56 raters were included. Fig 2 depicts these results, showing that index values reached a stable plateau after roughly 30 Table 4. Correlation coefficients for relationships between Likert ratings and mElo scores and stimulus variables for full, half and quarter participant sets. Likert ratings mElo scores grip strength PC1 of body size grip strength PC1 of body size full set 0.40 0.60 0.44 0.65 ½ set 0.37 0.57 0.41 0.60 ¼ set 0.32 0.53 0.38 0.57 https://doi.org/10.1371/journal.pone.0190393.t004 Table 4. Correlation coefficients for relationships between Likert ratings and mElo scores and stimulus variables for full, half and quarter participant sets. 8 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Why rate when you could compare? Fig 2. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–56, in increments of 1). The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g002 Fig 2. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–56, in increments of 1). Methods Target stimuli, stimuli measures, rating measures and statistical software were identical to Study 1, but mElo(M) scores (based on 1000 sequences) were not calculated. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 9 / 16 Why rate when you could compare? Fig 3. Violin plots depict the distribution of response times for paired comparison trials (left), and Likert trials (right). Individual values are depicted by small grey circles. Error bars depict within-subject 95% confidence intervals. Means are depicted by large black circles. Fig 3. Violin plots depict the distribution of response times for paired comparison trials (left), and Likert trials (right). Individual values are depicted by small grey circles. Error bars depict within-subject 95% confidence intervals. Means are depicted by large black circles. https://doi.org/10.1371/journal.pone.0190393.g003 https://doi.org/10.1371/journal.pone.0190393.g003 Rating participants. 96 participants (59 male, 37 female; mean age = 32.6, sd = 9.31) took part in and completed the rating study. These participants were recruited from mTurk (US res- idents only) and paid $3 USD for participation. Before completing any task, participants gave informed consent by ticking a check box stating that they had read and understood the proce- dure. Approval for their participation was given by the Faculty of Science Human Research Ethics Committee, at the University of Bristol. Rating procedure. Participants viewed each image three times, once within a Likert rating task, and twice within a paired comparison task. The order of these tasks was counterbalanced. The tasks were presented using Xperiment online presentation software (www.experiment. mobi). Unlike Study 1, no response times were recorded. For the Likert rating task, images were presented sequentially in random order. Each image was presented for 2 seconds before text appeared, reading, “How strong is this man?” accom- panied by a 7-point scale anchored by “very weak” at point 1 and “very strong” at point 7. Participants clicked a point on the scale to indicate a response. 82 responses were required to complete the task. The paired comparison task was presented in two blocks. In each block, each image was randomly paired with a different image and each pair was presented in random order. The paired images were presented side by side for 2 seconds before text appeared, reading, “Which man is stronger?”. Participants indicated a response by clicking on the button corresponding to either the left or right image. Methods 41 responses were required to complete each block, and 82 responses were required to complete the task. Why rate when you could compare? Table 5. Descriptive statistics for mean Likert ratings, Elo scores, mElo scores and Bradley-Terry scores for the 82 stimulus images. mean standard deviation median minimum maximum Likert 3.90 0.79 3.76 2.16 5.65 Elo 0 277.8 -34 -571 628 mElo 0 255.8 -20.7 -620.2 562.4 Bradley-Terry 0.012 0.019 0.0044 0.00019 0.096 https://doi.org/10.1371/journal.pone.0190393.t005 Table 6. Correlations between mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores for the 82 stimulus images. Likert Elo mElo Likert - Table 5. Descriptive statistics for mean Likert ratings, Elo scores, mElo scores and Bradley-Terry scores for the 82 stimulus images. mean standard deviation median minimum maximum Likert 3.90 0.79 3.76 2.16 5.65 Elo 0 277.8 -34 -571 628 mElo 0 255.8 -20.7 -620.2 562.4 Bradley-Terry 0.012 0.019 0.0044 0.00019 0.096 https://doi org/10 1371/journal pone 0190393 t005 rt ratings, Elo scores, mElo scores and Bradley-Terry scores for the 82 stimulus images. scriptive statistics for mean Likert ratings, Elo scores, mElo scores and Bradley-Terry scores for the 82 stimulus images. Table 5. Descriptive statistics for mean Likert ratings, Elo scores, mElo scores and Bradley-Terry scores for Table 6. Correlations between mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores for the 82 stimulus images. Likert Elo mElo Likert - Elo 0.92 - mElo 0.97 0.95 - Bradley-Terry 0.97 0.94 1 https://doi.org/10.1371/journal.pone.0190393.t006 Table 6. Correlations between mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores for the 82 stimulus images. Descriptive statistics. Table 5 displays descriptive statistics for Likert ratings of perceived strength, and Elo scores (from the original sequence), mElo scores, and Bradley-Terry scores for the sample of 82 stimulus images. Correlations between rating measures and stimuli measures. All rating measures were strongly correlated with each other (see Table 6), particularly mElo and Bradley-Terry scores (log-transformed), which were almost perfect correlated (r = 0.9992). All ratings measures were correlated with stimuli measures (grip strength and PC1 of body size) to a similar extent (see Table 7). Correlations between rating measures from Study 1 and Study 2. To examine whether lab participants and online participants produce similar results using these rating measures, we correlated ratings and scores from Studies 1 and 2 (see Table 8). The measures were strongly correlated with one another, indicating that mode of delivery (lab or online) did not have a great impact. Comparing Elo and mElo scores. Results All analyses shown incorporate responses from male and female rating participants together, as we were not interested in sex differences for this project. 10 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. https://doi.org/10.1371/journal.pone.0190393.g004 https://doi.org/10.1371/journal.pone.0190393.g004 Consistency of Likert ratings and mElo scores. To measure inter-rater consistency of Likert ratings, an average score intraclass correlation coefficient (model: two-way; type: consis- tency) was calculated. The result (ICC = 0.99, 95% CI: 0.985 < ICC < 0.994) indicates very high agreement between raters about the perceived strength of targets. To measure inter-trial consistency of perceived strength comparisons used to calculate mElo scores, we used the novel consistency indices described (formulas 5 and 6). The mean unweighted consistency index was 0.740, while the mean weighted consistency index was 0.837. Again, note that these values are not directly comparable with ICC values. The unweighted value indicates that 74.0% of trial outcomes were concordant with the predictions of preceding Elo scores, and the weighted value indicates that the direction of 83.7% of all points exchanged was predicted by preceding Elo scores. Both values indicate high consistency of judgements between trials. How many raters are needed to establish stable consistency indices?. As in Study 1, to demonstrate how many raters are needed before estimates of consistency become stable, the weighted consistency index was re-calculated multiple times, starting with data from just the first rater (according to the original sequence), then adding the second rater, then the third rater, and so on until all 96 raters were included. Fig 5 depicts these results, showing that index values changed little after about 40 raters. Again, this could also be used as an indication of the minimal number of raters required to produce relatively stable rankings of mElo scores. Comparing computation times for Bradley-Terry and mElo scores. To compare the time it takes to compute scores via mElo and Bradley-Terry methods, we timed each procedure on the same laptop computer (2.2 GHz processor speed, 8 GB RAM), using R’s system.time function. The elapsed time for computing mElo scores for the entire data set was 5 seconds. The elapsed time for computing Bradley-Terry scores for the entire data set was 904 seconds. Fig 4 shows Elo scores from the original sequence and mElo scores for individual stimulus images on the same plot for comparison. Table 7. Correlations between stimuli measures (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores) for the 82 stimulus images. Likert Elo mElo Bradley-Terry grip strength 0.38 0.40 0.43 0.43 PC1 of body size 0.61 0.59 0.62 0.62 https://doi.org/10.1371/journal.pone.0190393.t007 Table 8. Correlations between rating measure scores from Study 1 and Study 2. Study 2 Likert Elo mElo Bradley-Terry Study 1 Likert 0.98 Elo 0.91 0.85 mElo 0.96 0.91 0.97 Bradley-Terry 0.96 0.92 0.97 0.97 https://doi.org/10.1371/journal.pone.0190393.t008 PLOS ONE | https://doi org/10 1371/journal pone 0190393 January 2 2018 11 / 16 Table 7. Correlations between stimuli measures (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores) for the 82 stimulus images. Likert Elo mElo Bradley-Terry grip strength 0.38 0.40 0.43 0.43 PC1 of body size 0.61 0.59 0.62 0.62 https://doi.org/10.1371/journal.pone.0190393.t007 Table 7. Correlations between stimuli measures (grip strength and PC1 of body size) and rating measures (mean Likert ratings, Elo scores, mElo scores and log-transformed Bradley-Terry scores) for the 82 stimulus images. Table 8. Correlations between rating measure scores from Study 1 and Study 2. Study 2 Likert Elo mElo Bradley-Terry Study 1 Likert 0.98 Elo 0.91 0.85 mElo 0.96 0.91 0.97 Bradley-Terry 0.96 0.92 0.97 0.97 https://doi.org/10.1371/journal.pone.0190393.t008 Table 8. Correlations between rating measure scores from Study 1 and Study 2. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 11 / 16 Why rate when you could compare? Fig 4. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. Fig 4. Elo scores from the original sequence (grey circles) and mElo scores (black circles) are depicted for each of the stimulus images, arranged from left to right in order of decreasing mElo. The whiskers depict the range of Elo scores from the 100 sequences generated to calculate the mElo scores. Fig 4. Discussion Likert ratings, Elo scores and Bradley-Terry scores were closely correlated with mElo scores, indicating that they were tracking perceptions similarly. Each measure of perceived strength PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 12 / 16 Why rate when you could compare? Fig 5. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–96, in increments of 1). The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g005 Fig 5. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–96, in increments of 1). The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g005 Fig 5. Weighted consistency indices for the original sequence (small grey circles) and mean weighted consistency indices (large black circles) calculated for an increasing number of Study 1 raters (1–96, in increments of 1). The whiskers represent inter-quartile ranges of indices from the 100 sequences generated to calculate the mean index. https://doi.org/10.1371/journal.pone.0190393.g005 https://doi.org/10.1371/journal.pone.0190393.g005 was also similarly correlated with actual strength and strength-related measurements of the men depicted in the stimuli, indicating that these perceptions reliably track actual physical differences between men, as reported by Sell et al. [36]. In addition, these relationships were observed in both the laboratory, using a primarily UK undergraduate participant base, and online, using a more diversely aged US participant base. On the basis of these facts alone, there is little to separate one measure from another, and little reason to favour one above the other options. However, we argue that mElo scores represent a good alternative option to Elo scores, Bradley-Terry scores and Likert ratings, for distinct reasons. Figs 1 and 4 demonstrates the range of Elo scores that can result from different sequences of the same trial comparisons with the same outcomes, and the range of correlations with grip strength and PC1 of body size (Study 1) demonstrates why this is problematic. As argued ear- lier, this variation in final scores represents an undesirable feature in the context of our rating temporally stable features, and we proposed mElo scores to address this issue. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Discussion When multiple mElo scores are generated, each using the same trial data but using different randomly shuffled sequence orders, the results are far less variable. Each mElo score was almost perfectly corre- lated with all others, and there was no detectable variation between them when predicting actual strength and strength-related measurements. This was even the case for the one mElo score that was generated using 1000 different sequence orders, which indicates that, in this case at least, using 100 sequence orders is sufficient and saves computing time without cost. We argue that the mElo approach is conceptually simpler than standard Bradley-Terry models, and thus more likely to appeal to researchers who are new to pairwise comparison. Our results suggest that mElo is computationally simpler as well, calculating score in 5 seconds or less, whereas Bradley-Terry models took over 15 minutes. This could be related to the num- ber of stimuli used in our example, so studies using an even greater number of stimuli may suf- fer even greater time disparities. Although results from Likert ratings and mElo scores were largely similar for this task, the procedure for obtaining them was quite different, leading to a very divergent participant PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 13 / 16 Why rate when you could compare? experience. Participants completed pairwise comparison trials significantly quicker than rating trials, and the effect size for this difference was large (see Fig 3). Taking response time as a proxy of cognitive load [46], the speed difference between the trial types indicates that partici- pants found the pairwise comparisons to be easier. This is likely to be a particularly important consideration for more difficult tasks (e.g. rating characteristics which are not readily appar- ent, such as personality traits from faces [47], or kinship in monkeys [48]), or for participant groups that have either compromised capacity or limited experience with stimulus tasks, par- ticularly those involving novel concepts. In addition, because participants complete pairwise comparison tasks more quickly, they might be willing to be paid less to provide similarly useful information. This is not the only way in which pairwise comparisons may prove to be more efficient. Note that correlations with actual strength and strength-related measures deteriorated as more raters were excluded for both Likert ratings and mElo scores, despite strong correlations with ratings and scores from the full participant set. Acknowledgments This research was supported by a research project grant awarded to A.P.C. and I.S.P-V from the Leverhulme Trust. Thanks to Tim Fawcett for contributing idea to plot consistency by number of raters. Discussion This indicates that collecting data from more participants is likely to beneficial for both methods. However, also note that the mElo scores obtained from the halved set had strikingly similar associations with stimulus variables as Likert ratings from the full set did, and similarly mElo scores from the quartered set produced similar associations as Likert ratings from the halved set (see Table 2). If this pattern is typical of other data sets, this suggests that only half as many pairwise comparison participants are required to produce results equivalent to those from ratings tasks. This could represent a considerable savings for researchers both in time and participant payments. In conclusion, using pairwise comparisons in conjunction with the “EloChoice” package represents a simple alternative option for researchers interested in quantifying perceived char- acteristics of stimuli, offering participants a less demanding experience while requiring fewer of them, and representing a minimal learning cost even for researchers inexperienced with R. Author Contributions Conceptualization: Andrew P. Clark, Ian S. Penton-Voak. Data curation: Andrew P. Clark, Andy T. Woods, Christof Neumann. Formal analysis: Andrew P. Clark, Christof Neumann. Formal analysis: Andrew P. Clark, Christof Neumann. Funding acquisition: Andrew P. Clark, Ian S. Penton-Voak. Funding acquisition: Andrew P. Clark, Ian S. Penton-Voak. Investigation: Kate L. Howard, Andy T. Woods, Ian S. Penton-Voak. Methodology: Andrew P. Clark, Kate L. Howard, Andy T. Woods, Ian S. Penton-Voak, Chris- tof Neumann. Methodology: Andrew P. Clark, Kate L. Howard, Andy T. Woods, Ian S. Penton-Voak, Chris- tof Neumann. Project administration: Ian S. Penton-Voak. References 1. Brown TA, Cash TF, Noles SW. Perceptions of physical attractiveness among college students: selected determinants and methodological matters. The Journal of Social Psychology. 1986; 126(3): 305–316. 2. Perrett DI, May KA, Yoshikawa S. Facial shape and judgements of female attractiveness. Nature. 1994; 368(6468): 239–242. https://doi.org/10.1038/368239a0 PMID: 8145822 3. Brooks RC, Shelly JP, Jordan LA, Dixson BJW. The multivariate evolution of female body shape in an artificial digital ecosystem. Evolution and Human Behavior. 2015; 36(5): 351–358. 4. Likert R. A technique for the measurement of attitudes. Archives of Psychology. 1932; 22(140): 5–55. 5. Uebersax JS. Likert scales: dispelling the confusion. Statistical Methods for Rater Agreement; 2015. http://john-uebersax.com/stat/likert.htm. 6. Cunningham MR, Roberts AR, Barbee AP, Druen PB, Wu C-H. “Their ideas of beauty are, on the whole, the same as ours”: Consistency and variability in the cross-cultural perception of female physical attractiveness. Journal of Personality and Social Psychology. 1995; 68(2): 261–279. 7. Mellor D, Moore KA. The use of Likert scales with children. Journal of pediatric psychology. 2014; 39 (3): 369–379. https://doi.org/10.1093/jpepsy/jst079 PMID: 24163438 8. Dressler WW, Oths KS. Social survey methods. In: Bernard HR, Gravlee CC, editors. Handbook of methods in cultural anthropology. 2nd ed. Lanham, MD USA: Altamira Press; 2014. p. 497–515. 9. Bo¨ckenholt U. Thresholds and intransitivities in pairwise judgments: A multilevel analysis. Journal of Educational and Behavioral Statistics. 2001; 26(3): 269–82. 10. Thurstone LL. A law of comparative judgment. Psychological Review. 1927; 34(4): 273–286. 11. Bradley RA, Terry ME. Rank analysis of incomplete block designs: I. The method of paired compari- sons. Biometrika. 1952; 39(3/4): 324–345. 12. Glickman ME. Parameter estimation in large dynamic paired comparison experiments. Journal of the Royal Statistical Society: Series C (Applied Statistics). 1999; 48(3): 377–394. 13. Shev A, Hsieh F, Beisner B, McCowan B. Using Markov chain Monte Carlo (MCMC) to visualize and test the linearity assumption of the Bradley–Terry class of models. Animal Behaviour. 2012; 84(6): 1523–1531. PMID: 24052665 14. Maydeu-Olivares A, Bo¨ckenholt U. Structural equation modeling of paired-comparison and ranking data. Psychological methods. 2005; 10(3): 285. PMID: 16221029 15. Strobl C, Wickelmaier F, Zeileis A. Accounting for individual differences in Bradley-Terry models by means of recursive partitioning. Journal of Educational and Behavioral Statistics. 2011; 36(2): 135–53. 16. Tsukida K, Gupta MR. How to analyze paired comparison data. UWEETR-2011-0004. University of Washington Seattle Department of Electrical Engineering; 2011. 17. Zermelo E. Die Berechnung der Turnier-Ergebnisse als ein Maximumproblem der Wahrscheinlichkeits- rechnung. Project administration: Ian S. Penton-Voak. Resources: Andy T. Woods. Resources: Andy T. Woods. Software: Andy T. Woods, Christof Neumann. Software: Andy T. Woods, Christof Neumann. Supervision: Ian S. Penton-Voak. Supervision: Ian S. Penton-Voak. Validation: Christof Neumann. 14 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 Why rate when you could compare? Visualization: Andrew P. Clark, Christof Neumann. Writing – original draft: Andrew P. Clark, Kate L. Howard. Writing – review & editing: Andrew P. Clark, Ian S. Penton-Voak, Christof Neumann. Visualization: Andrew P. Clark, Christof Neumann. Writing – original draft: Andrew P. Clark, Kate L. Howard. Writing – review & editing: Andrew P. Clark, Ian S. Penton-Voak, Christof Neumann. References Mathematische Zeitschrift. 1929; 29(1): 436–460. 18. Elo AE. The rating of chess players past and present. New York: Arco; 1978. 19. Glickman ME. Parameter estimation in large dynamic paired comparison experiments. Applied Statis- tics. 1999; 48(3): 377–94. 20. Herbrich R, Minka T, Graepel T. Trueskill™: A Bayesian skill rating system. Advances in neural informa- tion processing systems. 2006; 19: 569–576. 21. Elliott LL. Reliability of judgments of figural complexity. Journal of Experimental Psychology. 1958; 56 (4): 335. PMID: 13587863 22. Mueser KT, Grau BW, Sussman S, Rosen AJ. You’re only as pretty as you feel: facial expression as a determinant of physical attractiveness. Journal of Personality and Social Psychology 1984; 46(2): 469– 478. 23. Glickman ME. A comprehensive guide to chess ratings. American Chess Journal. 1995; 3: 59–102. 24. Hvattum LM, Arntzen H. Using ELO ratings for match result prediction in association football. Interna- tional Journal of forecasting. 2010; 26(3): 460–70. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 15 / 16 Why rate when you could compare? 25. Doebler P, Alavash M, Giessing C. Adaptive experiments with a multivariate Elo-type algorithm. Behav- ior research methods. 2015; 47(2): 384–94. https://doi.org/10.3758/s13428-014-0478-7 PMID: 24878597 26. Ziemba B. The 2014–2015 NFL Season, Playoffs, and the Super Bowl. Wilmott. 2015; 2015(77): 24– 43. 27. Goodspeed R. Research note: An evaluation of the Elo algorithm for pairwise visual assessment sur- veys. Landscape and Urban Planning. 2017; 157: 131–7. 28. Luon Y, Aperjis C, Huberman BA. Rankr: A mobile system for crowdsourcing opinions. InInternational Conference on Mobile Computing, Applications, and Services 2011 Oct 24 (pp. 20–31). Springer Berlin Heidelberg. 29. Neumann C, Duboscq J, Dubuc C, Ginting A, Irwan AM, Agil M, Widdig A, Engelhardt A. Assessing dominance hierarchies: validation and advantages of progressive evaluation with Elo-rating. Animal Behaviour. 2011; 82(4): 911–921. 30. R Core Team. R: A language and environment for statistical computing (version 3.2.1). Vienna, Austria: R Foundation for Statistical Computing; 2015. https://www.R-project.org. 31. Albers P, de Vries H. Elo-rating as a tool in the sequential estimation of dominance strengths. Animal Behaviour. 2001; 61: 489–495. 32. Franz M, McLean E, Tung J, Altmann J, Alberts SC. Self-organizing dominance hierarchies in a wild pri- mate population. Proceedings of the Royal Society B: Biological Sciences. 2015; 282(1814): 20151512. https://doi.org/10.1098/rspb.2015.1512 PMID: 26336168 33. Reid DA, Nixon MS, Stevenage SV. Soft biometrics; human identification using comparative descrip- tions. Pattern Analysis and Machine Intelligence, IEEE Transactions on. 2014; 36(6): 1216–28. PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018 References 34. Hallgren KA. Computing inter-rater reliability for observational data: an overview and tutorial. Tutorials in quantitative methods for psychology. 2012; 8(1): 23. PMID: 22833776 35. Gwet KL. Handbook of inter-rater reliability: The definitive guide to measuring the extent of agreement among raters. Advanced Analytics, LLC; 2014. 36. Sell A, Cosmides L, Tooby J, Sznycer D, von Rueden C, Gurven M. Human adaptations for the visual assessment of strength and fighting ability from the body and face. Proceedings of the Royal Society B. 2009; 276: 575–584. https://doi.org/10.1098/rspb.2008.1177 PMID: 18945661 37. Lê S, Josse J, Husson F. FactoMineR: an R package for multivariate analysis. Journal of Statistical Software. 2008; 25(1): 1–18. 38. Neumann C. EloChoice: Preference rating for visual stimuli based on elo ratings (version 0.29). http:// cran.r-project.org/web/packages/EloChoice. 39. Zeileis A, Strobl C, Wickelmaier F, Komboz, B, Kopf J. psychotools: Infrastructure for Psychometric Modeling (version 0.4–2). http://cran.r-project.org/web/packages/psychotools. 40. Diedenhofen B, Musch J. cocor: A comprehensive solution for the statistical comparison of correlations. PLoS One. 2015; 10(4): e0121945. https://doi.org/10.1371/journal.pone.0121945 PMID: 25835001 41. Williams EJ. The comparison of regression variables. Journal of the Royal Statistical Society, Series B. 1959; 21(2): 396–399. 42. Gamer M, Lemon J, Fellows I, Singh P. irr: Various coefficients of interrater reliability and agreement (version 0.84). http://cran.r-project.org/package=irr. 43. Lakens D. The perfect t-test (version 0.1.0). 2015. http://github.com/Lakens/perfect-t-test. 44. Crump M, McDonnell J, Gureckis T. Evaluating Amazon’s Mechanical Turk as a tool for experimental behavioral research. PLoS One. 2013; 8(3): e57410. https://doi.org/10.1371/journal.pone.0057410 PMID: 23516406 45. Ramsey SR, Thompson KL, McKenzie M, Rosenbaum A. Psychological research in the internet age: The quality of web-based data. Computers in Human Behavior. 2016; 58: 354–360. 46. Luce RD. Response times: Their role in inferring elementary mental organization. New York: Oxford University Press; 1986. 47. Penton-Voak IS, Pound N, Little AC, Perrett DI. Personality judgments from natural and composite facial images: More evidence for a “kernel of truth” in social perception. Social Cognition. 2006; 24(5): 607–40. 48. Kazem AJ, Widdig A. Visual phenotype matching: cues to paternity are present in rhesus macaque faces. PLoS One. 2013; 8(2): e55846. https://doi.org/10.1371/journal.pone.0055846 PMID: 23451032 16 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0190393 January 2, 2018
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An Ideal Society? Neighbors of Diverse Origins Interact to Create and Maintain Complex Mini-Organs in the Skin
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Open access, freely available online Open access, freely available online Primer An Ideal Society? Neighbors of Diverse Origins Interact to Create and Maintain Complex Mini-Organs in the Skin Sarah E. Millar p Sarah E. Millar DOI: 10.1371/journal.pbio.0030372.g001 DOI: 10.1371/journal.pbio.0030372.g001 T he largest organ in the body, the skin provides protection against environmental insults and dehydration, and is an important gateway for sensory input. In addition to forming an environmental barrier, the skin has evolved to produce an amazing variety of appendages, including scales, feathers, hair follicles, sweat glands, and mammary glands (Figure 1). These organs arise from embryonic skin progenitor cells and endow wide-ranging properties, including regulation of body temperature, and the ability to fl y, nurse young, and attract mates. As the body’s primary frontier, the skin is both vulnerable to disease, such as melanoma, and a source of therapeutic promise, harboring accessible stem cells (SCs) that can regenerate skin and potentially other organs. T DOI: 10.1371/journal.pbio.0030372.g001 Figure 1. Histological and Schematic Depictions of Mature Skin (A) Hematoxylin/eosin-stained section of mouse skin at postnatal day 28, showing hair follicles in the anagen growth phase; major layers of the skin are indicated. (B) Schematic depiction of postnatal skin showing a hair follicle in the growth phase. Biological processes occurring in the skin are listed and SC locations are indicated. Dark blue arrows indicate the movements of stem and matrix cell progeny; pale blue arrows indicate SC self-renewal. Pink, epidermis and hair follicle outer root sheath; yellow, inner root sheath; green, matrix; red, hair follicle DP; light brown, hair shaft precursors; darker brown, hair shaft; violet circles, SCs; black ovals, dermal fi broblasts. g p y g Mammalian skin contains three major cell types: epithelial cells that form a stratifi ed epidermis containing specialized intermediate fi lament proteins called keratins; mesenchymal cells that form the underlying dermis and, together with epithelial cells, contribute to hair follicles and other appendages; and melanocytes that provide the skin and hair follicles with pigmentation. In addition, the skin is highly innervated, and contains populations of specialized cells including dentritic Langerhans cells (a type of antigen presenting cell), Merkel mechanoreceptor cells (which form complexes with sensory axons), and mast cells (which produce histamine). These different cell types have diverse origins, and undergo extensive interactions, migration, proliferation, and differentiation during embryonic development. Epidermal Origins The epidermis originates from the outer layer of the embryo, the surface ectoderm. BMPs activate the epidermal differentiation program and induce the expression of keratin proteins via several known transcription factors [1,2]. The surface ectoderm proliferates and migrates from the dorsal midline to cover the embryo [3], and persists as a simple epithelium until approximately embryonic day (E) 9.5 of mouse embryogenesis. At this stage basal cells begin to express keratins 5 and 14, presaging epidermal stratifi cation [2], which requires the activity of a key epidermal transcription factor that also regulates epidermal fate, proliferation, and adhesion [4–9]. By birth, the epidermis consists of a proliferative basal layer that differentiates to form suprabasal layers, and an outer, “cornifi ed,” enucleated shell that constitutes the epidermal barrier and is continuously shed and replenished. Epidermal Morphogenesis is controlled by a relatively small number of key intercellular signaling pathways that occur sequentially and in specifi c combinatorial fashions. Activation of these pathways is directed by secreted ligands, including members of the WNT, fi broblast growth factor, tumor necrosis factor, and Hedgehog families, and bone morphogenetic protein (BMP) and other transforming growth factor superfamily members, which act over relatively short distances and bind to specifi c receptor proteins on neighboring cells to regulate cell shape, size, adhesion, polarity, movements, proliferation, and transcriptional activity. The molecular events underlying skin development in mammals have been studied using a variety of systems and techniques. These include analysis of human genetic syndromes and spontaneously occurring and induced mouse mutants, production of transgenic and gene- targeted mouse models, in vitro culture of rodent and human skin cells, and grafting of human or rodent skin to immune- defi cient mice. Citation: Millar SE (2005) An ideal society? Neighbors of diverse origins interact to create and maintain complex mini-organs in the skin. PLoS Biol 3(11): e372. Citation: Millar SE (2005) An ideal society? Neighbors of diverse origins interact to create and maintain complex mini-organs in the skin. PLoS Biol 3(11): e372. Copyright: © 2005 Sarah E. Millar. 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. Abbreviations: BMP, bone morphogenetic protein; DP, dermal papilla; E[number], embryonic day [number]; GFP, green fl uorescent protein; SC, stem cell Sarah E. Abbreviations: BMP, bone morphogenetic protein; DP, dermal papilla; E[number], embryonic day [number]; GFP, green fl uorescent protein; SC, stem cell Citation: Millar SE (2005) An ideal society? Neighbors of diverse origins interact to create and maintain complex mini-organs in the skin. PLoS Biol 3(11): e372. Dermal Origins The dermis of the skin consists of loosely packed fi broblasts and has a remarkable variety of embryonic origins. Fate mapping of mammalian dermis, which traces the developmental path of dermal cells, has been achieved by introducing artifi cial genes into mice. Specifi cally, the bacterial Cre recombinase enzyme is introduced under the control of a promoter that is active in a particular embryonic region, in this case either lateral plate mesoderm or neural crest. This transgene is combined with a Cre-activatable lacZ reporter gene, resulting in lacZ expression in Cre-expressing embryonic cells and all of their descendants. This allows tracking of the fates of these descendants by X-gal staining to detect activity of the β-galactosidase enzyme encoded by lacZ. These experiments showed that the lateral plate mesoderm gives rise to ventral trunk dermis, while head dermis arises, at least in part, from neural crest, a transient population of migratory cells derived from the neural plate [11–14]. While fate mapping of dorsal trunk dermis in mammalian embryos has not yet been described, analysis of chick–quail chimeras revealed that in avian embryos the dorsal dermis arises from the dorsal regions of somites [15,16]. Embryonic dermal fi broblasts from different body regions have different inductive properties [17–19] and show position-specifi c differences in gene expression, even in the adult [20]. The molecular controls of mammalian dermal development remain relatively unexplored. Surgical removal of the DP and the contiguous lower dermal sheath prevents hair growth, indicating the importance of the DP as a key signaling center for the follicle [33]. DPs or dermal sheaths implanted at ectopic sites demonstrate a remarkable ability to induce the formation of new follicles, even from terminally differentiated epithelium such as the central cornea of the eye [34–36]. This inductive ability is lost if the DP cells are cultured in the absence of epithelial cells or without the addition of WNT proteins [37,38], suggesting that signals from neighboring epithelium are necessary for maintenance of DP function (see Figure 1B). Not surprisingly, these extensive epithelial–mesenchymal interactions require the activities of secreted ligands that allow communication between different cell types. WNT signals play key roles at the initiation of embryonic hair follicle development [39–42]. Fibroblast growth factor proteins and the tumor necrosis factor family member ectodysplasin also promote epithelial placode formation, while Sonic hedgehog controls proliferation of both embryonic and postnatal hair follicle epithelium [30]. Dermal Origins The BMP inhibitor Noggin is essential for hair follicle development [43], and acts in conjunction with WNT signaling and transforming growth factor β2 to control invasion of the dermis by hair follicle epithelial cells [44,45]. Differentiation of matrix cells toward either inner root sheath or hair shaft requires BMP signaling and numerous transcription factors [30,46–49]. The precise mechanisms by which these factors facilitate communication between cells of different types to produce an organ as complex as the hair follicle remain only partially understood. Pigmentation of the epidermis and hair follicles is supplied by melanocytes that manufacture melanin (black pigment) or phaeomelanin (yellow/orange pigment) and deposit it into melanosomes that are transferred to hair shaft or epidermal cells. Melanocytes differentiate from melanoblasts that arise from the neural crest. After undergoing an initial period of proliferation, melanoblasts migrate between the dermatome and overlying ectoderm, and from E10.5 of mouse embryogenesis migrate through the developing dermis. Starting at approximately E12.5, melanoblasts move from the dermis to the epidermis, and by E15.5 they begin to migrate into the developing hair follicles [21–24] (Figure 2). By two weeks of age melanoblasts are absent from haired regions of mouse epidermis [24]. In contrast, in human skin melanoblasts and melanocytes populate the epidermis as well as the hair follicles. Genetic analysis in mice has revealed requirements for several factors in melanoblast migration, proliferation, and/or survival [25,26]. Mutations in a variety of these genes are known to cause pigmentation and other neural-crest-related defects in humans as well as in mice [25]. DOI: 10.1371/journal.pbio.0030372.g002 Figure 2. Schematic Depiction of Directions of Melanoblast Migration in Embryonic Mouse Skin from E9.5 to E17.5 Pink, epithelium; black dots, dermal fi broblasts; blue ovals, melanoblasts; red dots, dermal condensate/DP. DOI: 10.1371/journal.pbio.0030372.g002 Epidermal Origins Millar is in the Departments of Dermatology and Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America. E-mail: millars@mail.med.upenn.edu Primers provide a concise introduction into an important aspect of biology highlighted by a current PLoS Biology research article. DOI: 10.1371/journal.pbio.0030372 PLoS Biology | www.plosbiology.org November 2005 | Volume 3 | Issue 11 | e372 November 2005 | Volume 3 | Issue 11 | e372 1873 revealed that signals from the dermis initiate hair follicle development by inducing formation of a regular array of local thickenings, or placodes, in the overlying surface epithelium [17]. Signals from each epithelial placode induce the clustering of a ball of underlying mesenchymal cells, the dermal condensate. Signaling from the dermal condensate to the epithelium results in downward growth of the epithelial cells (Figure 3), which surround the dermal condensate, thereafter known as the dermal papilla (DP) [17]. The DP directs the rapid proliferation of adjacent epithelial cells called matrix cells, which gradually exit from the proliferative compartment, and terminally differentiate to form the hair shaft and the inner root sheath that molds the shaft [32]. An outer root sheath that is contiguous with the epidermis surrounds the inner root sheath, and the follicle is bound by a dermal sheath. renewal continues throughout life, suggesting that this tissue harbors an SC population; maintenance of epidermal SCs in vivo has been recently shown to require the Rho guanosine triphosphatase Rac1 [10]. PLoS Biology | www.plosbiology.org Hair Follicle Growth: The Role of SCs These experiments identifi ed genes not previously associated with hair follicles, and revealed cell-type-specifi c expression for several genes affected in hair disorders. y Several recent reports have identifi ed cell populations in the skin that appear to be capable of differentiating into cell types other than hair follicle epithelium and epidermis, raising the exciting possibility that skin SCs could be used to regenerate organs beside the skin and hair follicles. For example, neural crest derivatives [14] and cells expressing a Nestin–green fl uorescent protein (GFP) transgene [61] have been isolated from the bulge regions of vibrissa (whisker) follicles and can differentiate in culture into neurons, smooth muscle cells, and melanocytes. Multipotent cells termed skin-derived precursor cells have also been derived from the dermis of adult skin, and several lines of evidence suggest that the endogenous niche for these cells is the hair follicle DP [13]. In a separate study, clonal cell lines established from dissected rat vibrissa DPs and dermal sheaths had extended proliferative capacities and could differentiate into fat- and bone-related lineages [62]. Although it is possible that the skin-derived precursor cells and dissected vibrissa preparations could have been contaminated with non- DP cells, these data suggest that DP cells not only possess remarkable inductive capacities but also reside in a niche provided by the surrounding follicular cells that enables them to maintain a large proliferative capacity and the ability to differentiate into multiple cell types. Importantly, the results of this study support and extend previous observations that the DP expresses genes originally associated with neural SCs [13]. However, Rendl et al. fi nd that the DP transcriptional profi le is clearly distinct from that of neural SCs, neural crest, or melanocytes, and is most similar to that of dermal fi broblasts. These data support the concept that DP cells and dermal fi broblasts arise from similar precursors. Consistent with this view, use of a Hoxb6- Cre transgene to label derivatives of lateral plate mesoderm resulted in apparent marking of ventral trunk DPs as well as ventral trunk dermal fi broblasts, suggesting that at least some DP cells have common origins with neighboring dermal fi broblasts ([11]; S. I. Candille and G. S. Barsh, personal communication). Hair Follicle Growth: The Role of SCs pure populations of these cells diffi cult. Recently, however, hair follicle bulge epithelial SCs have been isolated by utilizing a specifi c promoter to target GFP to adult bulge epithelium [52], or by taking advantage of the rarely cycling properties of epithelial bulge cells to target GFP expression to this population [55]. These approaches permit the use of fl uorescence-activated cell sorting (FACS) to isolate and transcriptionally profi le pure bulge epithelial SC populations. The hair follicle undergoes cycles of growth and regression throughout life. At the onset of a new cycle of hair growth (anagen), signals from the DP are thought to stimulate the transient proliferation of epithelial SCs in the permanent bulge region of the follicle [50]. Bulge SC descendants give rise to all the epithelial layers of the regenerating hair follicle [51–55] (see Figure 1B). The extent to which these cells also contribute to epidermis in normal and pathological situations is currently not clear. Epithelial SCs coexist in the bulge with a population of melanocyte SCs that proliferates transiently at anagen onset and gives rise to differentiated melanocytes that populate the hair follicle bulb [56]. Hair graying in genetic mouse models and in humans is associated with loss of the melanocyte SCs [57]. Forced expression of a stabilized form of the WNT pathway effector â-catenin in hair follicle epithelial SCs causes the onset of a new cycle of growth of pigmented hair, suggesting that WNT signaling is critical for epithelial SC proliferation, and that secreted factors downstream of β-catenin can activate melanocyte SCs [58–60]. In the current issue of PLoS Biology, Rendl et al. [63] take similar approaches to systematically analyze the transcriptional profi le of the DP and four surrounding cell types: dermal fi broblasts, melanocytes, epithelial matrix cells, and outer root sheath cells, from mouse hair follicles four days after birth. At this time point, embryonic hair follicles have reached a late stage in their differentiation and are beginning to synthesize hair shafts. Rendl et al. marked each skin cell population with a unique combination of fl uorescent tags, using a clever mix of cell-type-specifi c transgenic expression of red and green fl uorescent proteins, together with immunolabeling of specifi c antigens. Each of the different cell populations was found to possess a distinct molecular signature. DOI: 10.1371/journal.pbio.0030372.g003 Figure 3. Successive Stages of Hair Follicle Development in Embryonic Mouse Skin at E14.5–E15.5 Sections were stained with Hoechst dye to reveal the nuclei. The epithelial–mesenchymal boundary is marked by a dashed white line in each panel, and sites of hair follicle development are bracketed. The directions of inductive signals are indicated for each stage (green arrows). Left: placode stage; note larger size and columnar appearance of placode nuclei compared with those in adjacent epithelium. Middle: induction of the dermal condensate (DC). Right: formation of a germ stage hair follicle with a well-developed dermal condensate. air Follicle Development in Embryonic Mouse Skin at E14.5–E15.5 t dye to reveal the nuclei. The epithelial–mesenchymal boundary is marked by a dashed white line in each panel, and e bracketed. The directions of inductive signals are indicated for each stage (green arrows). Left: placode stage; note ce of placode nuclei compared with those in adjacent epithelium. Middle: induction of the dermal condensate (DC). air follicle with a well-developed dermal condensate. PLoS Biology | www.plosbiology.org Interactions between Neighboring Cells Control Hair Follicle Development While epidermal cells, dermal fi broblasts, and melanocytes have differing embryonic origins and migration pathways [27–29], these cells interact extensively during development of the skin and its appendages [17,19,30,31]. Experiments in which epithelium and mesenchyme from different body regions, different developmental stages, or different species were combined and allowed to develop at ectopic sites DOI: 10.1371/journal.pbio.0030372.g002 Figure 2. Schematic Depiction of Directions of Melanoblast Migration in Embryonic Mouse Skin from E9.5 to E17.5 Pink, epithelium; black dots, dermal fi broblasts; blue ovals, melanoblasts; red dots, dermal condensate/DP. PLoS Biology | www.plosbiology.org November 2005 | Volume 3 | Issue 11 | e372 1874 DOI: 10.1371/journal.pbio.0030372.g003 Figure 3. Successive Stages of Hair Follicle Development in Embryonic Mouse Skin at E14.5–E15.5 Sections were stained with Hoechst dye to reveal the nuclei. The epithelial–mesenchymal boundary is marked by a dashed white line in each panel, and sites of hair follicle development are bracketed. The directions of inductive signals are indicated for each stage (green arrows). Left: placode stage; note larger size and columnar appearance of placode nuclei compared with those in adjacent epithelium. Middle: induction of the dermal condensate (DC). Right: formation of a germ stage hair follicle with a well-developed dermal condensate. Hair Follicle Growth: The Role of SCs Thus, differences in gene expression in DP cells compared with neighboring dermis may refl ect the infl uences of follicular matrix and outer root sheath cells, rather than being the result of a distinct embryonic origin of the DP. Interestingly, WNT signaling and inhibition of BMP signaling are key elements in induction and maintenance of both hair follicles [39–43] and neural crest [1], and are featured in the DP signature. The re-utilization of these Relatively few cells reside in specialized niches such as the hair follicle DP and bulge, making molecular analyses of November 2005 | Volume 3 | Issue 11 | e372 1875 13. Fernandes KJ, McKenzie IA, Mill P, Smith KM, Akhavan M, et al. (2004) A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 6: 1082–1093. pathways in the complex signaling environment of the hair follicle may account in part for the unusual gene expression pattern in the DP, for the remarkable inductive properties of DP cells, and perhaps for their apparent ability to maintain multipotency. 14. Sieber-Blum M, Grim M, Hu YF, Szeder V (2004) Pluripotent neural crest stem cells in the adult hair follicle. Dev Dyn 231: 258–269. y 15. Mauger A (1972) The role of somitic mesoderm in the development of dorsal plumage in chick embryos. I. Origin, regulative capacity and determination of the plumage-forming mesoderm. J Embryol Exp Morphol 28: 313–341. y 15. Mauger A (1972) The role of somitic mesoderm in the dev p y The approach employed by Rendl et al. has produced a remarkably comprehensive picture of gene expression in different components of the skin and hair follicles in the early postnatal period. These data will likely form the basis for a multitude of further investigations into the molecular nature of mesenchymal–epithelial interactions in the hair follicle, and the functions of hair-follicle-expressed genes in both normal development and disease. Similar approaches could be employed to begin to address outstanding questions in skin and appendage biology. Profi ling of different follicular compartments during the hair growth cycle may identify putative inductive signals produced by the DP, and could provide clues as to how cycling activity of epithelial and melanocyte SCs is coordinated. Profi ling of different cell compartments in various developing appendages might begin to reveal the molecular mechanisms by which apparently similar sets of signaling pathways direct the formation of diverse organs. References 1. Meulemans D, Bronner-Fraser M (2004) Gene-regulatory interactions in neural crest evolution and development. Dev Cell 7: 291–299. 1. 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Oliver RF (1966) Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. J Embryol Exp Morphol 15: 331–347. g J y p p 34. Reynolds AJ, Jahoda CA (1992) Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis. Development 115: 587–593. p 35. Reynolds AJ, Lawrence C, Cserhalmi-Friedman PB, Christiano AM, Jahoda CA (1999) Trans-gender induction of hair follicles. Nature 402: 33–34. Hair Follicle Growth: The Role of SCs As the basic mechanisms of hair follicle development and hair growth are conserved between mice and humans [30], these studies are directly relevant to human hair follicle biology. However, some important aspects of human hair biology, such as the follicular response to androgens at puberty and in male pattern baldness, are absent in mice [64]. 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Bio-activities and phytochemical investigation of &lt;i&gt;Cnestis palala&lt;/i&gt; (Lour.) Merr.
African Journal of Traditional Complementary and Alternative Medicines
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Introduction Cnestis palala (Lour.) Merr., belongs to the Connaraceae family and in Thailand is commonly called Maa tai mi-phaak-laak and Ngonkai (local name in Thai: Kra phaak lak (Trat), Kalingping (Ratchabury), Mataimaithonglak (Chon Buri), Mataiwai or Masakkalat (Lampang), Mataiwai Ngonkai (Central), Ndonkipa, Kadoling (Nong Khai). In Peninsula, it is called Ngonkai nuai or Maa daeng (Smitinand, 2001). C. palala is a climbing shrub, laden with a velvety scarlet-orange fruit and has a wide repartition in South East Asia particularly in the Andaman Islands, Myanmar, Laos, Vietnam, Thailand, Malaysia, Sumatra, Borneo, Philippines and Sulawesi (Bunyapraphatsara and Lemmens, 2003). In the past, some plants in Cnestis genus such as C. glabra, C. polyphylla and C. ferruginea exhibited interesting biological activities; anti-diarrheal (Akindele et al., 2006), anti-oxidant (Akindele et al., 2010), anti-plasmodia (Bero et al., 2009), anti-inflammatory (Akindele and Adeyemi, 2007), anti-bacterial and anti-malarial activities (Dan et al., 2006). In traditional medicines; the root of C. palala was used for treatment of stomach ache, malaria, urinary trouble and snakebite; the seed was used for poisoning rat and stray dog. However, the seeds were reported to be toxic and some Malays and Thais use the seed for poisoning stray dogs. From previous studies, 50% ethanol root extract and aerial parts were tested for poisoning in mice within which the mice died at 1000 mg/kg (Bhakuni et al., 1988). In Thailand, it has been reported about its poisoning fresh seed against students in Pangnga province. After 10 hours of eating fresh seed, the children developed symptoms like dizziness, headache, nausea and vomiting (Jiratchariyakul, 2001). In Japan it has been reported for acute toxicity that at 75% ethanol extracts of freshly harvested seeds in dogs present with the poisoning symptoms and subsequently died at 347 mg/kg dosage after 24-25 hr. An unusual amino acid, L-methionine sulfoximine has been isolated from fresh seeds and also reported its toxicity in dogs (Murakoshi et al., 1993). In addition, some plants in Cnestis genus presented with methionine sulfoximine (i.e. C. glabra, C. polyphylla and C. ferruginea), exhibited poisoning in various organism (Jeannoda et al., 1985a; Garon et al., 2007). So, some biological activities such as anti-microbial, anti-cancer and anti-malarial activities of this plant has not been reported except for one compound, methionine sulfoximine from ethanol seed extract (Murakoshi et al., 1993). Introduction Thus, the study of biological activities and phytochemical investigation of this plant will be useful in getting more information and proof on the traditional uses of this plant in order to have a database for further study in new medicine from natural products. This is the first report of the chemical constituents in all parts of this plant used in carrying out an experimental approach to assess the anti-microbial, anti-fungal, anti-malarial, anti-tuberculosis and cytotoxicity activities. Moreover, we described the isolation and structure elucidation, together with the evaluation of biological activities of isolated pure compounds. Abstract Background: In traditional medicines, the root of Cnestis palala was used for the treatment of stomach ache, malaria, urinary track disorders and snakebite. The seed was used for poisoning rat and stray dogs. However, the bioactivities and chemical constituents have not been reported. So, this will be the first report. Material and Methods: Biological investigations of C. palala extracts against bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, and Pseudomonas aeruginosa), fungi (Trichophyton mentagrophytes, Trichophyton rubrum and Microsporum gypseum), yeast (Candida albicans), tuberculosis (Mycobacterium tuberculosis), malaria (Plasmodium falciparum) and cancer cell lines (MCF-7 and HT-29) were evaluated. The chromatographic and spectroscopic techniques were used for the isolation and identification of pure compounds. Results: The results showed that the ethanol leaf and bark extracts were active against S. aureus and S. epidermidis. The hexane leaf and seed extracts and petroleum ether bark and root extracts showed strongly inhibition values against MCF-7. Moreover, the petroleum ether bark extract exhibited high inhibition value against HT-29. Seven compounds were isolated as β-sitosterol-glucoside (CP1), hydroquinone (CP2), β-sitosterol (CP3), mixture of fatty acids (CP4) and ethyl caffeate (CP5), scopoletin (CP6) and 2-nonenal (CP7). The CP2 was strongly active against S. aureus and S. epidermidis, the MIC was showed 30.10 µg/ml and 15.05 µg/ml and the MBC was showed 15 µg/ml and 7.5 µg/ml, respectively. Conclusion: These results suggested that C. palala is a great potential source of anti-microbial agents. Hence, this is the first study, which will be the database for chemical constituents and bioactivities of C. palala. Keywords: Cnestis palala, Connaraceae, Phytochemistry, Bioactivities, Herbal medicines. Sukanya Dej-adisai*, Kittiya Tinpun, Chatchai Wattanapiromsakul, Niwat Keaw Department of Pharmacognosy and Pharmaceutical Botany,Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, 90112 Thailand. *E-mail: sukanya.d@psu.ac.th dejadisai@yahoo.com Isolation procedure of plant material Isolation of ethanol extract of C. palala leaf Ethanol extract from leaf (40 g) was isolated with silica gel column (vacuum liquid chromatography, VLC). The column was eluted with the mixture of methylene chloride (CH2Cl2) and ethyl acetate (EtOAc). The ratios between CH2Cl2 and EtOAc were 100:0 to 0:100. The solvent polarity was increasing until it yielded 42 fractions (500 ml/each). Fractions were pooled as guided with TLC fingerprint and evaporated to dryness under reduced pressure to give the following fraction L1-6. The fraction L5 (6.654 g) from VLC added on a silica gel column and eluted by the mixture of CHCl3, acetone and MeOH with the ratio 100:0:0 to 50:40:10, respectively. The fractions were combined into six major groups depending on their TLC profiles, to furnish compound CP1 137.5 mg (The %yield = 0.022 % based on dried weight of crude leaf extract). g p p g p , p g ( y g ) The fraction L1 (1.207 g) was further isolated by silica gel column. The column was eluted with the mixture of CH2Cl2 and EtOAc using the ratio 100:0 to 60:40, respectively. The fractions were combined into 10 fractions (H1-H10) based on TLC fingerprint. The fractions H1- H10 was submitted to flash column chromatography, being eluted with petroleum ether and EtOAc, in increasing polarity afforded compound CP3 (25.7 mg; % yield = 0.064% based on dried weight of crude leaf extract) and compound CP4 (7.28 mg; % yield = 0.018% based on dried weight of crude leaf extract). g The fraction L3 were pre-fractionated by column chromatography (CC) on silica gel eluting with the mixture of chloroform (CHCl3) and methanol (MeOH) with the ratio 100:0 to 50:50 to get 54 fractions. The fractions were combined into 8 fractions (F1-F8). After crystallization (F8) in CHCl3 and MeOH, compound CP2 (220.7 mg; %yield = 0.55% based on dried weight of crude leaf extract) was obtained as white needle. According to TLC analysis, the CP2 was a major component of ethanol leaf extract. The fraction F5 (297.5 mg) was purified by silica gel column and eluted by the mixture of hexane and EtOAc with the ratio 100:0 to 60:40 to get 24 fractions (Le1-24). The Le10-18 (100.8 g) was subjected to SephadexLH-20 column eluted with MeOH 100% to furnish 25 fractions (Le1a-Le25a). Fractions Le2a (40.23 mg) were subjected to RP-18 CC eluted by the mixture of MeOH and H2O with the ratio 50:50. Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Thani Province, Thailand. The voucher specimen was identified by comparison with authentic sample and was deposited at the herbarium of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand with herbarium number SKP 053 03 16 01. After cleaning, plant materials were dried at 60°C in hot air oven and ground to powder and kept for extraction at room temperature. Isolation procedure of plant material Isolation procedure of plant material Isolation of ethanol extract of C. palala leaf Isolation of ethanol extracts of C. palala seed Ethanol seed extract (5 g) was isolated in the mixture of CHCl3, acetone, MeOH by the ratio 100:0:0 to 20:70:10, respectively. The extracts were evaporated under reduced pressure to gain 64 fractions and combined into 15 major groups (SE1-15), the fraction SE2 to provide compound 7 (CP7) 80 mg (the % yield = 6.0 % based on dried weight of crude seed extract). CP1 (25 mg) was also isolated from fraction SE6. So, CP1 could be isolated from leaf, wood and seed of this plant. Isolation of ethanol extract of C. palala wood The EtOH extract (20 g) was dissolved in the mixture of EtOH and H2O by the ratio 2:8 and further extracted by partition with hexane (3 x 250 ml) to get hexane and aqueous extracts. Aqueous extract was diluted with H2O (250 ml) and then extracted with CHCl3 (3 x 250 ml), EtOAc (3 x 250 ml), and n-BuOH (3 x 250 ml), respectively. Solvent extracts were then evaporated to dryness under reduced pressure to give hexane extract (2.468g), CHCl3 extract (698.2 mg), EtOAc extract (5.973 g), n-BuOH extract (8.312g) and remaining aqueous extract (3.447g). The CHCl3 fraction (698 mg) was subjected to RP-18 column in the mixture of MeOH and H2O by the ratio 5:5 and the fractions were subjected to silica gel in the mixture of CHCl3 and MeOH by the ratio 100:0 to 50:40 and obtaining 8 main fractions (WCC A-H). The WCC A fraction (138.1 mg) was further fractionated by CC on silica gel using increasingly polar in the mixtures of hexane and EtOAc by the ratio 100:0 to 60:60. The fractions were combined into 4 major groups (WCC A1-4) depending on their TLC profiles and elution with hexane and EtOAc. The WCC A2 fractions were combined and applied to SephadexLH-20 column eluted with 100% MeOH, affording compound CP6 3.48 mg (The %yield = 0.01% based on dried weight of crude extract wood). However, CP1 and CP3 were also isolated from hexane and EtOAc wood extracts. The EtOH extract (20 g) was dissolved in the mixture of EtOH and H2O by the ratio 2:8 and further extracted by partition with hexane (3 x 250 ml) to get hexane and aqueous extracts. Aqueous extract was diluted with H2O (250 ml) and then extracted with CHCl3 (3 x 250 ml), EtOAc (3 x 250 ml), and n-BuOH (3 x 250 ml), respectively. Solvent extracts were then evaporated to dryness under reduced pressure to give hexane extract (2.468g), CHCl3 extract (698.2 mg), EtOAc extract (5.973 g), n-BuOH extract (8.312g) and remaining aqueous extract (3.447g). The CHCl3 fraction (698 mg) was subjected to RP-18 column in the mixture of MeOH and H2O by the ratio 5:5 and the g j y fractions were subjected to silica gel in the mixture of CHCl3 and MeOH by the ratio 100:0 to 50:40 and obtaining 8 main fractions (WCC A-H). Isolation procedure of plant material Isolation of ethanol extract of C. palala leaf Preparative TLC was used to provide CP5, 8.84 mg (The %yield = 0.022 % based on dried weight of crude leaf extract). Isolation of ethanol extract of C. palala wood The WCC A fraction (138.1 mg) was further fractionated by CC on silica gel using increasingly polar in the mixtures of hexane and EtOAc by the ratio 100:0 to 60:60. The fractions were combined into 4 major groups (WCC A1-4) depending on their TLC profiles and elution with hexane and EtOAc. The WCC A2 fractions were combined and applied to SephadexLH-20 column eluted with 100% MeOH, affording compound CP6 3.48 mg (The %yield = 0.01% based on dried weight of crude extract wood). However, CP1 and CP3 were also isolated from hexane and EtOAc wood extracts. Materials and methods Plant material ected from Krabi Plant Production Materials Technical Service Center, Krabi Province and Rajjaprabha Dam, Surat 27 Extraction, Isolation and Identification: Extraction procedure Dried leaf, wood, pod, bark, root and seed powders weighing 6, 3.5, 0.30, 1.5, 1.48 and 0.052kg, respectively were used. They were macerated at room temperature for three days with petroleum ether, hexane and ethanol consecutively (3 times). The filtrates obtained from each step were concentrated under reduced pressure, afforded the petroleum ether, hexane and ethanol extracts, respectively. Biological studies Agar disc diffusion method; determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were followed using Lorian (2005); Yasunaka et al., (2005); Phongpaichit et al. (2006); Kuete et al. (2007); Niyomkam (2007); Kummee and Intaraksa (2008); Ghalem and Mohamed (2009). The gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Propionibactelium acnes and gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa were obtained from the culture collection of the Thailand Institute of Scientific and Technology Research. The dermatophytes employed in this study, 28 Extraction The dried powder of leaf, stem, fruit, bark, root and seed from C. palala were exhaustively extracted with petroleum ether, hexane and ethanol by maceration, respectively. Activity against gram negative bacteria The gram negative bacteria were treated with C. palala crude extracts by disc diffusion method; all crude extract did not show activity against E. coli and P. aeruginosa (at 2.5 mg/ml). While, negative standard norfloxacin was presented inhibition zone against E. coli and P. aeruginosa, 30.8±0.5 mm and 25.10±0.9 mm, respectively. Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum gyseum and Candida albicans were obtained from the culture collection of the Specialized Centre for Medical Mycology, Federal University of the Department of Medical Science, Ministry of Public Health, Thailand. Activity against gram positive bacteria The crude extracts of fruit, root and seed were not tested for activity against S. aureus and S. epidermidis. Although, all parts of crude extract were not tested for activity against P. acnes, the crude ethanol leaf, stem and bark extracts showed low effect against S. aureus and S. epidermidis as showed in Table 1. Cytotoxic activity Sulforhodamine B (SRB) assay was used for cytotoxic activity determination and was supported by Assist. Prof. Dr. Supreeya Yuenyongsawad (Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University). In this study, the human breast adenocarcinoma cells (MCF-7), human colon adenocarcinoma cell line (HT-29) and normal cell (human gingiva fibroblasts cell) were obtained from National Cancer Institute, Bangkok, Thailand. Anti-malarial activity The anti-malarial activity assay was tested by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The micro-culture radioisotope technique based on the method described by Desjardins et al. (1979) was used in this assay. Plasmodium falciparum (K1, multidrug resistant strain) was used for evaluation. Screening of anti-fungal and yeast activities The results as showed in Table 2 indicated that some parts of the ethanol extract produced the inhibition zones against all fungi and yeast strains. The ethanol seed extract was observed strongly against T. rubrum, T. mentagrophytes and M. gypseum. The ethanol leaf, stem and bark extracts showed the inhibition zones against C. albicans. Anti-tubercular activity The anti-tubercular activity assay was tested by the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. The Green fluorescent protein (GFP)-based fluorescent detection assay as described by Changsen et al. (2003) was used for this assay. Mycobacterium tuberculosis strain H37Ra was used for evaluation. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for gram positive bacteria Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for gram positive bacteria From the screening of the activity against gram positive bacteria the ethanol leaf, stem and bark extracts displayed an inhibition zone against S. aureus and S.epidermidis as indicated. So, MIC and MBC were determined as showed in Table 3. 29 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Table 1: Screening of anti-gram positive bacteria activity from C. palala crude extracts. Plant part Average of growth inhibition zone (mm) at sample concentration 2.5 mg/ ml/ disc Petroleum ether extract Hexane extract Ethanol extract S. aureus S. epidermidis P. acnes S. aureus S. epidermidis P. acnes S. aureus S. epidermidis P. acnes Leaf - - - - - - 7.96±0.70 13.83±0.90 - Stem - - - - - - 7.05±0.43 7.00±0.21 - Bark - - - - - - 10.9±0.60 13.42±0.67 - Fruit - - - - - - - - - Root - - - - - - - - - Seed - - - - - - - - - Oxacillin 19.0±0.17 29.33±0.70 29.85±0.90 19.0±0.17 29.33±0.70 29.85±0.90 19.0±0.17 29.33±0.70 29.85±0.90 Standard for gram positive bacteria, concentration 1 µg/disk Table 2: Screening of anti-fungi and yeast activities from C. palala crude extracts. Plant part Average of growth inhibition zone (mm) atsample 2.5 mg/disc Petroleum ether extracts Hexane extracts Ethanol extracts TR TM MG CA TR TM MG CA TR TM MG CA Leaf - - - - - - - - - - - - Stem - - - - - - - - - - - 12.71 ± 0.56 Bark - - - - - - - - - - - 19.66 ± 0.56 Fruit - - - - - - - - - - - - Root - - - - - - - - - - - - Seed - - - - - - - - 54.98 ± 0.38 49.20 ± 0.85 45.74 ± 0.45 33.20 ± 0.14 amphotericin B - - - 16.40 ±0.65 - - - 16.40±0 .65 - - - 16.40 ± 0.65 ketoconazole 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - TR = T. rubrum, TM = T. standard for C. albicans, standard for T. rubrum, T. mentagrophytes and M. gypseum. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for gram positive bacteria mentagrophytes, MG = M. gypseum, CA = C. albicans, standard for C. albicans, standard for T. rubrum, T. mentagrophytes and M. gypseum. Table 1: Screening of anti-gram positive bacteria activity from C. palala crude extracts. Table 1: Screening of anti-gram positive bacteria activity from C. palala crude extracts. Table 2: Screening of anti-fungi and yeast activities from C. palala crude extracts. Plant part Average of growth inhibition zone (mm) atsample 2.5 mg/disc Petroleum ether extracts Hexane extracts Ethanol extracts TR TM MG CA TR TM MG CA TR TM MG CA Leaf - - - - - - - - - - - - Stem - - - - - - - - - - - 12.71 ± 0.56 Bark - - - - - - - - - - - 19.66 ± 0.56 Fruit - - - - - - - - - - - - Root - - - - - - - - - - - - Seed - - - - - - - - 54.98 ± 0.38 49.20 ± 0.85 45.74 ± 0.45 33.20 ± 0.14 amphotericin B - - - 16.40 ±0.65 - - - 16.40±0 .65 - - - 16.40 ± 0.65 ketoconazole 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - 58.80 ± 0.35 43.75 ± 0.66 40.43 ± 0.75 - TR = T. rubrum, TM = T. mentagrophytes, MG = M. gypseum, CA = C. albicans, standard for C. albicans, standard for T. rubrum, T. mentagrophytes and M. gypseum. Table 2: Screening of anti-fungi and yeast activities from C. palala crude extracts. A f th i hibiti ( ) t l 2 5 /di 30 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Table 3: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for gram positive bacteria Sample & standard drug MIC & MBC (µg/ml) S. epidermidis S. aureus MIC MBC MIC MBC Leaf 500 1000 1000 1500 Stem 2500 2500 1250 5000 bark 250 500 250 1000 oxacillin 0.126 0.54 0.24 0.97 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 In the present study, it can be observed that all microorganisms were susceptible to the ethanol leaf and bark extracts, with a variation in the MIC values from 0.03-4 µg/ml. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for gram positive bacteria We determined through the broth dilution method carried out in triplicate that the MIC cut point was established at 500 µg/ml. The results suggested that ethanol extract from bark was found to be more effective than the ethanol extract from leaf against all the organisms. Screening anti-malarial and anti-tuberculosis activities In traditional medicine, the decoction of C. palala root was used for the treatment of malaria (Murakoshi et al., 1993). Therefore the ethanol root extract of C. palala was investigated for anti-malarial activity. The results showed that the ethanol root extract of C. palala was inactive against P. falciparum at maximum concentration 10 µg/ml. f p µg The result of anti-tubercular activity showed that the ethanol root extract was inactive against Mycobacterium tuberculosis H37Ra at the maximum concentration 50 µg/ml. Determination Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) for fungi and yeast From the screening of the activity of C. palala crude extract against fungi and yeast displayed strong inhibition zone. Consequently, we have tested MIC and MFC of ethanol stem, bark and seed extracts for fungi and yeast as showed in Table 4. From the results, the MIC and MFC showed that the crude extracts were low potency against fungi and yeast. Table 4: Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) for fungi and yeast Sample ethanol extract & Standard MIC & MFC C. albicans T. rubrum T. mentagrophytes M. gypseum MIC MFC MIC MFC MIC MFC MIC MFC stem 3.75 15 - - - - - - bark 1.00 >30 - - - - - - seed 1.25 >40 5.00 >40 10 >40 5 >40 amphotericin B 0.5 15 - - - - - - ketoconazole - - 0.5 15 0.25 25 0.25 15 = mg/ml, , = µg/ml Table 4: Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) for fungi and yeast Structure determination of compound 1 (CP1) 1 13 g g g p From 1H-NMR spectrum in high field region at δ 0.68-2.75 ppm to the same class of phytosterols and in the region low field region at δ 3.95-5.03 was sevens protons of the sugar moiety. The EI-Mass spectrum was proposed 396 (m/z) [Aglycone (sterol) + H – H2O)] that suggested molecular ion peak of an only aglycone of sterol that the sugar was hydrolyzed. The HMBC spectrum showed correlations of the long rang coupling between 1H and 13C. The CP1 was melting point at 275–277oC. From 1H-NMR spectrum in high field region at δ 0.68-2.75 ppm to the same class of phytosterols and in the region low field region at δ 3.95-5.03 was sevens protons of the sugar moiety. The EI-Mass spectrum was proposed 396 (m/z) [Aglycone (sterol) + H – H2O)] that suggested molecular ion peak of an only aglycone of sterol that the sugar was hydrolyzed. The HMBC spectrum showed correlations of the long rang coupling between 1H and 13C. The CP1 was melting point at 275–277oC. 1 13 g p On the basis of IR, 1H-NMR,13C-NMR, 2D-NMR spectral data of CP1 and the comparison of its spectral data with the previous report (Moghaddam et al., 2007), it was identified as β-sitosterol-D-glucoside (Daucosterin). Structure determination of compound 2 (CP2) The compound 2 (CP2) is colorless prismatic needles and it presented brown spot on TLC (chloroform: methanol (9:1, Rf= 0.49). The UV spectrum in methanol revealed absorption maxima (λmax, MeOH) at 224 and 294, the FT-IR spectrum absorptions bands provided at 3172 cm-l (-OH stretching), 3030 cm-1 (=CH stretching), 2012-1651 cm-1 (phenyl ring substitution overtones), 1517 and 1467 cm-1 (aromatic ring stretching), 1193 and 1092 cm-1 (-C-O- stretching), 833 and 760 (phenyl ring substitution bands). The EI mass spectrum performed a molecular ion at 110 m/z (62.9, 64, 68.9, 81, 82, 83, 94.9, 107.9, 108.9, 109.9, 110.9 and 111.9), suggesting a molecular formula as C6H6O2. The two signals of 1H NMR spectrum of CP2 exhibited resonances (acetone-d6) at δ 6.65 ppm (2H, s) δ 7.61 ppm (1H, s). The 13C-NMR spectrum provided two carbons at δ 116.59 ppm and 151.18 ppm. NMR and 13C-NMR spectrum of CP2 suggested aromatic derivative that the symmetry in the chemical structure. Its UV spectral data and the comparison with previous report (Sabrahanian et al., 1973) suggested that CP2 was hydroquinone. pp From 1H NMR and 13C-NMR spectrum of CP2 suggested aromatic derivative that the symmetry i IR, MS, 1H-NMR, 13C-NMR, spectral data and the comparison with previous report (Sabrahanian et al., 1973) sugges Structure evaluations This study was the first report on the chemical investigation of C. palala except for those reported in the ethanol seed extract, which was found L-methionine sulfoximine, which was very toxic in organism from pervious study (Murakoshi et al., 1993; Garon et al., 2007). The isolation of pure compound through chromatography techniques and characterized by spectroscopy techniques. In the first study, the active components in ethanol extract of leaf and bark against gram positive bacteria were isolated (bioassay guided fractionation) and in the second, the components of all several parts of C. palala were investigated also. Chemical compounds from ethanol extract of leaf and bark Compound 1-5 (CP1-5) were isolated from the leaf extract and in this study could not purify the compound from und 1-5 (CP1-5) were isolated from the leaf extract and in this study could not purify the compound from the bark Screening of cytotoxic activity The results of cytotoxic activity against MCF-7 and HT-29 of the extracts from C. palala were depicted in Table 5. The cytotoxic activities against MCF-7 from hexane extract of leaf and seed; petroleum ether extract of bark and root showed % inhibition values of 79.97±6.09, 83.91±13.74, 93.58±1.87 and 83.36±7.44, respectively. Pronounced cytotoxic activity of HT-29 was exhibited by the petroleum ether bark extracts with % inhibition value 92.16±2.38 and other fractions showed low effect to both cell lines. In addition, the results of both cell lines showed that the most active extract was non-polarity fraction, because of the petroleum ether and hexane extracts were shown to be highly active against MCF-7 and HT-29 cell lines. This result suggested that the active constituents should be non-polarity compounds. Although several members of the Cnestis genus have already been reported as having cytotoxic activity in methanol, ethanol and aqueous extracts (Jeannoda et al., 1985a; Garon et al., 2007). It was found that the L-methionine sulfoximine was the potent cytotoxic compound in Cnestis (Jeannoda et al., 1985b; Garon et al., 2007). Some species such as Cnestis ferruginea is abundant in West Africa and in root applications, used in traditional medicine to treat diverse conditions were studied from sub- chronic toxicity in methanol root extract. The results showed that possible side effects identified as induced delayed platelet anomaly, anemia in females, weight reduction and sterility in males (Ishola et al., 2012). From the screening of cytotoxic activity against MCF-7 and HT-29 cell lines possessed a high activity accordingly. Our study involved the investigation of cytotoxic activity against normal cells as presented in Table 5. The petroleum ether and hexane extracts not being active against normal cells suggested that they were not toxic against normal cells in high concentration (25 µg/ml). Consequently, these fractions were investigated in isolation from the active compound. 31 The compound 3 (CP3) is a white crystalline compound. It displayed a positive anisaldehyde-sulphuric and 50 % sulphuric acid reagent as shown through the violet color. Its melting point was at 93-95 oC. The UV spectrum in chloroform showed absorption maxima (λmax, CHCl3) 216 and 243 nm, the FT-IR spectrum absorptions bands appeared at 3423 cm-1(-OH stretching), 2936.8 cm-1(-CH2 stretching), 2867.38 cm-1(=CH stretching), 1710.1 cm-1(-C=C-), 1465.9 cm-1 (-CH2 bending), 1382.5 cm-1 (-CH3 bending) 1053.6 cm-1 (-C-O- stretching). The EI mass spectrum exhibited a molecular ion at 414 m/z (396, 381, 353, 329, 303, 272, 255, 231, 213, 199, 173, 163, 161, 145, 133, 107, 95, 85 and 71), corresponding to the molecular formula C29H50O. Structure determination of compound 1 (CP1) 13 , , ), p y p ( , ) The 13C-NMR spectrum (125 MHz, pyridine-d5) showed thirty-five carbons at δ 12.04, 12.22, 19.09, 19.32, 19.47, 19.99, 21.38, 23.54, 24.57, 26.66, 28.57, 29.65, 30.35, 32.19, 32.25, 34.36, 36.44, 37.58, 37.03, 39.46, 40.08, 42.60, 46.22, 50.51, 56.41, 56.96, 63.02, 71.91, 75.39, 78.49, ) p y p ( ) The 13C-NMR spectrum (125 MHz, pyridine-d5) showed thirty-five carbons at δ 12.04, 12.22, 19.09, 19.32, 19.47, 19.99, 21.38, 23.54, 24.57, 26.66, 28.57, 29.65, 30.35, 32.19, 32.25, 34.36, 36.44, 37.58, 37.03, 39.46, 40.08, 42.60, 46.22, 50.51, 56.41, 56.96, 63.02, 71.91, 75.39, 78.49, 78.31, 78.66, 102.70, 141.07 and 121.92 ppm. Based on the information obtained from the HMQC spectrum all protonated carbons of CP1 were assigned. The long-rang C-H correlation of CP1 could observed from HMBC spectrum. The C NMR spectrum (125 MHz, pyridine d5) showed thirty five carbons at δ 12.04, 12.22, 19.09, 19.32, 19.47, 19.99, 21.38, 23.54, 24.57, 26.66, 28.57, 29.65, 30.35, 32.19, 32.25, 34.36, 36.44, 37.58, 37.03, 39.46, 40.08, 42.60, 46.22, 50.51, 56.41, 56.96, 63.02, 71.91, 75.39, 78.49, 78.31, 78.66, 102.70, 141.07 and 121.92 ppm. Based on the information obtained from the HMQC spectrum all protonated carbons of CP1 were assigned. The long-rang C-H correlation of CP1 could observed from HMBC spectrum. 78.31, 78.66, 102.70, 141.07 and 121.92 ppm. Based on the information obtained from the HMQC spectrum all protonated carbons of CP1 were assigned. The long-rang C-H correlation of CP1 could observed from HMBC spectrum. 78.31, 78.66, 102.70, 141.07 and 121.92 ppm. Based on the information obtained from the HMQC spectrum all assigned. The long-rang C-H correlation of CP1 could observed from HMBC spectrum. 1 g g g p From 1H-NMR spectrum in high field region at δ 0.68-2.75 ppm to the same class of phytosterols and in the region low field region at δ 3.95-5.03 was sevens protons of the sugar moiety. The EI-Mass spectrum was proposed 396 (m/z) [Aglycone (sterol) + H – H2O)] that suggested molecular ion peak of an only aglycone of sterol that the sugar was hydrolyzed. The HMBC spectrum showed correlations of the long rang coupling between 1H and 13C. The CP1 was melting point at 275–277oC. Structure determination of compound 1 (CP1) The compound 1 (CP1) was isolated as white powder but presented violet color after spraying with anisaldehyde-sulphuric acid reagent and 50 % sulphuric acid reagent and heating on an electric oven at 110oC. The UV spectrum in methanol demonstrated absorption maxima (λmax, MeOH) 204, 230 and 244 nm and the structures according to FT-IR presented functional group to be as follows: 3600-3400 cm-1 (-OH stretching), 2934 cm-1 (-C-H stretching), 2850 cm-1 (=C-H sp2 stretching), 1640 cm-1 (-C=C- stretching), 1450 and 1379 cm-1 (-C-H bending), 1073 and 1023 cm-1 (-C-O- stretching). The EI mass spectrum exhibited a molecular ion at 396 m/z, consistent with formula of C29H49O6. g) p 9 9 6 From the 1H-NMR spectrum (500 MHz, pyridine-d5), the high field region indicated six methyl groups at δ 0.68 (3H, s), δ0.88 (3H, d, J = 6.6 Hz), δ 0.87 (3H, d, J = 6.9 Hz), δ 0.90 (3H, m , J = 7.3 Hz), δ 0.96 (3H, s), δ 0.99 (3H, d , J = 6.5 Hz) and the low field region showed methine proton at δ 3.95 (1H, m), δ 4.01 (1H, t , J = 7.9 Hz), δ 4.25 (1H , m), δ 4.22 (1H , m), δ 4.52 (1H, dd, J = 11.6 Hz, 2.1 Hz, β), δ 4.38 (1H, dd , J=11.7 Hz, 5 .2 Hz,α), respectively and one olefinic proton at δ 5.36 (1H, broad s). g p From the 1H-NMR spectrum (500 MHz, pyridine-d5), the high field region indicated six methyl groups at δ 0.68 (3H, s), δ0.88 (3H, d, J = 6.6 Hz), δ 0.87 (3H, d, J = 6.9 Hz), δ 0.90 (3H, m , J = 7.3 Hz), δ 0.96 (3H, s), δ 0.99 (3H, d , J = 6.5 Hz) and the low field region showed methine proton at δ 3.95 (1H, m), δ 4.01 (1H, t , J = 7.9 Hz), δ 4.25 (1H , m), δ 4.22 (1H , m), δ 4.52 (1H, dd, J = 11.6 Hz, 2.1 Hz, β), δ 4.38 (1H, dd , J=11.7 Hz, 5 .2 Hz,α), respectively and one olefinic proton at δ 5.36 (1H, broad s). N = not active against normal cell. Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 From the 1H-NMR spectrum (500 MHz, chloroform-d), its presence was indicated six methyl groups at δ 0.72 (3H, s), δ 0.85 (3H, d, J = 6.7 Hz), δ 0.87 (3H, d, J = 6.7 Hz), δ 0.89 (3H, t, J = 7.4 Hz), δ 0.96 (3H, d, J = 6.5 Hz), 1.05 (3H, s) and methine proton at δ 3.56 (1H, m), and one olefinic proton at δ 5.39 (1H, broad m), respectively. ( , ) p y The 13C-NMR spectrum (125 MHz chloroform-d5), showed twenty nine carbons at δ 11.86, 11.99, 18.79, 19.04, 19.40 .07, 28.26, 29.16, 31.60, 31.65, 31.93, 33.95, 36.15, 36.50, 37.02, 39.88, 42.30, 42.31, 45.83, 50.13, 56.06, 56.77, 121.70 1 13 ( ) p y MR spectrum (125 MHz chloroform-d5), showed twenty nine carbons at δ 11.86, 11.99, 18.79, 19.04, 19.40, 19.83, 21.0 29.16, 31.60, 31.65, 31.93, 33.95, 36.15, 36.50, 37.02, 39.88, 42.30, 42.31, 45.83, 50.13, 56.06, 56.77, 121.70 and 140.70 13 From 1H-NMR and 13C-NMR spectra pattern were similar to that of β-sitosterol glucoside (CP1), but the chemical shift at 3.95-5.03 (the region of sugar moiety) does not appear. The comparison of its 1H-NMR and 13C-NMR spectra with previously report (Patra et al., 2010), CP3 was identified as β-sitosterol. Structure determination of compound 4 (CP4) Compound 4 (CP4) was white powder; it showed violet color when spayed with 50% sulphuric acid and sh The 1H-NMR spectrum of CP4 displayed the mixtures of fatty compounds, the1H-NMR spectrum directe (1H ) 1 54 (2H ) h l h l (CH ) i l δ 1 20 1 33 (29H ) 0 85 (2H ) Compound 4 (CP4) was white powder; it showed violet color when spayed with 50% sulphuric acid and showed one spot on TLC. The 1H-NMR spectrum of CP4 displayed the mixtures of fatty compounds, the1H-NMR spectrum directed δ (Chloroform-d); δ 3.62 ppm (1H, t), 1.54 ppm (2H, m), the large methylene (CH2) proton signal δ 1.20-1.33 ppm (29H, m), 0.85 ppm (2H, t). 1 The 1H-NMR spectrum of CP4 displayed the mixtures of fatty compounds, the1H-NMR spectrum directed δ (Chloroform-d); δ 3.62 ppm (1H, t), 1.54 ppm (2H, m), the large methylene (CH2) proton signal δ 1.20-1.33 ppm (29H, m), 0.85 ppm (2H, t). 1 The H-NMR spectrum of CP4 displayed the mixtures of fatty compounds, the H-NMR spectrum directed δ (Chloroform-d); δ 3.62 ppm (1H, t), 1.54 ppm (2H, m), the large methylene (CH2) proton signal δ 1.20-1.33 ppm (29H, m), 0.85 ppm (2H, t). 1 From 1H-NMR spectrum suggested that CP4 are long-chain carbon, when analyzed of long-chain carbon mixture of two major saturated fatty acids, as hexadecanoic acid (72.16%), octadecanoic acid (32.83%) and unkno From 1H-NMR spectrum suggested that CP4 are long-chain carbon, when analyzed of long-chain carbon by GC-MS, they performed the mixture of two major saturated fatty acids, as hexadecanoic acid (72.16%), octadecanoic acid (32.83%) and unknown (5.74%). Structure determination of compound 6 (CP6) CP6 was obtained as a yellow needle, the UV-wavelength at 365 nm showed violet color on the TLC plats. This compound CP6 presented UV absorbance (λmax, methanol) at 207, 223, 297 and 348 nm, the FT-IR spectrum of CP6 showed absorption bands at 3436 cm-1 (due to a hydroxyl group), 1683 cm-1 (corresponding to carbonyl group (δ-lactone)), 1615 cm-1 (corresponding to CH=CH group) 1507 and 1424 cm-1 (corresponding to aromatic benzene ring), 1365 cm-1 (-CH3 bending) and 1020 cm-1 (-C-O bending). The GC-MS presented molecular ion peak at m/z 192.1 (42, 51.1, 69, 79, 92, 105.1, 121.1, 149.1, 164.1, 177, 192.1), corresponding to C10H8O4. , , , , , , , , ), p g 10 8 4 From the 1H NMR spectrum (500 Mz, DMSO-d6) was indicated one methoxy groups at δ 3.80 (3H, s), the four olefinic proton at δ 6.20 (1H, d, J = 9.08 Hz), 6.76, (1H, s), 7.2, (1H, s) and 7.88 (d, 1H, J = 9.08 Hz). The 13C-NMR spectrum showed eleven carbons at δ 56.1, 102.9, 109.7, 110.5, 111.6, 144.6, 145.5, 149.7, 151.6 and 160.8. Based on the information obtained from the HMQC, all protonated carbons of CP6 were assigned. The comparisons of 1H NMR and 13C-NMR spectra of CP6 with previous report (Silva et al., 2001), from spectroscopy techniques suggested that the CP6 was scopoletin (7-(hydroxy-6-methoxycoumarin), it showed its melting point as 203-204oC. It is simple coumarin group. It has been shown to exert several biological activities, such as: anti-cholinesterasic (Orhan et al., 2008), anti-nociceptive (Ribas et al., 2008), anti- inflammatory (Kim et al., 2004), anti-thyroid, anti-oxidant, anti-hyperglycemic (Panda and Kar, 2006), hypouricemic (Ding et al., 2005) and anti- tumor activities (Liu et al., 2001). Compound from ethanol wood extract Ethanol wood extract of C. palala showed the weakest activity for microorganisms but was active against snake-bite in preliminary study (Jiratchariyakul, 2001). So, this part was investigated for chemical constituents. From the experiment, we noted that a major compound in this part was β-sitosterol-glucoside. However, compound 6 (CP6) was also isolated from ethanol wood extract. Structure determination of compound 5 (CP5) The compound 5 (CP5) was a yellowish powder, with UV-visible absorption in methanol at λmax 207, 299, 300, 328 nm, the structures according to FT-IR spectra presented functional group to be as follows: 3399 cm-1 (-OH stretching), 2927 cm-1 (-CH2 stretching), 2850 cm-1(=CH stretching), 2298 cm-1 (Overton of aromatic ring), 1699 cm-1 (-C=O bending), 1602 cm-1 (-C=C- of aromatic ring), 1447 cm-1 (-CH2 bending), 1369 cm-1 (-CH3 bending), 1264 1117, 1034 and 1180 cm-1 (-C-O- stretching) and 981-701 cm-1 (signal of aromatic). The EI-MS spectrum of CP5 showed the molecular ion peak at m/z 208, corresponding to C11H12O4. p p g The 1H -NMR spectrum of CP5 showed one methyl group at δ 1.22 ppm (3H, t), one methylene group at δ 4.15 ppm (2H, q) and offered olifinic proton at δ 6.25 ppm, (1H, d, J = 16.06 Hz), δ 6.88 ppm (1H, d, J = 8.3 Hz), δ 6.99 ppm (1H, dd, J = 2.07 Hz, J = 8.03 Hz), δ 7.03 ppm (1H, d, p p g The 1H -NMR spectrum of CP5 showed one methyl group at δ 1.22 ppm (3H, t), one methylene group at δ 4.15 ppm (2H, q) and offered olifinic proton at δ 6.25 ppm, (1H, d, J = 16.06 Hz), δ 6.88 ppm (1H, d, J = 8.3 Hz), δ 6.99 ppm (1H, dd, J = 2.07 Hz, J = 8.03 Hz), δ 7.03 ppm (1H, d, J = 2.07 Hz) and δ 7.55 ppm (1H, d, J = 16.81 Hz). The 13C-NMR spectrum of CP5 displayed eleven signals at the δ 14.40, 59.8, 114.15, 114.91, 115.0, 121.47, 125.60, 145.13, 145.74, 148.61 and 166.68 ppm. h li i h 1 hibi d h li f l fi i i id h i d trum of CP5 displayed eleven signals at the δ 14.40, 59.8, 114.15, 114.91, 115.0, 121.47, 125.60, 145.13, 145.74, 148.6 The 13C-NMR spectrum of CP5 displayed eleven signals at the δ 14.40, 59.8, 114.15, 114.91, 115.0, 121.47, 125.60, 145.13, 145.74, 148.61 and 166.68 ppm. Structure determination of compound 5 (CP5) h li i h 1 hibi d h li f l fi i i id h i d Moreover, the coupling constants in the 1H NMR spectrums exhibited the coupling of olefinic proton in side chain proton, presented trans form, the internal molecule showed coupling aromatic ring, the presented Jortho coupling at 8.03 Hz, Jmeta coupling at 2.07 Hz and Jpara coupling was not shown in the 1H NMR. Its 1H and 13C NMR spectra when compared with previous report (Chiang et al., 2005) suggested that CP5 was ethyl caffeate. It is a derivative of caffeic acid. From literature, it presented an anti-inflammatory and anti-oxidant activity (Chiang et al., 2005). Structure determination of compound 3 (CP3) The compound 3 (CP3) is a white crystalline compound. It displayed a positive anisaldehyde-sulphuric and 50 % sulphuric acid reagent as shown through the violet color. Its melting point was at 93-95 oC. The UV spectrum in chloroform showed absorption maxima (λmax, CHCl3) 216 and 243 nm, the FT-IR spectrum absorptions bands appeared at 3423 cm-1(-OH stretching), 2936.8 cm-1(-CH2 stretching), 2867.38 cm-1(=CH stretching), 1710.1 cm-1(-C=C-), 1465.9 cm-1 (-CH2 bending), 1382.5 cm-1 (-CH3 bending) 1053.6 cm-1 (-C-O- stretching). The EI mass spectrum exhibited a molecular ion at 414 m/z (396, 381, 353, 329, 303, 272, 255, 231, 213, 199, 173, 163, 161, 145, 133, 107, 95, 85 and 71), corresponding to the molecular formula C29H50O. 32 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Table 5: Determination of cytotoxic activity with MCF-7, HT-29, and Normal cell lines. Table 5: Determination of cytotoxic activity with MCF-7, HT-29, and Normal cell lines. Table 5: Determination of cytotoxic activity with MCF-7, HT-29, and Normal cell lines. Table 5: Determination of cytotoxic activity with MCF-7, HT-29, and Normal cell lines. Plant part % inhibition (at 25 µg/ml) MCF-7 cell line HT-29 cell line Normal cell Petroleum ether extract Hexane extract Ethanol extract Petroleum ether extract Hexane extract Ethanol extract Petroleum ether extract Hexane extract Ethanol extract Leaf 60.98±16.42 79.97±6.09 44.67±19.51 1.89±14.52 5.877±5.63 35.76±8.45 N 17.04±6.09 N wood 31.48±13.77 60.67±20.08 27.36±46.63 56.45±4.76 34.75±6.83 41.09±8.26 N N N Bark 93.58±1.87 66.33±11.60 38.14±20.92 92.16±2.38 38.05±9.57 25.33±7.26 2.66±0.8 N N Fruit 35.88±13.35 1.65±5.99 -2.64±14.86 29.17±10.57 26.25±37.21 41.09±24.99 N N N Seed 27.42±12.51 83.91±13.74 36.24±20.96 45.93±17.96 41.00±10.22 37.99±24.16 N 20.68±10.76 N Root 83.36±7.44 57.13±8.77 40.05±15.07 43.65±0.08 53.01±11.64 59.45±2.9 N N 25.3±4.47 N = not active against normal cell. 33 Ethanolic seed extract from C. palala showed a chemical constituent likely comparable with the compound in leaf and wood extract. The chemical structures were presented β-sitosterol, β-sitosterol-glucoside and Compound 7 (CP7). Determination of anti-bacterial activity of isolated compounds All isolated compounds from ethanol leaf extract obtained in the present study as CP1-5 were studied for activity against gram positive bacteria (S. aureus and S. epidermidis), the results are showed in Table 6. Table 6: Anti-gram positive bacterial activity of isolated compounds from C. palala Pure compounds & Standard MIC & MBC (µg/ml) S. aureus S. epidermidis MIC MBC MIC MBC β-sitosterol-glucoside (CP1) 500 >500 500 >500 Hydroquinone (CP2) 15.05 30.10 7.25 15.05 β-sitosterol (CP3) 500 >500 500 >500 Mixture of fatty acids (CP4) N N N N Ethyl caffeate (Le11) 500 >500 250 >500 Oxacilline (Std.) 0.25 1.00 0.25 1.00 N = inactive against S.aureus and S.epidermidis Table 6: Anti-gram positive bacterial activity of isolated compounds from C. palala s & Standard MIC & MBC (µg/ml) Table 6: Anti-gram positive bacterial activity of isolated compounds from C. pa The anti-microbial activities of substances were assayed against two gram-positive bacteria (S. aureus and S. epidermidis). The MIC obtained in the initial assessment of the CP2 demonstrated a very promising activity from the ethanolic leaf extract against S. aureus and S. epidermidis. It also exhibited strong activity against S. epidermidis which presented MIC and MBC as 7.25µg/ml and 15.05µg/ml, respectively. Conversely, CP2 exhibited activity against S. aureus that showed MIC and MBC as 15.05µg/ml and 30.10µg/ml respectively. The other compounds showed weak activity against the S. aureus and S. epidermidis when displayed with the MIC and MBC showing a high concentration. The known anti-microbial mechanisms associated to each group of chemical to which the isolated compounds belong may explain the anti- microbial potency of the crude extracts from leaf of C. palala. The mechanisms of hydroquinone against microorganism could be suggested as having likely mechanisms action of quinone group (Stern et al., 1996; Kim et al., 2010) which actions are known to be complex irreversibly with nucleophilic amino acids in proteins, often leading to the inactivation of proteins and loss of function membrane. The influence of the position of hydroxyl groups on the aromatic ring, which seemed to play an important role to the agglutination of organism, was examined. From previous study, Himejima et al., (2004), studied agglutination and adherence of hydroquinone against Streptococcus mutans that its action mechanism was same as S. aureus and S. epidermidis. Hydroquinone altered the sucrose-induced agglutination of S. Structure determination of compound 7 (CP7) The CP7 presented white powders and showed violet color after spay with anisaldehyde-sulphuric acid reagent on the TLC plats. The FT- IR spectra of CP7 showed absorption bands at 2919 cm-1 (-C-H stretching), 2851 cm-1 (=C-H sp2 stretching), 1738 cm-1 (-C=O- stretching), 1471, 1417, 1392, 1281, 1266, 1247, 1221, 1197, 1179 and 1113 cm-1 (-C-H bending). The1H -NMR spectrum of CP7 showed one terminal methyl group at δ 0.84–0.86 ppm, (3H, m), the signal of the methylene (CH2) protons at δ 1.2–1.3 ppm ((CH2)m, m), at δ 1.56–1.59 ppm ((CH2)2, m), ), protons attached to the allylic carbons at δ 2.0–2.1 ppm, protons attached to the bis- allylic carbons at δ 2.2–2.3 ppm, δ 4.10–4.13 ppm (1H, dd, J = 6 Hz), at δ 4.25–4.28 ppm (1H, dd, J = 4.5 Hz) and olefinic protons at δ 5.2–5.3 ppm (2H, m). From 1H -NMR spectrum of CP7 showed a signal of the mixture with long chain hydrocarbon. Therefore, GC-MS was used for structure determination of this compound. hy presented many peaks on the chromatogram, and MS library predicted a compound in the GC-chromatogram a corresponding to C10H8O4, that showed as 2-Nonenal. From all data, CP7 was concluded as 2-nonenal. p The gas chromatography presented many peaks on the chromatogram, and MS library predicted a compou molecular ion peak 139 m/z, corresponding to C10H8O4, that showed as 2-Nonenal. From all data, CP7 was conclude Determination of anti-bacterial activity of isolated compounds mutans by forming the hydroquinone bridge, while neither resorcinol nor catechol exhibited any activity, presumably because of their steric hindrance. The result clearly indicates that the para-dihydroxy substitution is essential in making bridges between S. mutans cells. This study is the first report of the activity of hydroquinone against S. aureus and S. epidermidis. Compound from ethanol seed extract Ethanolic seed extract from C. palala showed a chemical constituent likely comparable with the compound in leaf and wood extract. The chemical structures were presented β-sitosterol, β-sitosterol-glucoside and Compound 7 (CP7). 34 In the phytochemical study, the isolation of compounds was done by chromatography techniques and characterization by spectroscopy techniques. The active components in ethanol leaf and bark extracts against gram positive bacteria were investigated and the components of all several parts from C. palala were investigated also. The ethanol leaf extract was performed β-sitosterol-glucoside (CP1) 0.022 %, hydroquinone (CP2) 0.55%, β-sitosterol (CP3) 0.064%, the mixture of fatty acids (CP4) 0.018% and ethyl caffeate (CP5) 0.022 %, respectively (Fig 1). From the Acknowledgements The authors are thankful to the Plant Genetic Conservation Project under The Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (RSPG project), Faculty of Pharmaceutical Sciences, Prince of Songkla University for financial support of this research. References Conclusions This study is the first report on the biological activities and phytochemical investigation in all parts of this plant, whose plant extracts have great potential as anti-microbial compounds against microorganisms. Thus, they can be used in the treatment of infectious diseases caused by microorganisms. Moreover, the synergistic effect from the association of antibiotics with plant extracts against resistant bacteria leads to new choices for the treatment of infectious diseases. This effect enables the use of the respective antibiotics when it is no longer effective by itself during therapeutic treatment. In addition, this study will provide a database for C. palala as touching the contents of chemical constituents and biological activities. Discussion Cnestis palala (Connaraceae Family) was said to be adaptive for poisonings in dogs, rats and other organism. In this study, the phytochemical investigation and screening of biological activities of leaf, wood, bark, fruit, seed and root in petroleum ether, hexane and ethanol extracts from Cnestis palala were done. The eighteen fractions were screened by biological activities against gram negative and gram positive bacteria, fungi, yeast and cytotoxicity on cancer cell lines. The ethanol root extract was screened with anti-malarial and anti-tuberculosis activities followed as traditional uses. From the results obtained, the ethanol leaf and bark extracts presented activity against gram positive bacteria (S. epidermidis and S. aureus) and all extracts were inactive against the gram negative bacteria, fungi and yeast. The results of cytotoxic activity with MCF-7 cancer cell lines demonstrated that hexane extract of leaf and seed; petroleum ether extract of bark and root showed % inhibition values of 79.97±6.09, 83.91±13.74, 93.58±1.87 and 83.36±7.44, respectively. Then petroleum ether bark extract exhibited the potential cytotoxicity to HT-29 cancer cell line with % inhibition value as 92.13±2.38 and other fractions showed low potency to both cancer cell lines. In the phytochemical study, the isolation of compounds was done by chromatography techniques and characterization by spectroscopy techniques. The active components in ethanol leaf and bark extracts against gram positive bacteria were investigated and the components of all several parts from C. palala were investigated also. The ethanol leaf extract was performed β-sitosterol-glucoside (CP1) 0.022 %, hydroquinone (CP2) 0.55%, β-sitosterol (CP3) 0.064%, the mixture of fatty acids (CP4) 0.018% and ethyl caffeate (CP5) 0.022 %, respectively (Fig 1). From the 35 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 results of CP1-5 against S. aureus and S. epidermidis were suggestive that hydroquinone (CP2) showed the strongest inhibition against S. aureus and S. epidermidis, the MIC and MBC of S. aureus and S. epidermidis were presented as 7.5 µg/mland 15 µg/ml and 15.05 µg/mland 30.10 µg/ml, respectively. This is the first report of anti-bacterial activity of hydroquinone against S. aureus and S. epidermidis. Conversely, other compounds CP1, CP3, CP5 and the mixture of fatty acids displayed the lowest activity against S. epidermidis and S. aureus. , , y p y y g p Furthermore, in the results we proposed the phytochemical study from other parts of C. palala, wood, fruit and seed extracts. The ethanol wood extract provided the scopoletin (CP6), the ethanol seed extract was including β-sitosterol, β-sitosterol-glucoside and the mixture of long chain hydrocarbon (the GC-MS library was predicted as 2-Nonenal). β it t l l id D t i (CP1)) β sitosterol gl coside; Da costerin (CP1)) β-sitosterol-glucoside; Daucosterin (CP1)) Hydroquinone (CP2) β-sitosterol (CP3) The mixture of fatty acids (CP4) Ethyl caffeate (CP5) Scopoletin (CP6) 2-Nonenal (CP7) Figure 1: Chemical structure of isolated compounds from C. palala. Octadecanoic acid Hexadecanoic acid CP1)) Hydroquinone (CP2) The mixture of fatty acids (CP4) Scopoletin (CP6) 2-Nonenal (CP7) Figure 1: Chemical structure of isolated compounds from C. palala. Octadecanoic acid Hexadecanoic acid β-sitosterol-glucoside; Daucosterin (CP1)) Hydroquinone (CP2) β-sitosterol (CP3) Ethyl caffeate (CP5) Octadecanoic acid Hexadecanoic acid The mixture of fatty acids (CP4) Hexadecanoic acid β-sitosterol (CP3) Scopoletin (CP6) Scopoletin (CP6) Scopoletin (CP6) Ethyl caffeate (CP5) Ethyl caffeate (CP5) 2-Nonenal (CP7) 2-Nonenal (CP7) Figure 1: Chemical structure of isolated compounds from C. palala. References 36 Dej-adisai et al., Afr J Tradit Complement Altern Med. (2015) 12(3):27-37 Liu, X.L., Zhang, L., Fu, X.L., Chen, K., and Qian, B.C. (2001). Effect of scopoletin on PC3- cell proliferation and apopto 3. Liu, X.L., Zhang, L., Fu, X.L., Chen, K., and Qian, B.C. (2001). Effect of scopoletin on PC3- cell proliferation and apoptosis. Act 4. Lorian, V. (2005). 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Abstract. Conservative mapping of data from one horizontal grid to another should preserve certain integral or mean properties of the original data. This may be essential in some model applications, including ensuring realistic exchange of energy and mass between coupled model components. It can also be essential for certain types of analysis, such as evaluating how far a system 5 is from an equilibrium state. For some common grids, existing remapping algorithms may fail to perfectly represent the shapes and sizes of grid cells, which leads to errors in the remapped fields. A procedure is presented here that enables users to rely on the mapping weights generated by remapping algorithms but corrects for their deficiencies. With this procedure, for a given pair of source and destination grids, a single set of remapping weights can be applied to remap any variable, including those with grid cells that are partially or fully masked. 10 10 1 Introduction When analyzing climate data from different sources, it is often necessary, as an initial step, to map the data to a common grid, a procedure commonly referred to as remapping or “regridding” the data. For some purposes it is essential when remapping the data that the global mean (or, alternatively, the global integral) of the field be preserved. Conservative remapping algorithms are meant to guarantee this. In practice, remapping occurs in two steps: 1) given a source and destination grid, mapping “weights” 15 are computed, and then 2) a sparse matrix multiplication of the source data by the weights yields the values of the field on the destination grid. Our focus here is on the second step: given the weights needed for remapping conservatively, we provide guidance on how they should be applied. Appendix A lists some remapping packages that can be used to generate weights (i.e., to execute step 1). It should be noted that many (perhaps all) of these packages slightly misrepresent the true grid cell shapes meant to guarantee this. In practice, remapping occurs in two steps: 1) given a source and destination grid, mapping “weights” 15 are computed, and then 2) a sparse matrix multiplication of the source data by the weights yields the values of the field on the destination grid. Our focus here is on the second step: given the weights needed for remapping conservatively, we provide guidance on how they should be applied. Appendix A lists some remapping packages that can be used to generate weights (i.e., to execute step 1). It should be noted that many (perhaps all) of these packages slightly misrepresent the true grid cell shapes 15 for certain common grids, so care must be taken to correct for this. Moreover, most packages provide inadequate guidance on 20 how to handle fields that are partially masked or for three dimensional fields how to account for variations in the thickness of individual layers. The main purpose here is to clearly explain how to compensate for a remapping algorithm’s inaccurate representation of grid cell shapes and to account for missing or partially masked data when that is necessary. for certain common grids, so care must be taken to correct for this. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 1 Introduction A different relationship must be satisfied to preserve a field’s global mean (denoted by an overbar): A different relationship must be satisfied to preserve a field’s global mean (denoted by an overbar): Fs = P i FsifsiAsi P i fsiAsi = P j FdjfdjAdj P j fdjAdj = Fd . Fs = P i FsifsiAsi P i fsiAsi = P j FdjfdjAdj P j fdjAdj = Fd . (2) (2) (2) In the next section, we introduce the formulas that apply to remapping data when fs = fd = 1 everywhere (i.e., when there In the next section, we introduce the formulas that apply to remapping data when fs = fd = 1 e is no partial or complete masking of any cells). This is followed in section 3 by a description of the more general procedure 45 when a field might be partially masked. In section 4 we provide recipes that should be followed in remapping different types of two-dimensional and three-dimensional fields. That is followed by a brief discussion of how to preserve certain properties when interpolating data in the vertical. In the summary, we include some discussion of when conservative remapping may be inappropriate (or at least inadvisable). 1 Introduction Moreover, most packages provide inadequate guidance on 20 how to handle fields that are partially masked or for three dimensional fields how to account for variations in the thickness of individual layers. The main purpose here is to clearly explain how to compensate for a remapping algorithm’s inaccurate representation of grid cell shapes and to account for missing or partially masked data when that is necessary. The objective in remapping conservatively is to preserve certain physically important characteristics of the climate system The objective in remapping conservatively is to preserve certain physically important characteristics of the climate system. For a climate in global thermodynamic equilibrium, for example, the mean net flux of energy at the top of the atmosphere is 25 zero. In properly formulated models run with all externally-imposed conditions unchanging over time, the net flux at the top of For a climate in global thermodynamic equilibrium, for example, the mean net flux of energy at the top of the atmosphere is 25 zero. In properly formulated models run with all externally-imposed conditions unchanging over time, the net flux at the top of 1 1 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. the atmosphere will indeed approach and fluctuate about zero as the system approaches equilibrium. When the fluxes from a simulation of this kind are mapped to a different grid, we would like to preserve this important characteristic of the simulation. This can only be done if the remapping algorithm is conservative. The fundamental relationship that must be satisfied to preserve the integral of a field over the global area is: 30 (1) r2 s X i FsifsiAsi = r2 d X j FdjfdjAdj , X i X j where F is the quantity that is mapped from a source grid to a destination grid, A is the grid cell area (expressed as as a solid angle and globally spanning 4π steradians), f is the grid cell fraction where F is defined (i.e., the “unmasked fraction” of a cell), and the subscripts, s and d, distinguish among the source and destination cells, respectively. 1 Introduction The i and j indices apply to where F is the quantity that is mapped from a source grid to a destination grid, A is the grid cell area (expressed as as a solid angle and globally spanning 4π steradians), f is the grid cell fraction where F is defined (i.e., the “unmasked fraction” of a cell), and the subscripts, s and d, distinguish among the source and destination cells, respectively. The i and j indices apply to p g g p y j pp y source and destination cells, respectively, and the sums are over the entire domain. In certain modeling applications, the radius 35 of the earth, r, for the source and destination grids may differ slightly, and this is accounted for by including the squares of rs and rd in (1). If there is a mismatch in radii, the values of Fd must be scaled so that the integral is preserved. The sums are over all grid cells in the domain. For source cells that are completely masked or where data are undefined or “missing”, fs = 0. For the most common case when the value in a source cell is representative of the entire cell extent and when there are no missing data, fs = 1 for all grid cells. Note that when remapping conservatively, if fs = 1 for all source cells, then we require 40 source and destination cells, respectively, and the sums are over the entire domain. In certain modeling applications, the radius 35 of the earth, r, for the source and destination grids may differ slightly, and this is accounted for by including the squares of rs and rd in (1). If there is a mismatch in radii, the values of Fd must be scaled so that the integral is preserved. The sums are over all grid cells in the domain. For source cells that are completely masked or where data are undefined or “missing”, fs = 0. For the most common case when the value in a source cell is representative of the entire cell extent and when there are no missing data, fs = 1 for all grid cells. Note that when remapping conservatively, if fs = 1 for all source cells, then we require 40 that fd = 1 for all destination cells. 2 Remapping without masking 50 (5) This identity can be used to prove that for the case fs = fd = 1, (3) preserves the global mean. The proof is obtained by substituting (4) into (3), then substituting the result into (2), reversing the order that the summations are performed, and 65 making use of (5). Importantly, a consistent set of areas must be used in evaluating (1), (2), and (5). 65 Noting that the fractional areas of the source cells contributing to a destination cell must add up to the area of the destination cell, we obtain a second useful identity: Noting that the fractional areas of the source cells contributing to a destination cell must add up to the area of the destination cell, we obtain a second useful identity: X i ωijAsi = Adj for all j. X i ωijAsi = Adj for all j. (6) (6) As discussed below, this identity holds only when the fractional contributions, ω, and cell areas, A, are consistently defined 70 (i.e., based on the same cell shapes). As discussed below, this identity holds only when the fractional contributions, ω, and cell areas, A, are consistently defined 70 (i.e., based on the same cell shapes). The most difficult step in remapping conservatively is to devise an algorithm to calculate the fractional contributions, ωij. Even when efficiently done, the calculation of ωij dominates the remapping execution time. In computing ωij, existing general remapping algorithms (see Appendix A) require the locations of grid cell vertices be specified (for both source and destination The most difficult step in remapping conservatively is to devise an algorithm to calculate the fractional contributions, ωij. Even when efficiently done, the calculation of ωij dominates the remapping execution time. In computing ωij, existing general remapping algorithms (see Appendix A) require the locations of grid cell vertices be specified (for both source and destination grids), and the remapping algorithm must make assumptions as to how the cell vertices are connected. The usual assumption is 75 that the cell edges run along great circles even though for a cartesian longitude by latitude grid, the latitude cell bounds follow latitude circles, not great circles. For commonly used grids of this kind, the ωij calculated by the remapping algorithm are, in general, only approximations of the true fractional contributions because the assumed cell shapes are incorrect. 2 Remapping without masking 50 Consider first the simple case in which fs = fd = 1 for all grid cells. Weights, wij, that preserve the global mean will be used to calculate Fdj through a matrix multiplication: Consider first the simple case in which fs = fd = 1 for all grid cells. Weights, wij, that preserve the global mean will be used to calculate Fdj through a matrix multiplication: Fdj = X i wijFsi . (3) Fdj = X i wijFsi . (3) Fdj = X i wijFsi . (3) These weights would need to be scaled by r2 s /r2 d, to preserve the global integral, rather than the global mean. Initially, we shall suppose that it is the global mean that should be preserved. At the end of the section 3, we show that the destination field that 55 preserves the global mean can be simply scaled to preserve the integral. d suppose that it is the global mean that should be preserved. At the end of the section 3, we show that the destination field that 55 preserves the global mean can be simply scaled to preserve the integral. suppose that it is the global mean that should be preserved. At the end of the section 3, we show that the destination field that 55 preserves the global mean can be simply scaled to preserve the integral. 2 2 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. Conceptually, the remapping weights are determined by overlaying the destination grid onto the source grid and calculat- ing what fraction of each source cell overlaps each destination cell. These fractional contributions, here denoted ω, are then multiplied by the ratio of the source cell area to the destination cell area to yield: (4) 60 wij = ωijAsi/Adj . 0 Each distinct portion of a source cell must contribute to one and only one destination cell and all portions must contribute to some destination cell. This implies that within any source cell i, the fractional contributions ωij must sum to unity: X ωij = 1 for all i (5) Each distinct portion of a source cell must contribute to one and only one destination cell and all portions must contribute to some destination cell. This implies that within any source cell i, the fractional contributions ωij must sum to unity: X j ωij = 1 for all i. 2 Remapping without masking 50 Despite this apparent problem, some users may choose to calculate the destination values according to The true fractional areas of the source cells contributing to a destination cell may not add up to the true area of the destination 95 cell because the true areas may be inconsistent with the shapes of cells assumed in generating the fractional contributions, ωij. Despite this apparent problem, some users may choose to calculate the destination values according to F ∗ dj = X i w∗ ijFsi . (11) It follows that X i FsiA∗ si = X j F ∗ djA∗ dj . 100 (12) The sum on the left side of the equation represents the true global integral. Thus, when true areas are used in constructing the weights, the remapped field can preserve the true global mean. The sum on the left side of the equation represents the true global integral. Thus, when true areas are used in constructing the weights, the remapped field can preserve the true global mean. Both alternatives for computing remapping weights, (4) or (9), rely on the same fractional contributions, ωij, which are based on approximate cell shapes constructed by the remapping algorithm. With the first option, the “approximate-area option”, cell areas are based on cells bounded by great circle segments, and the weights (wij) are defined by (4). In this case the areas are 105 consistent with the assumed cell perimeters, so (6) holds. With the second option, the “true-area option”, the true grid cell areas must be supplied to the remapping algorithm, and the weights (w∗ ij) are defined by (9). In this case the areas may be Both alternatives for computing remapping weights, (4) or (9), rely on the same fractional contributions, ωij, which are based on approximate cell shapes constructed by the remapping algorithm. With the first option, the “approximate-area option”, cell areas are based on cells bounded by great circle segments, and the weights (wij) are defined by (4). In this case the areas are 105 consistent with the assumed cell perimeters, so (6) holds. With the second option, the “true-area option”, the true grid cell areas must be supplied to the remapping algorithm, and the weights (w∗ ij) are defined by (9). 2 Remapping without masking 50 This means the destination grid cell values will be somewhat misrepresented, and the remapping errors will generally exceed the formally derived error estimates. 80 After computing the fractional contributions, ωij, remapping codes can generate the remapping weights using (4). This ensures that X i FsiAsi = X j FdjAdj , X i FsiAsi = X j FdjAdj , (7) (7) where the areas are calculated based on the algorithm’s assumed cell shapes. These areas may differ somewhat from the true cell areas. If they do, then in general 85 where the areas are calculated based on the algorithm’s assumed cell shapes. These areas may differ somewhat from the true cell areas. If they do, then in general 85 cell areas. If they do, then in general 85 X i FsiAsi ̸= X i FsiA∗ si , (8) X i FsiAsi ̸= X i FsiA∗ si , (8) 3 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. where the asterisk distinguishes a true cell area (A∗) from an approximate cell area (A) generated by the remapping algorithm. The sum on the right hand side of the equation represents the true integral on the original source grid, but it is the sum on the left hand side that is preserved by (7). g pp g y pp g g The sum on the right hand side of the equation represents the true integral on the original source grid, but it is the sum on the left hand side that is preserved by (7). In order to preserve the true global mean, some packages can, as an option, use true cell areas (provided by the user) in calculating the remapping weights, 90 90 w∗ ij = ωijA∗ si/A∗ dj , w∗ ij = ωijA∗ si/A∗ dj , (9) but it should be noted that in this case and in contrast with (6), but it should be noted that in this case and in contrast with (6), X i ωijA∗ si ̸= A∗ dj . (10) (10) X i ωijA∗ si ̸= A∗ dj . The true fractional areas of the source cells contributing to a destination cell may not add up to the true area of the destination 95 cell because the true areas may be inconsistent with the shapes of cells assumed in generating the fractional contributions, ωij. 2 Remapping without masking 50 In this case the areas may be inconsistent with the assumed cell perimeters used to generate the fractional contributions (ωij), so there may be a mismatch between the source areas and the destination areas, as noted in (10). 110 For the purpose of evaluating the relative merits of the two options, we now consider an example of a simple source grid 110 and an idealized temperature field. For this example, suppose both the source and destination grids are spherical coordinate grids with the same latitude spacing (15◦) but with the destination grid having half the longitudinal resolution of the source grid (60◦vs. 30◦spacing). Suppose the grids are aligned such that each destination cell completely contains exactly two source cells. The cell areas are given in Table 1. For the source cells nearest the pole, the true cell areas (with latitude cell bounds For the purpose of evaluating the relative merits of the two options, we now consider an example of a simple source grid 110 and an idealized temperature field. For this example, suppose both the source and destination grids are spherical coordinate grids with the same latitude spacing (15◦) but with the destination grid having half the longitudinal resolution of the source grid (60◦vs. 30◦spacing). Suppose the grids are aligned such that each destination cell completely contains exactly two source cells. The cell areas are given in Table 1. For the source cells nearest the pole, the true cell areas (with latitude cell bounds For the purpose of evaluating the relative merits of the two options, we now consider an example of a simple source grid 110 and an idealized temperature field. For this example, suppose both the source and destination grids are spherical coordinate grids with the same latitude spacing (15◦) but with the destination grid having half the longitudinal resolution of the source grid (60◦vs. 30◦spacing). Suppose the grids are aligned such that each destination cell completely contains exactly two source cells. The cell areas are given in Table 1. For the source cells nearest the pole, the true cell areas (with latitude cell bounds following latitude circles) is 0.0178r2, whereas the approximate cell areas (assuming all cell sides follow great circles) is 115 0.0171r2. Thus, there is a 4% error in approximating the cell area. 2 Remapping without masking 50 125 If, however, we remap this temperature field based on an algorithm that assumes cell boundaries are defined by great circle segments when computing cell areas (the approximate-area option), the destination values will lie on the dashed brown curve in Figure 1. And if the algorithm relies on the true cell areas when computing weights (the true-area option), the descrepancy is much larger, with the resulting destination values given by the dashed blue curve in Figure 1. Neither option correctly remaps values and also would lie on the black line. 125 If, however, we remap this temperature field based on an algorithm that assumes cell boundaries are defined by great circle segments when computing cell areas (the approximate-area option), the destination values will lie on the dashed brown curve in Figure 1. And if the algorithm relies on the true cell areas when computing weights (the true-area option), the descrepancy is much larger, with the resulting destination values given by the dashed blue curve in Figure 1. Neither option correctly remaps the field but the approximate-area option appears to be far superior to the true-area option 130 the field, but the approximate-area option appears to be far superior to the true-area option. 130 Under each option, a global integral can be preserved, according to (12) and (7), but only when true areas are used and the destination field is obtained using (11) can we be certain to preserve the global integral as calculated on the original grid. Since the primary purpose in applying a conservative remapping scheme is to preserve the true global mean (i.e., the mean calculated with true areas), the approximate-area option would seem to be unacceptable. In Figure 1, although the destination the field, but the approximate-area option appears to be far superior to the true-area option. 130 Under each option, a global integral can be preserved, according to (12) and (7), but only when true areas are used and the destination field is obtained using (11) can we be certain to preserve the global integral as calculated on the original grid. Since the primary purpose in applying a conservative remapping scheme is to preserve the true global mean (i.e., the mean calculated with true areas), the approximate-area option would seem to be unacceptable. In Figure 1, although the destination the field, but the approximate-area option appears to be far superior to the true-area option. 2 Remapping without masking 50 130 Under each option, a global integral can be preserved, according to (12) and (7), but only when true areas are used and the destination field is obtained using (11) can we be certain to preserve the global integral as calculated on the original grid. Since the primary purpose in applying a conservative remapping scheme is to preserve the true global mean (i.e., the mean calculated with true areas), the approximate-area option would seem to be unacceptable. In Figure 1, although the destination values shown for the approximate area option appear to nearly coincide with the source values (“truth”) they are in fact 135 values shown for the approximate-area option appear to nearly coincide with the source values (“truth”), they are in fact 135 systematically underestimated and lead to a global mean temperature 0.45 K cooler than the true mean. On the other hand, it would seem equally unsatisfactory to adopt the true-area option (dashed blue curve of Figure 1), which produces remapped values at some latitudes differing by more than 30 K from the true values. The true-area option does indeed preserve the true global mean, but the pattern of the destination field can hardly be considered a good representation of the values shown for the approximate-area option appear to nearly coincide with the source values (“truth”), they are in fact 135 systematically underestimated and lead to a global mean temperature 0.45 K cooler than the true mean. On the other hand, it would seem equally unsatisfactory to adopt the true-area option (dashed blue curve of Figure 1), which produces remapped values at some latitudes differing by more than 30 K from the true values. The true-area option does indeed preserve the true global mean, but the pattern of the destination field can hardly be considered a good representation of the systematically underestimated and lead to a global mean temperature 0.45 K cooler than the true mean. On the other hand, it would seem equally unsatisfactory to adopt the true-area option (dashed blue curve of Figure 1), which produces remapped values at some latitudes differing by more than 30 K from the true values. The true-area option does indeed preserve the true global mean, but the pattern of the destination field can hardly be considered a good representation of the source field Thus for different reasons both options might be considered unacceptable 140 source field. 2 Remapping without masking 50 In general these errors increase toward the poles and as grid following latitude circles) is 0.0178r2, whereas the approximate cell areas (assuming all cell sides follow great circles) is 115 0.0171r2. Thus, there is a 4% error in approximating the cell area. In general these errors increase toward the poles and as grid 4 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. cell longitudinal width increases. For the destination grid, with twice the cell widths, the error quadruples to 17%, whereas halving the cell width shrinks the error to 1%. It can be shown that for small longitudinal cell widths, the fractional error in the approximate cell areas is proportional to the square of the cell widths. cell longitudinal width increases. For the destination grid, with twice the cell widths, the error quadruples to 17%, whereas halving the cell width shrinks the error to 1%. It can be shown that for small longitudinal cell widths, the fractional error in the approximate cell areas is proportional to the square of the cell widths. 120 In this example, correctly remapping a source field to the destination grid is trivial since each destination value is determined 120 solely by the contributions from the two source cells that alone occupy it. Consider a temperature that varies linearly with latitude and is independent of longitude. Then, the destination field is identical to the source field but with half the longitudinal resolution. The temperature dependence on latitude for the case considered is given by the black line labeled “source grid (truth)” in Figure 1. Of course when correctly remapped, the destination values should in this case be the same as the source values and also would lie on the black line 125 values and also would lie on the black line. 125 If, however, we remap this temperature field based on an algorithm that assumes cell boundaries are defined by great circle segments when computing cell areas (the approximate-area option), the destination values will lie on the dashed brown curve in Figure 1. And if the algorithm relies on the true cell areas when computing weights (the true-area option), the descrepancy is much larger, with the resulting destination values given by the dashed blue curve in Figure 1. Neither option correctly remaps values and also would lie on the black line. 2 Remapping without masking 50 Thus, for different reasons, both options might be considered unacceptable. 140 It is interesting and somewhat disconcerting to note that with the true-area option, the results of remapping depend on the units used to express the temperature. The dashed blue curve in Figure 1 shows results when temperature is expressed in kelvins (K). The figure also shows that converting the temperature to degrees Celsius (◦C), remapping that field, and then converting it back to kelvins results in considerably smaller errors (crosses in Figure 1). It can be shown that in general, the errors in source field. Thus, for different reasons, both options might be considered unacceptable. 140 It is interesting and somewhat disconcerting to note that with the true-area option, the results of remapping depend on the units used to express the temperature. The dashed blue curve in Figure 1 shows results when temperature is expressed in kelvins (K). The figure also shows that converting the temperature to degrees Celsius (◦C), remapping that field, and then converting it back to kelvins results in considerably smaller errors (crosses in Figure 1). It can be shown that in general, the errors in source field. Thus, for different reasons, both options might be considered unacceptable. 140 It is interesting and somewhat disconcerting to note that with the true-area option, the results of remapping depend on the units used to express the temperature. The dashed blue curve in Figure 1 shows results when temperature is expressed in kelvins (K). The figure also shows that converting the temperature to degrees Celsius (◦C), remapping that field, and then converting it back to kelvins results in considerably smaller errors (crosses in Figure 1). It can be shown that in general, the errors in destination values, when computed using the true-area option, are approximately proportional to the magnitude of the values 145 themselves. Since, on average, we can reduce the mean of the absolute values of a field by removing the global mean before remapping, we can use this strategy to reduce the errors. Our example of converting the temperature units from K to ◦C was an approximate application of this strategy which reduced the error because 0◦C is much closer to the mean than 0◦K. 2 Remapping without masking 50 If we were to adopt the more general approach of removing the global mean before remapping, we arrive at the following formula: destination values, when computed using the true-area option, are approximately proportional to the magnitude of the values 145 themselves. Since, on average, we can reduce the mean of the absolute values of a field by removing the global mean before remapping, we can use this strategy to reduce the errors. Our example of converting the temperature units from K to ◦C was an approximate application of this strategy which reduced the error because 0◦C is much closer to the mean than 0◦K. If we were to adopt the more general approach of removing the global mean before remapping, we arrive at the following formula: destination values, when computed using the true-area option, are approximately proportional to the magnitude of the values 145 themselves. Since, on average, we can reduce the mean of the absolute values of a field by removing the global mean before remapping, we can use this strategy to reduce the errors. Our example of converting the temperature units from K to ◦C was an approximate application of this strategy which reduced the error because 0◦C is much closer to the mean than 0◦K. If we were to adopt the more general approach of removing the global mean before remapping, we arrive at the following formula: F ∗ dj = Fs + X i w∗ ij(Fsi −Fs), 150 (13) 5 5 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. where here and in what follows an overbar indicates a global mean that must be computed using the true areas, not the approximate areas that remapping algorithms might generate. This variant of the true-area option will subsequently be referred to as the “true-area (centered)” approach, to distinguish it from the “true-area (uncentered)” approach which relies on (11). where here and in what follows an overbar indicates a global mean that must be computed using the true areas, not the approximate areas that remapping algorithms might generate. This variant of the true-area option will subsequently be referred to as the “true-area (centered)” approach, to distinguish it from the “true-area (uncentered)” approach which relies on (11). 2 Remapping without masking 50 Given the shortcomings of both the centered and uncentered variants of the true-area option, we reconsider the approximate- 175 area option, which relies on the remapping algorithm to construct cell shapes and areas assuming perimeters coincide with great circle segments. The fundamental problem with this approach, as expressed by (8), is that the true global mean (as calculated on the source grid using true areas) is not generally preserved. In the fields we have examined, we have found that the difference between the true mean and the mean of the field remapped using the approximate area approach is quite small. Given the shortcomings of both the centered and uncentered variants of the true-area option, we reconsider the approximate- 175 area option, which relies on the remapping algorithm to construct cell shapes and areas assuming perimeters coincide with great circle segments. The fundamental problem with this approach, as expressed by (8), is that the true global mean (as calculated on the source grid using true areas) is not generally preserved. In the fields we have examined, we have found that the difference between the true mean and the mean of the field remapped using the approximate area approach is quite small. Given the shortcomings of both the centered and uncentered variants of the true-area option, we reconsider the approximate- 175 area option, which relies on the remapping algorithm to construct cell shapes and areas assuming perimeters coincide with great circle segments. The fundamental problem with this approach, as expressed by (8), is that the true global mean (as calculated on the source grid using true areas) is not generally preserved. In the fields we have examined, we have found that the difference between the true mean and the mean of the field remapped using the approximate area approach is quite small. It seems reasonable, therefore, to simply adjust all values in the field by a uniform amount to correct for the small mismatch 180 in means. For the case considered in Figure 1, 0.45 K can be added to each of the destination grid cell values obtained with the approximate-area option. This straight-forward adjustment eliminates the flaw in the approximate-area option that led us to discard it originally. This “global adjustment” to the destination field means that local destination field values will be influenced by remote field values. 2 Remapping without masking 50 An objection to using (13) is that a change in Fs anywhere in the domain will change the mean and thereby impact the destination values everywhere in the domain. One would not expect a remote change in a field to affect a local destination 155 value, so (13) would also seem to be less than ideal. It should be noted that if weights generated with the approximate-area option were used in (13), the resulting destination field would be identical to that obtained with (3). This is because for these weights, (6) holds. Yet another shortcoming of the true-area option is that its application to a spatially uniform source field can result in a destination field with non-zero spatial variance, which is obviously unrealistic. Consider, for example, a source field that 160 everywhere has the value 1. For the grid defined earlier (see Table 1), application of (11) results in a destination field with the same mean (equal to 1), but with non-zero, area-weighted variance equal to 0.064, and a maximum absolute deviation from the true value of 0.13. These unrealistic variations may in some applications be unacceptable. Algorithms that maintain the uniformity of an originally constant field are said to be “consistent” (e.g., Ullrich and Taylor (2015)), so the true-area option i h b d ib d “ i ” might be described as “not consistent”. 165 For the true-area option, use of (11) or (13) can sometimes result in a destination field with nonphysical values. Consider, for example, a possible result of mapping to a destination grid the ice-free fraction (i.e., the fraction of a grid cell area that is is ice-free). As in the first example above, suppose ice-free fraction is independent of longitude and a linear function of latitude, varying from 0 in the polar-most latitude band to 1 in the latitude band adjacent to the equator. Application of (11) results in a value of 1.06 for the latitude band nearest the equator, and application of (13) results in a value of 1.01. Clearly, the 170 remapping algorithm in both cases yields a nonphysical result with the ice-free fraction exceeding 1 in the tropics. In contrast, the approximate-area option generates destination values that never exceed the maximum or minimum source values. The approximate-area approach is said to be a monotone method (e.g., Ullrich and Taylor (2015)), whereas the true-area approach is not. 2 Remapping without masking 50 This is undesirable, but as noted earlier, the true-area (centered) approach is similarly impacted. On the other hand, a virtue of the approximate-area option is that unlike the true-area option, a change in units (from, for example, 185 6 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. kelvins to ◦C) does not affect the accuracy of the result. In addition, a source field that has no variations (i.e., is everywhere the same) will map to a destination field that is also constant. Both of these results follow because when applying the approximate- area option, (6) holds. kelvins to ◦C) does not affect the accuracy of the result. In addition, a source field that has no variations (i.e., is everywhere the same) will map to a destination field that is also constant. Both of these results follow because when applying the approximate- area option, (6) holds. Since neither of the cell-area choices offered by remapping codes is without shortcomings, it is worth further examining the characteristics of their errors to determine which approach results in the more realistic representation of the original field. 190 For the temperature field considered earlier, Figure 2 shows the remapping error when approximate cell areas are used (with and without a global mean correction) and when the true cell areas are used with global mean removed and then reapplied following (13), labeled the “true area (centered) option” in the figure. By design, the correction to preserve the global mean under the approximate-area option simply offsets the curve by the same amount everywhere. With this correction and ignoring for the moment the dotted curve, the “approximate-area (corrected uniformly) option” (as it will be identified subsequently) 195 has the smallest root-mean-squared error (RMS error, calculated with grid cell area weighting). The RMS errors are: 0.15 K for the approximate-area (corrected uniformly) option, 0.48 K for the approximate-area (uncorrected) option, and 0.63 K for the true-area option (centered). In this example, although the correct global mean is preserved under two of the methods, the approximate-area (corrected uniformly) option leads to an RMS error a factor of 4 smaller than that resulting from the true-area (centered) option. 200 In Figure 3 we consider for a spherical coordinate (cartesian longitude by latitude) grid how the remapping errors depend on grid cell size. 2 Remapping without masking 50 The source data were taken from Figure 1 with values independent of longitude location and 15◦latitude spacing. Source grids with longitudinal resolution from 0.5◦to 30◦were considered, and in each case the destination grid had half the longitudinal resolution of the source. As discussed above, the errors arise because of inaccuracies in the representation of grid cell shapes by the remapping algorithm (which assumes the cell latitude bounds follow great circles, not latitude circles). It 205 is not surprising then that the finer the resolution, the smaller the errors (because for small grid cells, the great circles deviate very little from latitude circles). Figure 3 shows that under all options the RMS error is proportional to the square of the grid cell’s longitude width and that for any given resolution, the approximate-area option error is about 1/4 the size of the true-area (centered) option error and more than two orders of magnitude smaller than the error in uncentered true-area option. From when applying that option can sometimes push values outside the limits. This issue can be addressed with a refined correction, 215 which will be described in a part of the next section. 7 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3 Remapping of partially or fully masked cells We now consider the more general procedure for conservative remapping when there might be undefined elements in the source array (e.g., missing or masked elements) or when grid cell values might be defined for only a fractional portion of the source cell (for example only over the land portion of a cell). For this purpose, we will adopt and generalize the form of the 220 approximate-area option because, as discussed above, it was found to be more accurate than the true-area options and because with this option we can simplify some subsequent formulas using (6). Moreover, where the field is constant on the source grid, the approximate-area option, unlike the true-area option, does not introduce unrealistic spatial variations in a region. 220 The key to handling data that may represent conditions on only a portion of each grid cell is to specify for each cell the “unmasked” fraction, and when remapping is performed, generate the appropriate destination unmasked fractions. Although 225 sometimes the unmasked fractions are binary (either 0 or 1) and might be inferred from special bit strings assigned to any “missing” data, if the data are remapped, the unmasked fractions will in general no longer be binary, and thus information will be lost unless the unmasked fractions are carried as an additional field along with the data field. The key to general remapping then is to carefully account for the unmasked fractions and to ensure that they are consistently defined on the destination grid. “unmasked” fraction, and when remapping is performed, generate the appropriate destination unmasked fractions. Although 225 sometimes the unmasked fractions are binary (either 0 or 1) and might be inferred from special bit strings assigned to any “missing” data, if the data are remapped, the unmasked fractions will in general no longer be binary, and thus information will be lost unless the unmasked fractions are carried as an additional field along with the data field. The key to general remapping then is to carefully account for the unmasked fractions and to ensure that they are consistently defined on the destination grid. 230 Generalizing (3) to account for fully or partially masked fields requires modification of the weights defined by (4). This is 230 done by replacing in (4) the ratio of areas by the ratio of unmasked portions of the cell areas. 3 Remapping of partially or fully masked cells Alternatively and equivalently, we can explicitly include the unmasked fractions in (3), which then becomes ˆFdj = X i wij(fsi/ ˆfdj)Fsi , (14) ˆFdj = X i wij(fsi/ ˆfdj)Fsi , (14) where the ‘hat’ atop Fdj indicates that this is a preliminary estimate of the destination value which might be corrected subse- p dj p y g quently to preserve the global mean. Note that the source unmasked fractions, fsi, must be set to 0 wherever there are missing 235 data. When this is done, missing data need not be treated specially because whatever value has been assigned to indicate the data value is missing, the value will invariably get multiplied by fsi, yielding 0 for the product. This ensures that missing values have no impact on the remapped fields. quently to preserve the global mean. Note that the source unmasked fractions, fsi, must be set to 0 wherever there are missing 235 data. When this is done, missing data need not be treated specially because whatever value has been assigned to indicate the data value is missing, the value will invariably get multiplied by fsi, yielding 0 for the product. This ensures that missing values have no impact on the remapped fields. For some applications, destination fractions may have been imposed as part of the definition of the destination grid. For the purposes of remapping a field, however, it is essential that the destination fractions in (14) be defined such that 240 ˆfdj = X i wijfsi . (15) The remapping formula (14) can then be written The remapping formula (14) can then be written ˆFdj = P i wijfsiFsi P i wijfsi . (16) ˆFdj = P i wijfsiFsi P i wijfsi . (16) Thus, the destination value is simply a weighted mean of the contributing grid cell values. This ensures that the destination Thus, the destination value is simply a weighted mean of the contributing grid cell values. This ensures that the destination value will not lie outside the maximum and minimum values of the contributing cells. This further implies that if all source 5 ll t ib ti t d ti ti ll h th l th d ti ti ll ill l b i d th t l N t th t p y g g g value will not lie outside the maximum and minimum values of the contributing cells. 3 Remapping of partially or fully masked cells This further implies that if all source 245 cells contributing to some destination cell have the same value, the destination cell will also be assigned that value. Note that if in (16) the sum in the denominator is zero, then the destination value should be designated as missing. value will not lie outside the maximum and minimum values of the contributing cells. This further implies that if all source 245 cells contributing to some destination cell have the same value, the destination cell will also be assigned that value. Note that if in (16) the sum in the denominator is zero, then the destination value should be designated as missing. 8 8 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. As shown earlier, use of approximate areas in computing the weights in (16) does not in general preserve the true global mean of F. As in the simpler case, a global adjustment to the ˆFdj values must be applied, but here we allow the correction to vary spatially, 50 Fdj = ˆFdj −γj γ ( ˆFd −Fs), (17) where the global mean of γ in the denominator above ensures Fd = Fs. With masking, the global mean quantities (indicated by overbars) must be computed with area weights proportional to only the unmasked area of grid cells, For example, ˆFd = P j ˆFdjfdjA∗ dj P f A∗ . (18) where the global mean of γ in the denominator above ensures Fd = Fs. With masking, the global mean quantities (indicated by overbars) must be computed with area weights proportional to only the unmasked area of grid cells, For example, where the global mean of γ in the denominator above ensures Fd = Fs. With masking, the global mean quantities (indicated by overbars) must be computed with area weights proportional to only the unmasked area of grid cells, For example, ˆFd = P j ˆFdjfdjA∗ dj P j fdjA∗ dj . (18) Note that in this formula, the unmasked fraction, fd, is not necessarily identical to the unmasked fraction, ˆfd, which appears 255 in (14). Sometimes, for example, the remapped data must conform to a destination grid with an imposed masked region. In that case, the already defined fractions, fd, can be used in (18). 3 Remapping of partially or fully masked cells This, however, could result in some destination field values calculated with (16) being masked. With those values no longer contributing to the destination field, the correction to the mean given by (17) must compensate, and this will alter the destination values, Fdj, globally, not just locally. It is therefore advisable Note that in this formula, the unmasked fraction, fd, is not necessarily identical to the unmasked fraction, ˆfd, which appears 255 in (14). Sometimes, for example, the remapped data must conform to a destination grid with an imposed masked region. In that case, the already defined fractions, fd, can be used in (18). This, however, could result in some destination field values calculated with (16) being masked. With those values no longer contributing to the destination field, the correction to the mean given by (17) must compensate, and this will alter the destination values, Fdj, globally, not just locally. It is therefore advisable to assign destination masked fractions consistent with (15) and avoid imposing externally defined destination masked fractions. 260 In (17), the correction coefficient , γj/γ, can vary spatially, but in the simplest case is set to 1 for all j, which adjusts each cell value by the same amount ( ˆFd −Fs). Sometimes this can lead to nonphysical results. Suppose, for example, that a positive definite quantity (such as the liquid water content of air) were mapped to a target grid using (16) and that the resulting global mean were greater than Fs. In this case, any cell with ˆFdj = 0 would, after a simple adjustment with γj = 1, become negative, which must be ruled out on physical grounds. 265 More generally, a uniform adjustment of the destination field may result in values that lie outside the range of source values. Returning to our earlier example, we see in Figure 2 that a uniform correction to the temperature field of 0.45 K results in a positive 0.1 K error in the equator-most cell, and the remapped temperature there is warmer than the warmest temperature found in the original field (290 K). Thus, the remapped field, which before correction was monotonic, is no longer so. which must be ruled out on physical grounds. 265 More generally, a uniform adjustment of the destination field may result in values that lie outside the range of source values. 3 Remapping of partially or fully masked cells Returning to our earlier example, we see in Figure 2 that a uniform correction to the temperature field of 0.45 K results in a positive 0.1 K error in the equator-most cell, and the remapped temperature there is warmer than the warmest temperature found in the original field (290 K). Thus, the remapped field, which before correction was monotonic, is no longer so. More generally, a uniform adjustment of the destination field may result in values that lie outside the range of source values. Returning to our earlier example, we see in Figure 2 that a uniform correction to the temperature field of 0.45 K results in a positive 0.1 K error in the equator-most cell, and the remapped temperature there is warmer than the warmest temperature found in the original field (290 K). Thus, the remapped field, which before correction was monotonic, is no longer so. 270 (19) γj = (Fdj Fmin) (Fmax Fdj) , (19) with Fmin = min[Fsi] and Fmax = max[Fsi]. The value of µ should be chosen just large enough to prevent in the destination field extremes beyond the source field limits. When µ is larger than necessary, the adjustments will unevenly be distributed among relatively few grid cells and may lead to a less accurate result. When the values of Fs span multiple orders of magnitude, the correction, if evenly distributed, would have a much larger relative impact on the smallest values. Consider, for example, 275 atmospheric smoke concentration, which might be found in a limited region. Any correction to ensure the mean is conserved, should mainly be applied to the high concentration region. In this case, then, µ should be large enough that the low-level, background concentrations of smoke (all near 0) would be unaffected. Note that one could devise an even more versatile adjustment by using two different exponents for the two factors in (19) the correction, if evenly distributed, would have a much larger relative impact on the smallest values. Consider, for example, 275 atmospheric smoke concentration, which might be found in a limited region. Any correction to ensure the mean is conserved, should mainly be applied to the high concentration region. In this case, then, µ should be large enough that the low-level, background concentrations of smoke (all near 0) would be unaffected. 3 Remapping of partially or fully masked cells In this example involving coarse resolution grids (with longitude widths ≥30◦), the uncorrected global mean of the remapped field is 0.45 K cooler than the true global mean. For finer resolution grids, the magnitude of the correction is considerably 290 smaller: when, for example, the same temperature field is mapped from a source grid with cell widths of 4 degrees longitude to a grid with widths of 8 degrees, the global mean is less than 0.01 K cooler than the true mean. When it is not essential to preserve the true mean, a user might choose to accept such a small error in global mean in order to avoid the correction procedure described above. field is 0.45 K cooler than the true global mean. For finer resolution grids, the magnitude of the correction is considerably 290 smaller: when, for example, the same temperature field is mapped from a source grid with cell widths of 4 degrees longitude to a grid with widths of 8 degrees, the global mean is less than 0.01 K cooler than the true mean. When it is not essential to preserve the true mean, a user might choose to accept such a small error in global mean in order to avoid the correction procedure described above. field is 0.45 K cooler than the true global mean. For finer resolution grids, the magnitude of the correction is considerably 290 smaller: when, for example, the same temperature field is mapped from a source grid with cell widths of 4 degrees longitude to a grid with widths of 8 degrees, the global mean is less than 0.01 K cooler than the true mean. When it is not essential to preserve the true mean, a user might choose to accept such a small error in global mean in order to avoid the correction procedure described above. A final adjustment to Fdj may be needed if the objective is to preserve the global integral of a field, rather than the global 295 mean. 3 Remapping of partially or fully masked cells (To conserve energy in a coupled climate model, for example, it is the surface heat flux between the atmospheric component and the ocean component that might need to be mapped from one grid to another, and it is the total flux which must be preserved.) Comparison of (1) and (2) indicates that to preserve the integral, the values of Fdj obtained using (17), which preserve the mean, should be scaled by the ratio of the global unmasked source area to the unmasked destination area: 295 c = r2 s P i fsiA∗ si r2 d P j fdjA∗ dj . (20) 300 c = r2 s P i fsiA∗ si r2 d P j fdjA∗ dj . 300 (20) If the destination unmasked grid-cell fractions have been defined such that the global mean unmasked fraction is preserved, the sums in the numerator and denominator of (20) are the same, and the formula simplifies to c = (rs/rd)2. When this is true and if rd = rs, the same destination field, without scaling, preserves both the mean and the integral. Note, however, if the destination unmasked fractions have been calculated with (15) and they have not been corrected to preserve the global mean unmasked fraction, then c must be calculated with (20). 305 unmasked fraction, then c must be calculated with (20). 305 3 Remapping of partially or fully masked cells Note that one could devise an even more versatile adjustment by using two different exponents for the two factors in (19) the correction, if evenly distributed, would have a much larger relative impact on the smallest values. Consider, for example, 275 atmospheric smoke concentration, which might be found in a limited region. Any correction to ensure the mean is conserved, should mainly be applied to the high concentration region. In this case, then, µ should be large enough that the low-level, background concentrations of smoke (all near 0) would be unaffected. Note that one could devise an even more versatile adjustment by using two different exponents for the two factors in (19) 9 9 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. As a rule in calculating γ, a relatively small value of µ, say 0.1 or 0.25, should be tried first, and then the destination values 280 should be checked to confirm that none exceeds the extremes found in the source field. If this condition is not met, the value of µ should be incrementally increased and γ recalculated until all destination field values fall within the extremes of the source field. In a few test cases, we found that the smaller the value of γ, the smaller the overall remapping error. In the example considered above, a correction with γ = 0.25 can be applied that eliminates the unrealistically high temper- As a rule in calculating γ, a relatively small value of µ, say 0.1 or 0.25, should be tried first, and then the destination values 280 should be checked to confirm that none exceeds the extremes found in the source field. If this condition is not met, the value of µ should be incrementally increased and γ recalculated until all destination field values fall within the extremes of the source field. In a few test cases, we found that the smaller the value of γ, the smaller the overall remapping error. I th l id d b ti ith 0 25 b li d th t li i t th li ti ll hi h t As a rule in calculating γ, a relatively small value of µ, say 0.1 or 0.25, should be tried first, and then the destination values 280 should be checked to confirm that none exceeds the extremes found in the source field. 3 Remapping of partially or fully masked cells If this condition is not met, the value of µ should be incrementally increased and γ recalculated until all destination field values fall within the extremes of the source field. In a few test cases, we found that the smaller the value of γ, the smaller the overall remapping error. In the example considered above, a correction with γ = 0.25 can be applied that eliminates the unrealistically high temper- ature near the equator which resulted from a uniform correction. The dotted curve in Figure 2 shows the result. In addition to 285 eliminating the artificial maximum, the correction slightly reduces the RMS error in the remapped field (see Figure 3), but this result does not carry over to all the resolutions considered. Larger values of γ would reduce the temperature correction near the extremes, which would be compensated by larger corrections to intermediate temperatures, as shown in Figure 4. I thi l i l i l ti id ( ith l it d idth ≥30◦) th t d l b l f th d In the example considered above, a correction with γ = 0.25 can be applied that eliminates the unrealistically high temper- ature near the equator which resulted from a uniform correction. The dotted curve in Figure 2 shows the result. In addition to 285 eliminating the artificial maximum, the correction slightly reduces the RMS error in the remapped field (see Figure 3), but this result does not carry over to all the resolutions considered. Larger values of γ would reduce the temperature correction near the extremes, which would be compensated by larger corrections to intermediate temperatures, as shown in Figure 4. In this example involving coarse resolution grids (with longitude widths ≥30◦), the uncorrected global mean of the remapped In the example considered above, a correction with γ = 0.25 can be applied that eliminates the unrealistically high temper- ature near the equator which resulted from a uniform correction. The dotted curve in Figure 2 shows the result. In addition to 285 eliminating the artificial maximum, the correction slightly reduces the RMS error in the remapped field (see Figure 3), but this result does not carry over to all the resolutions considered. Larger values of γ would reduce the temperature correction near the extremes, which would be compensated by larger corrections to intermediate temperatures, as shown in Figure 4. 4 Recipes for regridding Some conservative remapping packages (see Appendix A) have not been designed to handle the most general cases considered here (e.g., fields with missing values or grid cells that are partially masked). Those codes may nevertheless be relied on to provide weights defined by (4). For a given source and destination pair of grids, these weights can be calculated once, and then used to remap any field from the source grid to the destination grid. 310 used to remap any field from the source grid to the destination grid. 310 A step-by-step procedure for mapping variables conservatively is provided here. A step-by-step procedure for mapping variables conservatively is provided here. 10 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 1. Obtain from a remapping package the weights (wij) that apply when there is no masking or fractional weighting and that are generated based on the algorithm’s approximate reconstruction of grid cell shapes. 1. Obtain from a remapping package the weights (wij) that apply when there is no masking or fractional weighting and that are generated based on the algorithm’s approximate reconstruction of grid cell shapes. 2. Check that for all destination cells the weights satisfy (6), which with (4) can be rewritten: 2. Check that for all destination cells the weights satisfy (6), which with (4) can be rewritten: at for all destination cells the weights satisfy (6), which with (4) can be rewritten: X i wij = 1 for all j. (21) (21) 315 (It is prudent to include this step but not necessary if the weights returned by the remapping package are known to satisfy this requirement.) 3. Assign or calculate the source grid’s unmasked fractions, fsj. – If a source value is meant to represent conditions over the entire cell extent, set fsi = 1 for the cell. 320 – If unmasked fractions have been assigned to source cells prior to remapping, the pre-assigned values should be assigned to fs. – Wherever source cell data are missing, reset the unmasked fraction to 0 (fsi = 0). 4. Assign or calculate the destination grid’s unmasked fractions, fdj. 4. Assign or calculate the destination grid’s unmasked fractions, fdj. – If unmasked fractions have been assigned to destination cells prior to initiating the remapping procedure, fd should be set to the pre-assigned values. 4 Recipes for regridding As noted in the previous section, however, when destination masked values are not defined by (15), the destination field, Fd, will be impacted everywhere, so when possible, avoid applying external destination masked fractions different from ˆfd. 325 – If unmasked fractions have not been pre-assigned, generate preliminary estimates of the fractions with (15). When necessary or desirable, correct the values of ˆf to preserve the global mean by applying formulas analogous to (17) and (19): 330 fdj = ˆfdj −γj γ ( ˆfd −fs) (22) and γj = ˆf µ dj(1 −ˆfdj)µ . γj = ˆf µ dj(1 −ˆfdj)µ . (23) γj = ˆf µ dj(1 −ˆfdj)µ . (23) (23) This step ensures that the same destination field will preserve both the global integral and mean under the conditions discussed following (20). If the values of ˆf are corrected to preserve the true global mean using (22), a uniform adjustment (γj/γ = 1) cannot normally be applied as that could lead to some negative value of f or some values exceeding 1; rather, the adjustment should be made with γ defined by (19) and µ > 0. The global mean quantities ( ˆfd and fs) in the first equation above must be calculated using the true areas (A∗ dj and A∗ si) and without any fractional weighting. 335 11 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 5. Use (16) to calculate the preliminary destination values, ˆFdj. For any cell where the denominator in (16) is 0, designate the destination value as "missing". 340 6. Correct the destination values, ˆFdj, to preserve the true global mean and obtain the final destination field, Fd. In the next two sub-steps, when calculating (17), global means of Fsi and ˆFdj should be weighted by fsiA∗ si and fdjA∗ dj, respectively. 6. Correct the destination values, ˆFdj, to preserve the true global mean and obtain the final destination field, Fd. In the next two sub-steps, when calculating (17), global means of Fsi and ˆFdj should be weighted by fsiA∗ si and fdjA∗ dj, respectively. – Initially, impose a uniform correction to all values by applying (17) with γj = 1 for all j. If none of the resulting Fdj values have been shifted outside the extremes of the source field, consider the correction acceptable; otherwise proceed. 4 Recipes for regridding The destination grid and possibly the source grid (if its longitudinal resolution is coarse) should be subdivided into a number of sub-cell sectors, each of equal width. If the source cells have been subdivided, then each source cell value is replicated across each of its sub-cells, as is each unmasked fraction. Next, the weights are obtained 365 for mapping variables from the possibly subdivided source grid to the subdivided destination grid. Once the remapped field has been obtained for the fine-resolution destination grid, the sub-cells within each of the coarse-resolution destination cells are simply collected and averaged to obtain the final destination value. Each of the fine-resolution cells is contained within the boundaries of a single coarse destination cell, so no weighting is necessary. then each source cell value is replicated across each of its sub-cells, as is each unmasked fraction. Next, the weights are obtained 365 for mapping variables from the possibly subdivided source grid to the subdivided destination grid. Once the remapped field has been obtained for the fine-resolution destination grid, the sub-cells within each of the coarse-resolution destination cells are simply collected and averaged to obtain the final destination value. Each of the fine-resolution cells is contained within the boundaries of a single coarse destination cell, so no weighting is necessary. then each source cell value is replicated across each of its sub-cells, as is each unmasked fraction. Next, the weights are obtained 365 for mapping variables from the possibly subdivided source grid to the subdivided destination grid. Once the remapped field has been obtained for the fine-resolution destination grid, the sub-cells within each of the coarse-resolution destination cells are simply collected and averaged to obtain the final destination value. Each of the fine-resolution cells is contained within the boundaries of a single coarse destination cell, so no weighting is necessary. Care must be taken when the standard procedure for remapping is applied to a variable representing conditions within layers 370 of the atmosphere or ocean to ensure that mass-weighted means are preserved (as opposed to the usual area-weighted means). Additional complications might be encountered when a variable represents the ratio of two quantities (e.g., specific humidity 370 Care must be taken when the standard procedure for remapping is applied to a variable representing conditions within layers 370 of the atmosphere or ocean to ensure that mass-weighted means are preserved (as opposed to the usual area-weighted means). 4 Recipes for regridding – If necessary, next try applying (17) with γ defined by (19) and µ initially set to some relatively small value (e.g., 0.1 or 0.25). As before, if none of the resulting Fdj values has been shifted outside the extremes of the source field, consider the correction acceptable; otherwise repeat this step with µ increased (say doubled). Repeat this step, as necessary, until the correction becomes acceptable. Note that no correction of ˆFdj is needed if two conditions are met: 1) the remapping algorithm correctly represents the grid cell shapes (in which case the fractional contributions, ω, and cell areas will both have been correctly determined), and 2) the unmasked fractions on the destination grid are defined by (15). In what follows, the above recipe will be referred to as the “standard procedure”. The weights, wij, only depend on the 355 source grid and destination grid, so a single set of weights can be generated that can be applied in remapping any field. It should be noted that the approximate areas calculated by the remapping algorithm are of no interest once the mapping weights have been generated. In the subsequent mapping of a variable from the source grid to the destination grid, only the true areas of cells are needed. As we have seen, the errors in remapping data to or from grids defined by longitude and latitude coordinates arise because the 360 remapping algorithm constructs cell shapes with latitude sides that arc poleward following great circles rather than following latitude circles. The larger the longitudinal width of a cell, the larger its distortion. This suggests a simple strategy to improve the accuracy of the remapping algorithm. The destination grid and possibly the source grid (if its longitudinal resolution is coarse) should be subdivided into a number of sub-cell sectors, each of equal width. If the source cells have been subdivided, As we have seen, the errors in remapping data to or from grids defined by longitude and latitude coordinates arise because the 360 remapping algorithm constructs cell shapes with latitude sides that arc poleward following great circles rather than following latitude circles. The larger the longitudinal width of a cell, the larger its distortion. This suggests a simple strategy to improve the accuracy of the remapping algorithm. 4 Recipes for regridding Additional complications might be encountered when a variable represents the ratio of two quantities (e.g., specific humidity Care must be taken when the standard procedure for remapping is applied to a variable representing conditions within layers 370 of the atmosphere or ocean to ensure that mass-weighted means are preserved (as opposed to the usual area-weighted means). Additional complications might be encountered when a variable represents the ratio of two quantities (e.g., specific humidity 12 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. is the ratio of the mass of water vapor to the mass of air), where, rather than preserving the global mean ratio, it is better to preserve the two quantities themselves. The following guidelines may be helpful in treating these possibly troublesome variables. 375 (a) To remap a quantity representing a grid cell area fraction (e.g., cloud fraction, sea ice fraction, land fraction), the destination fractions should be calculated in the same way as unmasked fractions were calculated in step 4 above. (a) To remap a quantity representing a grid cell area fraction (e.g., cloud fraction, sea ice fraction, land fraction), the destination fractions should be calculated in the same way as unmasked fractions were calculated in step 4 above. (b) To conserve the total energy flowing through the surface of the earth), the heat flux must be expressed as a flux per unit area (“flux density” with units of, for example, W/m2, not W). Then the standard procedure is followed to remap the flux density to the destination grid where it is scaled by c, as defined by (20). (c) To remap the albedo (reflected radiation divided by incident radiation), which is undefined when the incident radiation is zero, it is best to conservatively remap the incident and reflected radiation flux densities (commonly termed “radiative fluxes”) and then form their quotient, rather than directly remapping the albedo. Destination values should be considered “missing” (undefined) where the remapped incident radiation is 0. (d) There are applications where the total volume of a space (which might be partially masked) should be preserved. For 385 grids constructed with height (or depth) used as a vertical coordinate, this can be achieved by calculating appropriate cell thicknesses on the destination grid. The standard procedure above is followed, applied to cell thickness. The resulting values must be scaled by c, as defined by (20). 4 Recipes for regridding The unmasked portion of a destination cell volume is the product of the remapped cell depth, the destination cell fraction that is unmasked (fdj), and the true destination cell area (A∗ dj). (d) There are applications where the total volume of a space (which might be partially masked) should be preserved. For 385 grids constructed with height (or depth) used as a vertical coordinate, this can be achieved by calculating appropriate cell thicknesses on the destination grid. The standard procedure above is followed, applied to cell thickness. The resulting values must be scaled by c, as defined by (20). The unmasked portion of a destination cell volume is the product of the remapped cell depth, the destination cell fraction that is unmasked (fdj), and the true destination cell area (A∗ dj). (e) For most 3-d quantities, remapping should preserve the mass-weighted mean, rather than the area-weighted mean. Prior 390 to remapping such variables, the mass field must be remapped conservatively. In order to preserve the total mass within a layer, the mass per unit area (M) of destination cells can be obtained following the standard remapping procedure. We consider two cases: models for which the bounds on layers can be expressed as a pressure and models for which the bounds on layers can be expressed as a distance. (e) For most 3-d quantities, remapping should preserve the mass-weighted mean, rather than the area-weighted mean. Prior 390 to remapping such variables, the mass field must be remapped conservatively. In order to preserve the total mass within a layer, the mass per unit area (M) of destination cells can be obtained following the standard remapping procedure. We consider two cases: models for which the bounds on layers can be expressed as a pressure and models for which the bounds on layers can be expressed as a distance. When the layer pressure thicknesses can be determined, the mass per unit area in the layer is M = ∆p/g (where ∆p is 395 the pressure thickness of the layer, which may vary with longitude and latitude, and g is the acceleration due to gravity). Preserving the mass within a layer is equivalent to preserving the pressure thickness of the layer. This is achieved following the standard remapping procedure with Fsi = ∆psi and scaling the result by c, as defined by (20). 4 Recipes for regridding When the layer thicknesses can be determined, the layer mass per unit area is equal to the product of cell density (ρ) and layer thickness (∆z), so that standard procedure is applied to ρ∆zsi. Again, the result is scaled by c, as defined by 400 (20). By this procedure we preserve global mass, but we should also like to define density and layer thickness on the d ti ti id h th t th i d t i M F d l ith if id l thi k ∆ it k When the layer pressure thicknesses can be determined, the mass per unit area in the layer is M = ∆p/g (where ∆p is 395 the pressure thickness of the layer, which may vary with longitude and latitude, and g is the acceleration due to gravity). Preserving the mass within a layer is equivalent to preserving the pressure thickness of the layer. This is achieved following the standard remapping procedure with Fsi = ∆psi and scaling the result by c, as defined by (20). When the layer thicknesses can be determined, the layer mass per unit area is equal to the product of cell density (ρ) and layer thickness (∆z), so that standard procedure is applied to ρ∆zsi. Again, the result is scaled by c, as defined by 400 (20). By this procedure we preserve global mass, but we should also like to define density and layer thickness on the destination grid such that their product is Mdj. For models with a uniform source-grid layer thickness, ∆z, it makes sense for the source-grid thickness to carry over to the destination grid. Then the density is given by ρdj = Mdj/∆z. If, instead, density is uniform in a source grid layer, then ∆zdj = Mdj/ρ. If, however, both density and thickness vary on 13 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. the source grid, then one can choose whether to preserve the global mean layer thickness or the global mean density. One of these fields can be remapped, preserving its mean, and then the other calculated by dividing Mdj by the first field. 405 (f) Once the mass per unit area is obtained for each destination grid cell, as just described in (e) above, the formula for preserving mass-weighted integrals or means can be derived. 4 Recipes for regridding For example, to remap temperature (T) in a layer such that the total internal energy is conserved within a layer, remap the temperature, weighted by each grid cell’s mass per unit area, and then divide by the cell mass per unit area on the destination grid (Mdj). For a pressure coordinate model 410 with a layer thickness (∆psi) that depends on location, the mass-weighted temperature U = T∆p, which is proportional to internal energy per unit area, is mapped to the destination grid following the standard procedure. The result, Udj, is then divided by the pressure thickness on the destination grid (defined, as described in (e), such that the global mass is preserved), yielding the temperature field consistent with conservation of internal energy: Tdj = Udj/∆pdj. For a height or depth coordinate model, ∆p is replaced by ρ∆z in the above formulas, with care taken to preserve the global 415 mass in the layer. Note that for layers of uniform mass thickness (either constant ∆p or constant ρ∆z), there is no need to consider mass, and instead, the simpler procedure described in (b) can be applied directly without regard to layer thickness. (f) Once the mass per unit area is obtained for each destination grid cell, as just described in (e) above, the formula for preserving mass-weighted integrals or means can be derived. For example, to remap temperature (T) in a layer such that the total internal energy is conserved within a layer, remap the temperature, weighted by each grid cell’s mass per unit area, and then divide by the cell mass per unit area on the destination grid (Mdj). For a pressure coordinate model 0 with a layer thickness (∆psi) that depends on location, the mass-weighted temperature U = T∆p, which is proportional to internal energy per unit area, is mapped to the destination grid following the standard procedure. The result, Udj, is then divided by the pressure thickness on the destination grid (defined, as described in (e), such that the global mass is preserved), yielding the temperature field consistent with conservation of internal energy: Tdj = Udj/∆pdj. For a height or depth coordinate model, ∆p is replaced by ρ∆z in the above formulas, with care taken to preserve the global 5 mass in the layer. 4 Recipes for regridding Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. (h) In remapping relative humidity (mixing ratio divided by saturation mixing ratio), one might want to preserve the rela- tionship between the remapped mixing ratio and the remapped temperature used to define saturation mixing ratio. That is, one might want the relative humidity on the destination grid to be defined by the ratio of a conservatively remapped mixing ratio divided by a saturation mixing ratio based on a conservatively remapped temperature. 5 Interpolating conservatively in the vertical The weights can then be obtained by overlaying the pressure bounds from the native grid onto the destination grid bounds and determining the fraction of each source cell that lies within the vertical extent of each destination cell. These fractions are used 455 to remap the data in the vertical through matrix multiplication. A difference from horizontal remapping is that the weights are not uniform across the other dimensions of the data; they can vary from one location to another and may evolve over time (e.g., in the atmospheric surface layer where surface pressure may vary in time). It is therefore not possible to calculate the weights once and for all as is done in horizontal remapping. Fortunately, calculating the weights for interpolating in the verticali determining the fraction of each source cell that lies within the vertical extent of each destination cell. These fractions are used 455 to remap the data in the vertical through matrix multiplication. A difference from horizontal remapping is that the weights are not uniform across the other dimensions of the data; they can vary from one location to another and may evolve over time (e.g., in the atmospheric surface layer where surface pressure may vary in time). It is therefore not possible to calculate the weights once and for all as is done in horizontal remapping. Fortunately, calculating the weights for interpolating in the vertical is computationally much less demanding than in the horizontal, so remapping 3-d fields remains practical. 460 5 Interpolating conservatively in the vertical When remapping a 3-d field both vertically and horizontally, the vertical dimension must be handled carefully to preserve a global mass-weighted integral. We consider here the specific case of interpolating from a model’s native vertical grid to surfaces of constant mass per unit area. If a field will ultimately be mapped to multiple horizontal destination grids, then performing 45 the vertical interpolation first will usually reduce the computational expense. Once vertical interpolation is completed, the remapping to a different horizontal grid proceeds as described in the previous sections. C d i h h i f i h d d i l i h h i l i i h i d fi h When remapping a 3-d field both vertically and horizontally, the vertical dimension must be handled carefully to preserve a global mass-weighted integral. We consider here the specific case of interpolating from a model’s native vertical grid to surfaces of constant mass per unit area. If a field will ultimately be mapped to multiple horizontal destination grids, then performing 445 the vertical interpolation first will usually reduce the computational expense. Once vertical interpolation is completed, the remapping to a different horizontal grid proceeds as described in the previous sections. Compared with the generation of weights needed to remap conservatively in the horizontal, it is much easier to define the of constant mass per unit area. If a field will ultimately be mapped to multiple horizontal destination grids, then performing 445 the vertical interpolation first will usually reduce the computational expense. Once vertical interpolation is completed, the remapping to a different horizontal grid proceeds as described in the previous sections. Compared with the generation of weights needed to remap conservatively in the horizontal, it is much easier to define the weights that will preserve integrals in the vertical. This is because the overlap of source and destination grid cells in the vertical is one dimensional, and only the cells thicknesses must be considered (not their shapes). For data stored on native model 450 levels, bounds defining the vertical extent of each grid cell are an essential component of the grid definition and should be known. Furthermore, the pressures associated with those interfaces should be derivable. Then the mass per unit area contained within the upper and lower bound of a layer can be calculated by dividing the pressure difference across the layer by g. 4 Recipes for regridding Note that for layers of uniform mass thickness (either constant ∆p or constant ρ∆z), there is no need to consider mass, and instead, the simpler procedure described in (b) can be applied directly without regard to layer thickness. 410 (g) The amount of a substance in a layer of the atmosphere or ocean is often expressed as a ratio. To remap quantities of this kind, separately remap the quantities represented by the numerator and denominator and then form their ratio, as in the following examples: (g) The amount of a substance in a layer of the atmosphere or ocean is often expressed as a ratio. To remap quantities of this kind, separately remap the quantities represented by the numerator and denominator and then form their ratio, as in the following examples: – for specific humidity, q (mass of water vapor divided by mass of air containing the water), preserve separately the mass of water vapor and the mass of the air. First conservatively remap the water vapor mass per unit area (qsiMsi). Then remap the mass of air per unit area (Msi), as described in (e) above. Finally, form the ratio of the two remapped fields to obtain the specific humidity on the destination grid. A similar procedure can be applied in remapping any mass fraction. – for water vapor mixing ratio (mass of water vapor divided by mass of dry air), preserve separately the mass of the water vapor and the mass of dry air. A similar procedure can be applied in remapping any mass mixing ratio. – for number concentration (number of particles divided by volume), preserve separately the number of particles and the volume. Then form their ratio. – for mass concentration (mass of substance divided by total volume of mixture), preserve separately the mass of the substance and the volume. Then form their ratio. – for mole concentration (number of moles per unit volume of, for example, a chemical species in the atmosphere or ocean), preserve separately the moles of the substance and the volume. Then form their ratio. – for volume mixing ratio (number of moles of a constituent divided by number of moles of all constituents combined; sometimes referred to as mole fraction), preserve separately the number of moles of the constituent of interest and the number of moles of all constituents combined. Then form their ratio. 14 https://doi.org/10.5194/gmd-2023-177 Preprint. 6 Summary and concluding remarks Most conservative remapping packages (see Appendix A) generate mapping weights based on grid cells that may differ slightly in shape from the true cell shapes. Typically, a remapping algorithm will construct cell polygons with edges that follow great circles and then use these to determine cell areas and mapping weights. On the other hand, many models and analysis grids are constructed on spherical grids with grid cell bounds that follow lines of constant longitude and lines of constant latitude 65 (not great circles). If data are mapped from or to a grid of this kind, the remapping algorithms can fail to preserve the true global mean or integral of a field. The algorithms preserve instead a global mean based on its approximate representation of cell shapes and areas, which generally differs from the true mean. This difference may especially matter when gauging whether Most conservative remapping packages (see Appendix A) generate mapping weights based on grid cells that may differ slightly in shape from the true cell shapes. Typically, a remapping algorithm will construct cell polygons with edges that follow great circles and then use these to determine cell areas and mapping weights. On the other hand, many models and analysis grids circles and then use these to determine cell areas and mapping weights. On the other hand, many models and analysis grids are constructed on spherical grids with grid cell bounds that follow lines of constant longitude and lines of constant latitude 465 (not great circles). If data are mapped from or to a grid of this kind, the remapping algorithms can fail to preserve the true global mean or integral of a field. The algorithms preserve instead a global mean based on its approximate representation of cell shapes and areas, which generally differs from the true mean. This difference may especially matter when gauging whether are constructed on spherical grids with grid cell bounds that follow lines of constant longitude and lines of constant latitude 465 (not great circles). If data are mapped from or to a grid of this kind, the remapping algorithms can fail to preserve the true global mean or integral of a field. The algorithms preserve instead a global mean based on its approximate representation of cell shapes and areas, which generally differs from the true mean. This difference may especially matter when gauging whether 465 15 https://doi.org/10.5194/gmd-2023-177 Preprint. 6 Summary and concluding remarks These recipes apply even when the remapping algorithm has correctly represented the shapes and areas of grid cells; when that is true, the steps involving correction of the mean can be skipped. when that is true, the steps involving correction of the mean can be skipped. Conservative remapping of the kind considered here must always operate on variables that are independent of the cell’s area. For example, rather than remap the area of snow cover in grid cells, the areas first must be converted to fractions, which p , p g ,i , can be conservatively remapped and then converted back to areas. Most variables reported from models are intensive, so such 485 conversions are rarely necessary. Conservative mapping is obviously required if it is important to preserve the global integrals (or means) of a field. When this is not essential, other methods of interpolating data to a destination grid may lead to a more physically consistent and realistic looking result. Consider, for example, the geopotential height and wind fields carried on a relatively coarse source grid. If these can be conservatively remapped and then converted back to areas. Most variables reported from models are intensive, so such 485 conversions are rarely necessary. Conservative mapping is obviously required if it is important to preserve the global integrals (or means) of a field. When this looking result. Consider, for example, the geopotential height and wind fields carried on a relatively coarse source grid. If these fields were mapped conservatively to a much finer resolution grid, box-fill contour plots of the resultant fields would look like 490 slightly blurred versions of the box-fill plots of the original fields. The sub-cells wholly contained within a given source cell would all share the same value; there would be no variation except for the relatively few cells at the borders of the original source cells. Thus, within the confines of each original source cell, the geopotential height and winds would be constant, and at the borders of original source cells, there would be large gradients. With non-zero wind values but no geopotential gradients within the confines of the original cell, the geostrophic balance generally prevailing outside the tropics would be upset. In 495 fields were mapped conservatively to a much finer resolution grid, box-fill contour plots of the resultant fields would look like 490 slightly blurred versions of the box-fill plots of the original fields. 6 Summary and concluding remarks The sub-cells wholly contained within a given source cell would all share the same value; there would be no variation except for the relatively few cells at the borders of the original source cells. Thus, within the confines of each original source cell, the geopotential height and winds would be constant, and at the borders of original source cells, there would be large gradients. With non-zero wind values but no geopotential gradients within the confines of the original cell, the geostrophic balance generally prevailing outside the tropics would be upset. In 495 general, when mapping from a coarse to a fine grid, simple bilinear interpolation should lead to more realistic gradients and more realistic looking results than conservative remapping. We have shown that with available remapping packages, careful application of the remapping weights, unmasked fractions, and in some cases layer masses can result in reasonably accurate results that preserve the true global integrals or means of interest. If grid cell shapes were correctly recreated by the remapping algorithms, the remaining inaccuracies could be reduced 500 further and the standard remapping procedure could be simplified (e.g., by eliminating the corrections needed to adjust the global means). This would seem to provide strong motivation for augmenting remapping packages with the option to correctly construct the commonly encountered longitude by latitude grids with cell edges conforming to the true grid cell shapes We have shown that with available remapping packages, careful application of the remapping weights, unmasked fractions, and in some cases layer masses can result in reasonably accurate results that preserve the true global integrals or means of We have shown that with available remapping packages, careful application of the remapping weights, unmasked fractions, and in some cases layer masses can result in reasonably accurate results that preserve the true global integrals or means of and in some cases layer masses can result in reasonably accurate results that preserve the true global integrals or means of interest. If grid cell shapes were correctly recreated by the remapping algorithms, the remaining inaccuracies could be reduced 500 further and the standard remapping procedure could be simplified (e.g., by eliminating the corrections needed to adjust the global means). This would seem to provide strong motivation for augmenting remapping packages with the option to correctly construct the commonly encountered longitude by latitude grids with cell edges conforming to the true grid cell shapes. interest. 6 Summary and concluding remarks Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. a model, having reached equilibrium, is conserving energy. If the global mean net top of the atmosphere (TOA) energy flux is in fact zero, as evaluated based on the original grid and correct cell areas, remapping that data and calculating the mean on a 470 new grid could lead to a different conclusion. Another limitation of many remapping packages is that although they may be able to treat gridded data where a binary mask applies (e.g., screening regions of missing data or limiting analysis to the ocean or land regions alone), they are not designed to conveniently handle data values that are representative of only a portion of a grid cell (i.e., are partially unmasked). Moreover, often the easiest option offered for handling such cases is to perform the computationally intensive recalculation of weights 475 Another limitation of many remapping packages is that although they may be able to treat gridded data where a binary mask applies (e.g., screening regions of missing data or limiting analysis to the ocean or land regions alone), they are not designed to conveniently handle data values that are representative of only a portion of a grid cell (i.e., are partially unmasked). Moreover, Here we have suggested how users can apply the weights provided by remapping packages, while avoiding or correcting their limitations. For a given pair of source and destination grids, the remapping weights need only be calculated once; the weights are independent of any full or partial masking of the source data. Each destination field can then be calculated via very sparse-matrix multiplications. The recipes appearing in section 4 provide step-by-step instructions on how to handle various 480 cases. These recipes apply even when the remapping algorithm has correctly represented the shapes and areas of grid cells; when that is true, the steps involving correction of the mean can be skipped. Conservative remapping of the kind considered here must always operate on variables that are independent of the cell’s area For example rather than remap the area of snow cover in grid cells the areas first must be converted to fractions which sparse-matrix multiplications. The recipes appearing in section 4 provide step-by-step instructions on how to handle various 480 cases. 6 Summary and concluding remarks If grid cell shapes were correctly recreated by the remapping algorithms, the remaining inaccuracies could be reduced 500 further and the standard remapping procedure could be simplified (e.g., by eliminating the corrections needed to adjust the global means). This would seem to provide strong motivation for augmenting remapping packages with the option to correctly construct the commonly encountered longitude by latitude grids with cell edges conforming to the true grid cell shapes. 16 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. Code availability. The calculations performed as part of this research were based on simple applications of the equations appearing in the manuscript, which were applied to completely artificial data (six temperatures that were assumed to decrease linearly from equator to pole). 505 Appendix A: Remapping packages The focus here has been on the second step of conservative remapping procedures: applying remapping weights that have been generated by the first step. In climate modeling, among the most commonly used packages used to generate these weights are the following: – C-Coupler2: This package was developed for use in coupling components of climate models (Liu et al., 2018) and includes a conservative remapping capability. 510 – Climate Data Operators (CDO): This package, designed to manipulate and analyze climate and weather prediction data, includes remapping capabilities based on the YAC package (see below). Documentation is available at https://code.mpimet.mpg.de/projects/cdo/wiki. – Earth System Modeling Framework (ESMF) Regrid Weight Generator (ERWG): This library contains a number of pack- 515 ages useful in the analysis of climate data, including a conservative remapping capability. Documentation is available at https://earthsystemmodeling.org/regrid/. – Model Coupling Toolkit (MCT): This package includes an application of the Jones (1999) method that has been paral- lelized (Larson et al., 2005). It is used by the OASIS coupler, and by a number of climate models. – netCDF Operators (NCO): This toolkit manipulates and analyzes data of interest to the geophysical community and 520 includes two options for creating remapping weights: TempestRemap and ESMF (ERWG). Documentation is available at https://sourceforge.net/projects/nco/, https://nco.sourceforge.net/nco.pdf, and https://acme-climate.atlassian.net/wiki/ spaces/DOC/pages/754286611/Regridding+E3SM+Data+with+ncremap – OASIS: This software was developed for coupling components of climate models and in its latest version, it relies on MCT to generate remapping weights (Craig et al., 2017). Documentation is available at http://www.cerfacs.fr/oa4web/oasis3- 525 mct_4.0/oasis3mct_UserGuide/node42.html and http://www.cerfacs.fr/oa4web/oasis3-mct_4.0/oasis3mct_UserGuide/ node48.html#subsec_mapdata. – Spherical Coordinate Remapping and Interpolation Package (SCRIP): This is the first library to implement the Jones (1999) approach to remap conservatively (see https://github.com/SCRIP-Project). Most other conservative remapping packages have been based on SCRIP. – TempestRemap: This is a conservative, consistent and monotone remapping package for arbitrary grid geometry with support for finite volumes and finite elements (see Ullrich and Taylor, 2015; Ullrich et al., 2016). 17 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. – Yet Another Coupler (YAC): A conservative remapping algorithm is included in this climate model component coupler (Hanke et al., 2016). Author contributions. The author was solely responsible for this contribution. 535 Competing interests. The author declares that he has no competing interests. Acknowledgements. I thank Paul Durack and Paul Ullrich for their helpful comments. This work was performed under the auspices of the U.S. Appendix A: Remapping packages DOE by Lawrence Livermore National Laboratory under contract DEAC52-07NA27344. It was supported by the Regional and Global Modeling Analysis (RGMA) program area under DOE’s Biological and Environmental Research (BER) Program. 18 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. References 540 Craig, A., Valcke, S., and Coquart, L.: Development and performance of a new version of the OASIS coupler, OASIS3-MCT_3.0, Geoscien- tific Model Development, 10, 3297–3308, https://doi.org/10.5194/gmd-10-3297-2017, 2017. Hanke, M., Redler, R., Holfeld, T., and Yastremsky, M.: YAC 1.2.0: new aspects for coupling software in Earth system modelling, Geosci- entific Model Development, 9, 2755–2769, https://doi.org/10.5194/gmd-9-2755-2016, 2016. Craig, A., Valcke, S., and Coquart, L.: Development and performance of a new version of the OASIS coupler, OASIS3-MCT_3.0, Geoscien- tific Model Development, 10, 3297–3308, https://doi.org/10.5194/gmd-10-3297-2017, 2017. Hanke, M., Redler, R., Holfeld, T., and Yastremsky, M.: YAC 1.2.0: new aspects for coupling software in Earth system modelling, Geosci- entific Model Development, 9, 2755–2769, https://doi.org/10.5194/gmd-9-2755-2016, 2016. Hanke, M., Redler, R., Holfeld, T., and Yastremsky, M.: YAC 1.2.0: new aspects for coupling software in Earth system modelling, Geosci- entific Model Development, 9, 2755–2769, https://doi.org/10.5194/gmd-9-2755-2016, 2016. Jones, P. W.: First-and second-order conservative remapping schemes for grids in spherical coordinates, Monthly Weather Review, 127, 545 2204–2210, https://doi.org/10.1175/1520-0493(1999)127<2204:FASOCR>2.0.CO;2, 1999. Larson, J., Jacob, R., and Ong, E.: The model coupling toolkit: A new Fortran90 toolkit for building multiphysics parallel coupled models, The International Journal of High Performance Computing Applications, 19, 277–292, https://doi.org/10.1177/1094342005056115, 2005.l Jones, P. W.: First-and second-order conservative remapping schemes for grids in spherical coordinates, Monthly Weather Review, 127, 45 2204–2210, https://doi.org/10.1175/1520-0493(1999)127<2204:FASOCR>2.0.CO;2, 1999. Jones, P. W.: First-and second-order conservative remapping schemes for grids in spherical coordinates, Monthly Weather Review, 127, 545 2204–2210, https://doi.org/10.1175/1520-0493(1999)127<2204:FASOCR>2.0.CO;2, 1999. Larson, J., Jacob, R., and Ong, E.: The model coupling toolkit: A new Fortran90 toolkit for building multiphysics parallel coupled models, The International Journal of High Performance Computing Applications, 19, 277–292, https://doi.org/10.1177/1094342005056115, 2005. Liu, L., Zhang, C., Li, R., Wang, B., and Yang, G.: C-Coupler2: a flexible and user-friendly community coupler for model coupling and nesting, Geoscientific Model Development, 11, 3557–3586, https://doi.org/10.5194/gmd-11-3557-2018, 2018. 550 Ullrich, P. A. and Taylor, M. A.: Arbitrary-order conservative and consistent remapping and a theory of linear maps: Part I, Monthly Weather Review, 143, 2419–2440, https://doi.org/10.1175/MWR-D-14-00343.1, 2015. Ullrich, P. A., Devendran, D., and Johansen, H.: Arbitrary-order conservative and consistent remapping and a theory of linear maps: Part II, Monthly Weather Review, 144, 1529–1549, https://doi.org/10.1175/MWR-D-15-0301.1, 2016. Ullrich, P. A. and Taylor, M. A.: Arbitrary-order conservative and consistent remapping and a theory of linear maps: Part I, Monthly Weather Review, 143, 2419–2440, https://doi.org/10.1175/MWR-D-14-00343.1, 2015. Ullrich, P. References 540 A., Devendran, D., and Johansen, H.: Arbitrary-order conservative and consistent remapping and a theory of linear maps: Part II, Monthly Weather Review, 144, 1529–1549, https://doi.org/10.1175/MWR-D-15-0301.1, 2016. Ullrich, P. A., Devendran, D., and Johansen, H.: Arbitrary-order conservative and consistent remapping and a theory of linear maps: Part II, Monthly Weather Review, 144, 1529–1549, https://doi.org/10.1175/MWR-D-15-0301.1, 2016. 19 19 230 240 250 260 270 280 290 300 310 320 0° - 15° 15° - 30° 30° - 45° 45-60 60° - 75° 75° - 90° destination value (K) latitude band source grid 'truth' true-area (K) approx. area (uncorrected) true-area (C) Figure 1. For the example described in the text, destination grid cell values resulting from different remapping options. The source data were defined on a longitude by latitude grid of 30◦by 15◦resolution and then mapped to a destination grid with half the longitudinal resolution but the same latitude spacing (i.e., 60◦by 15◦). The solid black line (source grid ‘truth’) also represents the destination values that would result from correctly remapping the data to the destination grid, applying an algorithm that correctly reconstructed the grid cell shapes. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 230 240 250 260 270 280 290 300 310 320 0° - 15° 15° - 30° 30° - 45° 45-60 60° - 75° 75° - 90° destination value (K) latitude band source grid 'truth' true-area (K) approx. area (uncorrected) true-area (C) source grid 'truth' true-area (K) approx. area (uncorrected) true-area (C) Figure 1. For the example described in the text, destination grid cell values resulting from different remapping options. The source data were defined on a longitude by latitude grid of 30◦by 15◦resolution and then mapped to a destination grid with half the longitudinal resolution but the same latitude spacing (i.e., 60◦by 15◦). The solid black line (source grid ‘truth’) also represents the destination values that would result from correctly remapping the data to the destination grid, applying an algorithm that correctly reconstructed the grid cell shapes. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 0° - 15° 15° - 30° 30° - 45° 45-60 60° - 75° 75° - 90° error (K) latitude band true-area (centered) approx. area (corrected uniformly) approx. area (uncorrected) approx. References 540 area (corrected; 𝜇=1/4) Figure 2. For the example described in the text, error in destination grid cell values resulting from different calculational options. -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 0° - 15° 15° - 30° 30° - 45° 45-60 60° - 75° 75° - 90° error (K) latitude band true-area (centered) approx. area (corrected uniformly) approx. area (uncorrected) approx. area (corrected; 𝜇=1/4) Figure 2. For the example described in the text, error in destination grid cell values resulting from different calculational options. ample described in the text, error in destination grid cell values resulting from different calculational options. Figure 2. For the example described in the text, error in destination grid cell values resulting from different calculational options. Figure 2. For the example described in the text, error in destination grid cell values resulting from different calculational options. 20 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1 2 4 8 16 32 64 normalzied RMS error destination cell width (deg longitude) true-area (uncentered) true-area (centered) approx. area (corrected uniformly) approx. area (corrected; 𝜇=1/4) Figure 3. For the example described in the text, dependence on the longitude cell width of the RMS error in destination cell values, with the error calculated over all latitudes and weighted by area. The RMS errors have been normalized by the true spatial standard deviation of the variable. Expressed in this way, an error equal to 1 means, for example, that the RMS error is as large as the spatial standard deviation of the variable, which in this example is 7.2 K. The mapping is always from a source grid with longitude cell widths half that of the destination grid but the same latitude resolution (15◦latitude bands for all longitudinal resolutions). 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1 2 4 8 16 32 64 normalzied RMS error destination cell width (deg longitude) true-area (uncentered) true-area (centered) approx. area (corrected uniformly) approx. area (corrected; 𝜇=1/4) Figure 3. For the example described in the text, dependence on the longitude cell width of the RMS error in destination cell values, with the error calculated over all latitudes and weighted by area. The RMS errors have been normalized by the true spatial standard deviation of the variable. Expressed in this way, an error equal to 1 means, for example, that the RMS error is as large as the spatial standard deviation of the variable, which in this example is 7.2 K. The mapping is always from a source grid with longitude cell widths half that of the destination grid but the same latitude resolution (15◦latitude bands for all longitudinal resolutions). 21 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 270 275 280 285 290 295 temperature correction (K) temperature 𝜇=4 𝜇=1 𝜇=1/4 𝜇=0 Figure 4. For the example described in the text, dependence of the temperature correction, (γµ/γµ)( ˆ Td −Ts), on temperature and the exponent µ. Curves shaped the same apply to any variable with the abscissa spanning the full range of the source data and the ordinate appropriately rescaled. https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 270 275 280 285 290 295 temperature correction (K) temperature 𝜇=4 𝜇=1 𝜇=1/4 𝜇=0 Figure 4. For the example described in the text, dependence of the temperature correction, (γµ/γµ)( ˆ Td −Ts), on temperature and the exponent µ. Curves shaped the same apply to any variable with the abscissa spanning the full range of the source data and the ordinate appropriately rescaled. 22 https://doi.org/10.5194/gmd-2023-177 Preprint. Discussion started: 4 September 2023 c⃝Author(s) 2023. CC BY 4.0 License. Table 1. True cell areas (A∗) and approximate cell areas (A) for source and destination spherical coordinate grids with longitudinal cell widths of 30◦and 60◦, respectively. The approximate areas are calculated assuming all bounds coincide with great circles. All areas are expressed as solid angles obtained by dividing the actual cell area by the square of Earth’s radius. For ease of comparison with destination cell areas, source cell areas have been doubled. source cells destination cells latitude band 2A∗ si 2Asi A∗ dj Adj 0◦−15◦ 0.271 0.277 0.271 0.297 15◦−30◦ 0.253 0.256 0.253 0.265 30◦−45◦ 0.217 0.216 0.217 0.213 45◦−60◦ 0.166 0.163 0.166 0.152 60◦−75◦. 0.105 0.101 0.105 0.090 75◦−90◦. 0.036 0.034 0.036 0.030 source cells destination cells latitude band 2A∗ si 2Asi A∗ dj Adj 0◦−15◦ 0.271 0.277 0.271 0.297 15◦−30◦ 0.253 0.256 0.253 0.265 30◦−45◦ 0.217 0.216 0.217 0.213 45◦−60◦ 0.166 0.163 0.166 0.152 60◦−75◦. 0.105 0.101 0.105 0.090 75◦−90◦. 0.036 0.034 0.036 0.030 23
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Testing Hypotheses About Glacial Dynamics and the Stage 11 Paradox Using a Statistical Model of Paleo-Climate
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Testing Hypotheses About Glacial Dynamics and the Stage 11 Paradox Using a Statistical Model of Paleo-Climate Conversely, poor model performance during MIS stage 11 and Termination V is consistent with arguments that the ‘stage 11 paradox’ represents a mismatch between orbital geometry and climate. Statistical orderings of simulation errors indicate that periods of reduced accuracy start with significant reductions in the model’s ability to simulate carbon dioxide, non-sea-salt sodium, and non-sea-salt calcium. Their importance suggests that the 20 t 11 d i t d b h i t h i d/ i i l ti th t ff t til ti f ~800,000 years suggest that changes in glacial cycles associated with the Mid-Brunhes event, which occurs near the 15 division between the out-of-sample period and the in-sample period, are not caused by changes in the dynamics of the climate system. Conversely, poor model performance during MIS stage 11 and Termination V is consistent with arguments that the ‘stage 11 paradox’ represents a mismatch between orbital geometry and climate. Statistical orderings of simulation errors indicate that periods of reduced accuracy start with significant reductions in the model’s ability to simulate carbon dioxide, non-sea-salt sodium, and non-sea-salt calcium. Their importance suggests that the 20 stage 11 paradox is generated by changes in atmospheric and/or oceanic circulation that affect ocean ventilation of https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Testing Hypotheses About Glacial Dynamics and the Stage 11 Paradox Using a Statistical Model of Paleo-Climate Robert K. Kaufmann1, Felix Pretis2 1Department of Earth and Environment, Boston University, Boston, Massachusetts, USA, 02215 2Department of Economics, University of Victoria, Victoria, BC, Canada; and Nuffield College, University of Oxford, Oxford, UK Correspondence to: Robert K. Kaufmann (Kaufmann@bu.edu) 1Department of Earth and Environment, Boston University, Boston, Massachusetts, USA, 02215 2Department of Economics, University of Victoria, Victoria, BC, Canada; and Nuffield College, University of Oxford, Oxford, UK Abstract To test hypotheses about glacial dynamics, the Mid-Brunhes event, and the stage 11 paradox, we evaluate 10 the ability of a statistical model to simulate climate during the previous ~800,000 years. Throughout this period, the model simulates the timing and magnitude of glacial cycles, including the saw-tooth pattern in which ice accumulates gradually and ablates rapidly, without nonlinearities or threshold effects. This suggests that nonlinearities and/or threshold effects do not play a critical role in glacial cycles. Furthermore, model accuracy throughout the previous Abstract To test hypotheses about glacial dynamics, the Mid-Brunhes event, and the stage 11 paradox, we evaluate 10 the ability of a statistical model to simulate climate during the previous ~800,000 years. Throughout this period, the model simulates the timing and magnitude of glacial cycles, including the saw-tooth pattern in which ice accumulates gradually and ablates rapidly, without nonlinearities or threshold effects. This suggests that nonlinearities and/or threshold effects do not play a critical role in glacial cycles. Furthermore, model accuracy throughout the previous ~800,000 years suggest that changes in glacial cycles associated with the Mid-Brunhes event, which occurs near the 15 division between the out-of-sample period and the in-sample period, are not caused by changes in the dynamics of the climate system. Conversely, poor model performance during MIS stage 11 and Termination V is consistent with arguments that the ‘stage 11 paradox’ represents a mismatch between orbital geometry and climate. Statistical orderings of simulation errors indicate that periods of reduced accuracy start with significant reductions in the model’s ability to simulate carbon dioxide, non-sea-salt sodium, and non-sea-salt calcium. Their importance suggests that the 20 ~800,000 years suggest that changes in glacial cycles associated with the Mid-Brunhes event, which occurs near the 15 division between the out-of-sample period and the in-sample period, are not caused by changes in the dynamics of the climate system. 1 Introduction When considered over the last eight-hundred thousand years, climate shows highly persistent movements. Most 25 notable are glacial cycles. During glaciations, temperature, greenhouse gas concentrations, and sea level remain below their sample mean for extended periods; during these same periods, land and sea ice remain above their sample means. These positions are reversed for extended periods known as inter-glacials. These persistent movements and complex climate dynamics create difficulties for statistical analyses of climate data over this long time-span. Using ordinary least squares to analyze time series that show persistent movements tends to indicate statistically meaningful relations 30 among time series when none are present (Yule, 1929; Engle and Granger, 1987). Monte Carlo simulations indicate a relation (based on t statistics) for about 85 percent of random pairings of time series with highly persistent movements (Hendry and Juselius, 2000). The difficulties posed by highly persistent movements and complex dynamics are greatly alleviated using the econometric methods of vector-autoregression, cointegration, and equilibrium correction. Using these methods, 35 Kaufmann and Juselius (2013), herein KJ2013, estimate a statistical model of climate over the previous 391 thousand years. The model, termed a cointegration vector autoregression (CVAR), specifies four exogenous variables for orbital geometry; eccentricity, obliquity, precession, and summer time insolation at 65o south to simulate ten endogenous When considered over the last eight-hundred thousand years, climate shows highly persistent movements. Most 25 notable are glacial cycles. During glaciations, temperature, greenhouse gas concentrations, and sea level remain below their sample mean for extended periods; during these same periods, land and sea ice remain above their sample means. These positions are reversed for extended periods known as inter-glacials. These persistent movements and complex climate dynamics create difficulties for statistical analyses of climate data over this long time-span. Using ordinary https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 2 2 variables that proxy various aspects of climate; Antarctic land and sea surface temperature, carbon dioxide and methane concentrations, land and sea ice, sea level, iron dust, and non sea-salt sulfate and calcium. The CVAR model explicitly represents long-run relations between climate and orbital geometry, which are given by ten cointegrating relations, and climate dynamics, which are given by the rates at which the climate system ‘equilibrium corrects’ from disequilibrium in the long-run (cointegrating) relations. 2 Methods The CVAR model described by KJ2013 is simulated in a dynamic simulation (equivalent to a dynamic forecast) 15 conditioned on orbital geometry alone over the 791 thousand years before the present (kyr BP). Simulated values (𝑥"#) are subtracted from the corresponding values from the proxy record (𝑥#) to calculate simulation errors 𝜀# = 𝑥# −𝑥"#. Simulation errors (𝜀t) are analyzed three ways. First, we compute the root mean square error (RMSE) to evaluate model accuracy over pre-defined periods. Second, simulation errors are analyzed to identify periods when the model fails systematically, either in a single time step (outlier) or during two or more consecutive time steps (persisting 20 errors). Third we examine the statistical ordering among simulation errors (and the explanatory power of simulations that are generated by conditioning the model on endogenous variables) to evaluate competing hypotheses for the ‘stage 11 paradox,’ which is a significant mismatch between orbital geometry and climate associated with marine isotope stage (MIS) 11, 424 – 375 kyr BP (Imbrie et al., 1993). The CVAR model described by KJ2013 is simulated in a dynamic simulation (equivalent to a dynamic forecast) 15 conditioned on orbital geometry alone over the 791 thousand years before the present (kyr BP). Simulated values (𝑥"#) are subtracted from the corresponding values from the proxy record (𝑥#) to calculate simulation errors 𝜀# = 𝑥# −𝑥"#. Simulation errors (𝜀t) are analyzed three ways. First, we compute the root mean square error (RMSE) to evaluate model accuracy over pre-defined periods. Second, simulation errors are analyzed to identify periods when the model fails systematically, either in a single time step (outlier) or during two or more consecutive time steps (persisting 20 errors). Third we examine the statistical ordering among simulation errors (and the explanatory power of simulations that are generated by conditioning the model on endogenous variables) to evaluate competing hypotheses for the ‘stage 11 paradox,’ which is a significant mismatch between orbital geometry and climate associated with marine isotope stage (MIS) 11, 424 – 375 kyr BP (Imbrie et al., 1993). 25 1 Introduction Davidson et al., (2016) apply a similar approach for a subset 5 of climate variables. These relations validate some basic hypotheses about the mechanisms that are postulated to drive glacial cycles (e.g. carbon dioxide affects temperature via radiative forcing), reproduce the main features of glacial cycles (e.g. the timing, magnitude, and saw-tooth pattern of changes in land ice volume), and separate observed deglaciations from skipped obliquity/precession beats (e.g. Huybers, 2012), which are peaks in insolation, including obliquity that do not generate 10 deglaciations (Huybers and Wunsch, 2005; Tzedakis et al., 2017). Subsequent analyses of the statistical model suggest a weak form of the Milankovitch hypothesis in which orbital geometry drives glacial cycles, with small perturbations imposed by internal climate dynamics (Kaufmann and Juselius, 2016). Conclusions that are based on a model conditioned solely on orbital geometry are notable because many climate models cannot simulate atmospheric concentrations of CO2 (Archer et al., 2000). This has lead to hypotheses that 15 orbital geometry and GHG are the ‘two primary forcings’ to the climate system (e.g. Yin and Berger, 2012). But KJ 2013 test and reject the hypothesis that carbon dioxide or methane is exogenous to the climate system; their concentrations are endogenous, driven by orbital geometry, which is exogenous to and is the primary driver of climate. Models that do simulate CO2 endogenously cannot simulate other aspects of climate jointly (e.g. ice volume) and so are simulated in absence of feedbacks (Brovkin et al., 2012) or in two steps (e.g. Ganopolski et al., 2016), which may 20 cause models to understate the effects of changes in orbital position (Pretis and Kaufmann, in review). 1. Nonlinearities, threshold effects, or phase-specific governing equations play an important role in the timing and magnitude of glacial cycles. 2. The Mid-Brunhes event (MBE), which refers to a climatic shift that occurs during the transition between marine isotope stage (MIS) 12 and MIS 11 (Jansen et al., 1986), changes the dynamics that drive glacial 35 cycles. 2. The Mid-Brunhes event (MBE), which refers to a climatic shift that occurs during the transition between marine isotope stage (MIS) 12 and MIS 11 (Jansen et al., 1986), changes the dynamics that drive glacial 35 cycles. 3. The ‘stage 11 paradox’ represents a mismatch between orbital geometry and climate. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 1 Introduction 3 Values for the RMSE and statistical differences between simulated values and values from the proxy record indicate that the model generally performs well during the in- and out-of-sample period. We interpret this general accuracy to indicate that: Values for the RMSE and statistical differences between simulated values and values from the proxy record indicate that the model generally performs well during the in- and out-of-sample period. We interpret this general accuracy to indicate that: 1. Nonlinear relations,threshold effects, and/phase-specific governing equations do not play a critical role in glacial cycles. 5 1. Nonlinear relations,threshold effects, and/phase-specific governing equations do not play a critical role in glacial cycles. 5 Glacial cycles are driven by the same dynamics before and after the MBE. 2. Glacial cycles are driven by the same dynamics before and after the MBE. 3. Terminations in general - and the ‘stage 11 paradox’ in particular - may be caused by changes in atmospheric circulation and/or the extent of sea ice, which affects the ventilation of the deep ocean and ultimately, affects the atmospheric concentration of carbon dioxide. These results and the methods used to obtain them are described in five sections. Section 2 describes the data and 10 methods used to generate and analyze the simulations. The results are described in section 3. Section 4 interprets the results relative to the three hypotheses described previously, and section 5 concludes. 2.1Model Data Non sea-salt calcium Ca has a terrestrial origin (mainly Patagonia) and may represent changes in temperature, moisture, vegetation, wind strength, glacial coverage, or changes in sea level in and around Patagonia (Basile et al., 1997), a locale thought to play an important role in glacial cycles. 20 the possible effect of iron-containing dust on biological activity and/or the effect of biological activity on atmospheric 15 CO2. Sea salt sodium Na is derived from the sea-ice surface and proxies the extent of winter sea-ice (Wolff et al 2003). It is included to represent the possible effect of sea ice on the flow of CO2 from the ocean to the atmosphere (Stephens and Keeling, 2000). Non sea-salt calcium Ca has a terrestrial origin (mainly Patagonia) and may represent changes in temperature, moisture, vegetation, wind strength, glacial coverage, or changes in sea level in and around Patagonia (Basile et al., 1997), a locale thought to play an important role in glacial cycles. 20 (Basile et al., 1997), a locale thought to play an important role in glacial cycles. 20 To make these data amenable to a statistical analysis, we convert them to a common time scale (EDC3) using conversions from Parrenin et al., (2007) and Ruddiman and Raymo (2003). Unevenly spaced observations are interpolated (linearly) to generate a data set in which each series has a time step of 1 kyr (Miller, 2019). To eliminate the effects on inverting matrices with elements that differ greatly in size (due to different units of measurement), each of the fourteen time series is standardized as follows: 25 To make these data amenable to a statistical analysis, we convert them to a common time scale (EDC3) using conversions from Parrenin et al., (2007) and Ruddiman and Raymo (2003). Unevenly spaced observations are interpolated (linearly) to generate a data set in which each series has a time step of 1 kyr (Miller, 2019). To eliminate the effects on inverting matrices with elements that differ greatly in size (due to different units of measurement), each of the fourteen time series is standardized as follows: 25 = (𝑦- −𝑦0)/3𝑉𝑎𝑟(𝑦), 𝑡= 1, … ,391 (1) (1) where is the value (in original units), is the average value over the in-sample period, and 𝑉𝑎𝑟(𝑦) is the variance over the in-sample period. ty y 2.1Model Data The four series used to represent orbital position, the six series used to represent climate, and the four series used to represent physical and biological mechanisms that link the six climate variables to each other and orbital geometry are the same as those used in KJ2013 (Table 1). KJ2013 uses four series to represent the effect of orbital geometry: 30 precession (Prec), obliquity (Obl), eccentricity (Ecc), and summer-time insolation at 65oS (SunSum). Observations for these time series are compiled back to 800 kyr BP from the same sources used by KJ 2013 (Paillard, 1996). KJ2013 uses these four measures of orbital geometry alone to simulate ten endogenous variables (six climate and four mechanisms); climate variables include land surface temperature (Temp), the atmospheric concentration of carbon dioxide (CO2) methane (CH4), sea surface temperature (SST), land ice volume (Ice), and sea level (Level). Variables 35 that capture mechanisms include iron dust (Fe), sea-salt sodium (Na), non sea-salt sulfate (SO4), and non sea-salt calcium (Ca); for additional details about the each series see Section I of the Supplemental Material. The four series used to represent orbital position, the six series used to represent climate, and the four series used to represent physical and biological mechanisms that link the six climate variables to each other and orbital geometry are the same as those used in KJ2013 (Table 1). KJ2013 uses four series to represent the effect of orbital geometry: precession (Prec), obliquity (Obl), eccentricity (Ecc), and summer-time insolation at 65oS (SunSum). Observations for these time series are compiled back to 800 kyr BP from the same sources used by KJ 2013 (Paillard, 1996). KJ2013 uses these four measures of orbital geometry alone to simulate ten endogenous variables (six climate and four mechanisms); climate variables include land surface temperature (Temp), the atmospheric concentration of carbon dioxide (CO2) methane (CH4), sea surface temperature (SST), land ice volume (Ice), and sea level (Level). Variables 35 that capture mechanisms include iron dust (Fe), sea-salt sodium (Na), non sea-salt sulfate (SO4), and non sea-salt calcium (Ca); for additional details about the each series see Section I of the Supplemental Material. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 4 Data for Temp, CO2, and CH4 are obtained from cores drilled into the Antarctic ice sheet. Carbon dioxide and methane are well-mixed gases and so measurements from Antarctic ice proxy global concentrations. 2.1Model Data Temp represents local conditions, but can be converted to global values by assuming that a scaling factor, which is derived from a limited set of observations can be applied across all observations (Masson-Delmotte et al., 2010; Masson-Delmotte et al., 2006). The 𝛿*+𝑂 data that are used to proxy ice volume, which also includes information about deep water temperature 5 (Chappell and Shackleton., 1986; Shackleton, 2000), are derived from 57 cores drilled by the Deep-Sea Drilling Project and Ocean Drilling Program across the globe (Lisiecki and Raymo, 2005). Sea surface temperature is constructed using alkenones from site PS2489-2/ODP1090 in the sub-Antarctic Atlantic. Data for sea level are reconstructed using oxygen isotope records from Red Sea sediments (Siddall et al., 2003). These six variables are linked to each other and orbital position via physical and biological mechanisms that are 10 represented by the four proxy variables. Fe is derived almost entirely from terrestrial sources and proxies changes in atmospheric circulation and a so-called iron fertilization effect, which may enhance the biotic uptake of CO2 (Martin, 1990). Sulfate SO4 originates mainly from marine biogenic emissions of dimethylsulphide (after removing sea-salt sources using the Na data), and so proxies marine biological activity (Cosme et al., 2005). It is included to represent These six variables are linked to each other and orbital position via physical and biological mechanisms that are 10 represented by the four proxy variables. Fe is derived almost entirely from terrestrial sources and proxies changes in atmospheric circulation and a so-called iron fertilization effect, which may enhance the biotic uptake of CO2 (Martin, 1990). Sulfate SO4 originates mainly from marine biogenic emissions of dimethylsulphide (after removing sea-salt sources using the Na data), and so proxies marine biological activity (Cosme et al., 2005). It is included to represent the possible effect of iron-containing dust on biological activity and/or the effect of biological activity on atmospheric 15 CO2. Sea salt sodium Na is derived from the sea-ice surface and proxies the extent of winter sea-ice (Wolff et al 2003). It is included to represent the possible effect of sea ice on the flow of CO2 from the ocean to the atmosphere (Stephens and Keeling, 2000). 2.2 Simulating the CVAR Model 30 Once the model is spun-up, the model is run continuously through the present; values from 792 30 kyr BP through 392 kyr BP constitute the out-of-sample period. Values from 391 kyr BP through the present constitute the in-sample period. 2.2 Simulating the CVAR Model 30 Π = 𝛼𝛽′ (3) 5 in which 𝛼 is a 10 × 𝑟 matrix of coefficients, which describe the rate at which the ten climate variables adjust back towards equilibrium after the system has been pushed away by exogenous shocks (i.e. changes in orbital geomtery); 𝑟 is the number of cointegration relations given by the reduced rank of the Π matrix; and 𝛽 is a 𝑟× 15 matrix of cointegration coefficients that define the r stationary deviations from long-run equilibrium relationships, the so called cointegration relations, 𝛽I𝑧#. Maximum likelihood estimates for the elements of the 𝛽 and 𝛼 matrices as reported by 10 KJ 2013 are given in section II of the Supplemental Material. The model in KJ2013 is estimated as a partial system (Johansen 1992, Harbo et al., 1998, Juselius 2006) where orbital variables are weakly exogenous. Here we simulate the estimated model model over the full time period using a dynamic simulation in an open model, conditioned on the (strongly) exogenous orbital variables 𝑤# (equivalent to a dynamic forecast). To simulate climate during the in- and out-of-sample periods, the ten endogenous variables x are expressed as a function of the exogenous 15 solar variables and shocks to the climate system by inverting Equation (2) into the moving average form: 𝑥# = 𝐶∑ 𝜀# # -S* + 𝐶∗(𝐿)𝜀# + 𝐶V𝑤# + 𝐶V ∗(𝐿)Δ𝑤# (4) conditioned on the (strongly) exogenous orbital variables 𝑤# (equivalent to a dynamic forecast). To simulate climate during the in- and out-of-sample periods, the ten endogenous variables x are expressed as a function of the exogenous 15 solar variables and shocks to the climate system by inverting Equation (2) into the moving average form: 𝑥# = 𝐶∑ 𝜀# # -S* + 𝐶∗(𝐿)𝜀# + 𝐶V𝑤# + 𝐶V ∗(𝐿)Δ𝑤# (4) (4) where 𝐶= 𝛽X(1 −Γ*)B*𝛼X; 𝛼X is a 10 × (10 −𝑟) matrix orthogonal to 𝛼 describing the stochastic trends and 𝛽X is a 10 × (10 −𝑟) matrix orthogonal to 𝛽 determining how the stochastic trends load into the climate variables; L is the lag operator (for example, 𝐿𝜀# = 𝜀#B*); 𝐶∗(𝐿) and 𝐶V ∗(𝐿) are stationary lag polynomials; 𝐶V is 10 × 4; and the 20 matrices are functions of the parameters (𝐴?, 𝐴*,Γ*,𝛼, 𝛽). 2.2 Simulating the CVAR Model 30 Based on the ten cointergating relations reported by KJ2013 r = 10, then C = 0, the in- and out-of-sample simulations are based on model (2) subject to (3) by setting 𝜀# = 0 which implies that the simulated variables, 𝑥"#, are calculated from the exogenous drivers, 𝐶V𝑤#, (𝐴?∆𝑤#), the dynamics attached to them, 𝐶V ∗(𝐿)∆𝑤#B*, (𝐴*∆𝑤#B*), and the internal climate dynamics 𝐶∗(𝐿)𝜀#(Γ*Δ𝑥"#B*, 𝛼𝛽IYZ[\). where 𝐶= 𝛽X(1 −Γ*)B*𝛼X; 𝛼X is a 10 × (10 −𝑟) matrix orthogonal to 𝛼 describing the stochastic trends and 𝛽X is a 10 × (10 −𝑟) matrix orthogonal to 𝛽 determining how the stochastic trends load into the climate variables; L is the lag operator (for example, 𝐿𝜀# = 𝜀#B*); 𝐶∗(𝐿) and 𝐶V ∗(𝐿) are stationary lag polynomials; 𝐶V is 10 × 4; and the 20 matrices are functions of the parameters (𝐴?, 𝐴*,Γ*,𝛼, 𝛽). Based on the ten cointergating relations reported by KJ2013 r = 10, then C = 0, the in- and out-of-sample simulations are based on model (2) subject to (3) by setting 𝜀# = 0 which implies that the simulated variables, 𝑥"#, are calculated from the exogenous drivers, 𝐶V𝑤#, (𝐴?∆𝑤#), the dynamics attached to them, 𝐶V ∗(𝐿)∆𝑤#B*, (𝐴*∆𝑤#B*), and the internal climate dynamics 𝐶∗(𝐿)𝜀#(Γ*Δ𝑥"#B*, 𝛼𝛽IYZ[\). The out-of-sample simulation is generated by allowing the model to ‘spin up’ between 800 kyr BP and 792 kyr BP, 25 which enables the endogenous variables to converge towards the values that are implied by the exogenous conditioning variables (Prec, Ecc, Obl, and SunSumS). During this ‘spin-up’ period, the model is initialized using observed values for Temp, SST, and Ice, which are available starting 800 kyr BP. The time series of CO2 CH4, Fe, Na, SO4, Ca, and Level have more recent start dates (Table 1). For these variables, the model is initialized with values that correspond to their sample mean. Once the model is spun-up, the model is run continuously through the present; values from 792 30 kyr BP through 392 kyr BP constitute the out-of-sample period. Values from 391 kyr BP through the present constitute the in-sample period. to their sample mean. Once the model is spun-up, the model is run continuously through the present; values from 792 30 kyr BP through 392 kyr BP constitute the out-of-sample period. Values from 391 kyr BP through the present constitute the in-sample period. to their sample mean. 2.2 Simulating the CVAR Model 30 The equations used to estimate the CVAR model in KJ2013 are given by: The equations used to estimate the CVAR model in KJ2013 are given by: (2) ∆𝑥# = 𝐴?∆𝑤# + 𝐴*∆𝑤#B + Γ*∆𝑥# + Π𝑧#B* ′ + 𝜀# ∆𝑥# = 𝐴?∆𝑤# + 𝐴*∆𝑤#B + Γ*∆𝑥# + Π𝑧#B* ′ + 𝜀# ∆𝑥# = 𝐴?∆𝑤# + 𝐴*∆𝑤#B + Γ*∆𝑥# + Π𝑧#B* + 𝜀# (2) in which 𝑥# is a 10 × 1 vector that includes the ten endogenous variables; Temp, CO ,CH , Ice, Fe, Na, Ca, SO , 2 4 4 in which 𝑥# is a 10 × 1 vector that includes the ten endogenous variables; Temp, CO ,CH , Ice, Fe, Na, Ca, SO 2 4 Level, and SST; is a 4 × 1 vector that includes the four exogenous variables Ecc, Prec, Obliq, and SunsumS; 𝑧I = 35 [𝑥# I,𝑤# I, 1], Γ*, 𝐴?, 𝐴*, are 10 × 14 matrices of short-run coefficients; Π is a 10 × 15 matrix of long-run coefficients, tw https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 5 ∆ is the first difference operator (∆𝑥# = 𝑥# −𝑥#B*), 𝜀- is an error term with mean value zero and variance Ω that is normally, independently, and identicially distributed. The condition that the conditional process (𝑥#|𝑤#) is nonstationary is formulated as a reduced rank hypothesis on the matrix Π (3) Π = 𝛼𝛽′ (3) 5 in which 𝛼 is a 10 × 𝑟 matrix of coefficients, which describe the rate at which the ten climate variables adjust back towards equilibrium after the system has been pushed away by exogenous shocks (i.e. changes in orbital geomtery); 𝑟 is the number of cointegration relations given by the reduced rank of the Π matrix; and 𝛽 is a 𝑟× 15 matrix of cointegration coefficients that define the r stationary deviations from long-run equilibrium relationships, the so called cointegration relations, 𝛽I𝑧#. Maximum likelihood estimates for the elements of the 𝛽 and 𝛼 matrices as reported by 10 KJ 2013 are given in section II of the Supplemental Material. The model in KJ2013 is estimated as a partial system (Johansen 1992, Harbo et al., 1998, Juselius 2006) where orbital variables are weakly exogenous. 2.3 Statistical Measures of Model Performance We use RMSE as a simple heuristic to compare the model’s predictive accuracy during the in- and out-of-sample periods. Because accuracy may vary over time, we use an indicator saturation technique [R-package gets Pretis et al., 2018; Castle et al., 2015] to identify periods during which the simulation significantly deviates from observations (i.e. simulation errors are statistically different from zero). Outliers refer to a statistically significant difference in the https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 6 simulated value of variable x relative to the observed value for a single time step, while persisting errors are statistically significant differences that persist for two or more consecutive time-steps. Outliers and persisting errors are evaluated for every possible time step. Here, we retain only those outliers or persisting errors that exceed the pα = 0.001 threshold. This tightly controls the false-positive rate of detected periods of model failure. The method used to identify outliers and persisting errors are summarized in Supplementary Section III. This approach is used to assess the time-varying performance of climate models (Pretis et al., 2015), the forecast accuracy of economic predictions (Ericsson 2017), as well as to detect volcanic eruptions in temperature reconstructions in both simulated climate data (Pretis et al., 2016) and proxy-reconstructions (Schneider et al., 2017). 2.4 Identifying Periods of Simulation Failures 10 To evaluate the distribution of outliers and persisting errors between the in- and out-of-sample periods and among marine isotope stages, we test whether their occurrence is different from a uniform random distribution (expected 20 under the null-hypothesis of equal performance) using a Pearson chi-square test (P), which is calculated as follows: To evaluate the distribution of outliers and persisting errors between the in- and out-of-sample periods and among marine isotope stages, we test whether their occurrence is different from a uniform random distribution (expected 20 under the null-hypothesis of equal performance) using a Pearson chi-square test (P), which is calculated as follows: 𝑃= ∑ _`aBbacd ba e fS* (5 𝑃= ∑ _`aBbacd ba e fS* (5) in which n is the number of periods (n=2; in-sample j = 1; out-of-sample j = 2; or nineteen marine isotope stages), is the number of outliers or persisting errors that are identified in period j, and Ej is the number of occurrences expec in period j. in which n is the number of periods (n=2; in-sample j = 1; out-of-sample j = 2; or nineteen marine isotope stages), Oj is the number of outliers or persisting errors that are identified in period j, and Ej is the number of occurrences expected in period j. 25 in which n is the number of periods (n=2; in-sample j = 1; out-of-sample j = 2; or nineteen marine isotope stages), Oj is the number of outliers or persisting errors that are identified in period j, and Ej is the number of occurrences expected in period j. 25 The number of occurrences expected in period j (Ej) is calculated based on the null hypothesis that outliers or persisting errors are distributed uniformly among periods. This null implies that the expected value (𝐸f) can be calculated as: 𝐸f = hia ∑hiaj k j × ∑ 𝑂f e fS* (6) The number of occurrences expected in period j (Ej) is calculated based on the null hypothesis that outliers or persisting errors are distributed uniformly among periods. 2.4 Identifying Periods of Simulation Failures 10 This null implies that the expected value (𝐸f) can be calculated as: hia ∑e ( ) 𝐸f = hia ∑hiaj k j × ∑ 𝑂f e fS* (6) (6) in which Yr is the number of thousand-year time steps in period j for which observed values are available and n is the number of periods for which observed values are available for the 791 kyr simulation period. P is evaluated against a 30 𝜒m distribution with n-1 degrees of freedom. If the test rejects the null hypothesis that outliers or persisting errors are distributed randomly among periods (i.e. some periods are simulated more/less accurately than others), the more accurate subsample is identified by the numerator of Equation (5) (𝑂f −𝐸f). A negative value during the in-sample period ((𝑂* −𝐸*) < 0) would indicate that the number of outliers or persisting errors detected during the in-sample in which Yr is the number of thousand-year time steps in period j for which observed values are available and n is the number of periods for which observed values are available for the 791 kyr simulation period. P is evaluated against a 30 𝜒m distribution with n-1 degrees of freedom. If the test rejects the null hypothesis that outliers or persisting errors are distributed randomly among periods (i.e. some periods are simulated more/less accurately than others), the more accurate subsample is identified by the numerator of Equation (5) (𝑂f −𝐸f). A negative value during the in-sample period ((𝑂* −𝐸*) < 0) would indicate that the number of outliers or persisting errors detected during the in-sample period is less than expected by a uniform random distribution. This result would suggest that the model generates a 35 more accurate simulation during the in-sample period. Equations (5) and (6) also are used to test whether outliers or persisting errors are distributed randomly across the nineteen marine isotope stages (n=19) that fall within the 791 kyr simulation. The first observation is 791 kyr BP, which falls in MIS 19. period is less than expected by a uniform random distribution. This result would suggest that the model generates a 35 more accurate simulation during the in-sample period. Equations (5) and (6) also are used to test whether outliers or persisting errors are distributed randomly across the nineteen marine isotope stages (n=19) that fall within the 791 kyr simulation. 2.4 Identifying Periods of Simulation Failures 10 If model performance does not change over time, we expect outliers and persisting errors to occur randomly throughout the sample and be equally likely in each sub-sample. We use this assumption to compare the distribution of outliers and persisting errors between in-sample and out-of-sample periods and among nineteen marine isotope stages. For each thousand-year time step, we count the number of variables that exhibit an outlier or persisting error. 15 Following this procedure, the maximum number of outliers or persisting errors for any single time-step is ten. These If model performance does not change over time, we expect outliers and persisting errors to occur randomly throughout the sample and be equally likely in each sub-sample. We use this assumption to compare the distribution of outliers and persisting errors between in-sample and out-of-sample periods and among nineteen marine isotope stages. For each thousand-year time step, we count the number of variables that exhibit an outlier or persisting error. 15 Following this procedure, the maximum number of outliers or persisting errors for any single time-step is ten. These sums (and values for individual variables) are assigned to the in- or out-of-sample period or individual marine isotope stages. stages. For each thousand-year time step, we count the number of variables that exhibit an outlier or persisting error. 15 Following this procedure, the maximum number of outliers or persisting errors for any single time-step is ten. These sums (and values for individual variables) are assigned to the in- or out-of-sample period or individual marine isotope stages. 2.4 Identifying Periods of Simulation Failures 10 The first observation is 791 kyr BP, which falls in MIS 19. period is less than expected by a uniform random distribution. This result would suggest that the model generates a 35 more accurate simulation during the in-sample period. Equations (5) and (6) also are used to test whether outliers or persisting errors are distributed randomly across the nineteen marine isotope stages (n=19) that fall within the 791 kyr simulation. The first observation is 791 kyr BP, which falls in MIS 19. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 7 2.5 Causes for Model Failure We expect the coefficients generally 20 to be statistically different from zero because simulation errors generally are correlated across variables, however, we are interested whether during the periods of simulation failure (as given by Di,t = 1), persisting errors for other endogenous variables propagate through the system, pre-dating/predicting persisting errors in the endogenous climate variable being modelled. Because the level of significance of selection in the first stage (pα ≃0.001) makes false- positives in Di,t unlikely (approximately 1 outlier to be expected spuriously on average), the detection of breaks in the 25 first stage probably has little effect on tests on Di,t in this second stage. We repeat this process without the simulation errors for sea level because the first observation for sea level (462 kyr BP) is much more recent than the other time series (Table 1), which limits the sample range when all ten simulation errors are analyzed using Equation (7). if For each simulation error for variable i ( ), we estimate Equation (7) ten times. In each, we eliminate the simulation errors for one of the ten endogenous variables interacted with its non-zero mean simulation dummy 30 ∑ 𝐷-,#Bf𝜃-,f𝜀-,#Bf u fS* .. This restriction is evaluated using an F-statistic that tests the null hypothesis that the persisting errors for the endogenous variable eliminated from Equation (7) have no information about the dependent variable beyond the additional variables included. These variables include the lagged values of simulation errors, the persisting simulation errors for the other endogenous variables, and the four exogenous variables for orbital geometry. Rejecting this null hypothesis allows us to state that the model’s inability to simulate the endogenous variable that is eliminated 35 from Equation (7) (as indicated by persisting errors) precedes the simulation errors for the endogenous climate variable on the left-hand side of Equation (7). εi ( ) q ( ) errors for one of the ten endogenous variables interacted with its non-zero mean simulation dummy 30 ∑ 𝐷-,#Bf𝜃-,f𝜀-,#Bf u fS* .. This restriction is evaluated using an F-statistic that tests the null hypothesis that the persisting errors for the endogenous variable eliminated from Equation (7) have no information about the dependent variable beyond the additional variables included. These variables include the lagged values of simulation errors, the persisting simulation errors for the other endogenous variables, and the four exogenous variables for orbital geometry. 2.5 Causes for Model Failure To evaluate the cause(s) for model failure, we test whether poor performance ‘starts’ with a specific variable(s) and whether this failure is communicated to the other variables through long- and short-run relations among endogenous 5 variables. To identify the variable(s) that initiates the poor performance, we formalize techniques that are used by previous analyses. Previous analyses estimate a regression equation that specifies a dependent variable as a function of lagged values for an independent variable thought to ‘precede’ the dependent variable. For example, Li et al., (1998) conclude that CO2 ‘precedes’ 𝛿*+𝑂 based on regression results that indicate 𝛿*+𝑂 is related to five lagged values of CO2. 10 But this approach is incomplete (from a statistical perspective) because it ignores the autocorrelation structure of the dependent variable. To account for this effect, we use a technique developed by Granger (1969) that is used to analyze relations among climate variables during the instrumental temperature record (e.g. Kaufmann and Stern, 1997; Stern and Kaufmann, 2014). For this application, we estimate the following regression: 𝜀-,# = 𝛼+ ∑ ∑ 𝜙-,f p fS* *? -S* 𝜀-,#Bf + ∑ ∑ 𝐷-,#Bf𝜃-,f p fS* *? -S* 𝜀-,#Bf + ∑ 𝜋f𝜔-,#Bf u fS? + 𝜂-,# (7) 15 i hi h D i i di t i bl th t l if th i l ti f i bl i d i i d t i t ti ti ll 𝜀-,# = 𝛼+ ∑ ∑ 𝜙-,f p fS* *? -S* 𝜀-,#Bf + ∑ ∑ 𝐷-,#Bf𝜃-,f p fS* *? -S* 𝜀-,#Bf + ∑ 𝜋f𝜔-,#Bf u fS? + 𝜂-,# (7) (7) in which Di,t is an indicator variable that equals one if the simulation error for variable i during period t is statistically different from zero (i.e. is a persisting error) (Di,t = 0 otherwise), 𝜂 is an error term (assumed to be normally distributed), and 𝛼, 𝜙, 𝜃, 𝜋, are regression coefficients that are estimated using ordinary least squares. The number of lags (s) is determined using the Akaike Information criterion (Akaike, 1973). Equation (7) is estimated ten times, once εi,t with the simulation error for each endogenous variable on the left-hand side. 3.1 Model Performance 5 5 For both the in- and out-of-sample periods, Figure 1 suggests that the model generally captures the timing and magnitude of persistent changes in climate that are described by glacial cycles, which frequently are summarized by changes in land ice volume (Ice). For this variable, the model generally simulates the timing and magnitude of glaciations and terminations, including the gradual accumulation of ice and its rapid ablation (i.e. the saw-tooth pattern). Furthermore, there are no skipped obliquity/precession beats (other than MIS 11). Finally, the model’s ability 10 to simulate glacial cycles during the out-of-sample period is inconsistent with speculation that the CVAR model’s ability to reproduce the ten climate/physical variables during the in-sample period simply reflects the model’s ability to reproduce the data used to estimate the coefficients. Instead, the ability of the model to simulate climate during the out-of-sample period suggests that its coefficients capture relations among orbital geometry and the ten climate/physical proxies that govern the climate system beyond the sample period. 15 3.1.1 In- vs. Out-of-Sample Comparisons Conversely, the number of persisting errors is not distributed randomly among the nineteen marine isotope stages, even if errors in MIS 11 are excluded (Table 2). Tests indicate that we cannot reject the null hypothesis that outliers are distributed randomly between the in- and out- 25 of-sample periods (Table 2). A test statistic χm(1) = 0.09 fails to reject (p > 0.76) the null hypothesis that as a group, outliers for the ten climate/physical variables are distributed randomly between the in- and out-of-sample periods. Conversely, a test statistic χm(1) = 52.5 rejects (p < 0.001) the null hypothesis that as a group, persisting errors for the ten climate/physical variables are distributed randomly between the in- and out-of-sample periods. 30 2.5 Causes for Model Failure Rejecting this null hypothesis allows us to state that the model’s inability to simulate the endogenous variable that is eliminated 35 from Equation (7) (as indicated by persisting errors) precedes the simulation errors for the endogenous climate variable on the left-hand side of Equation (7). i https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 8 8 3.1.1 In- vs. Out-of-Sample Comparisons The similarity between the model’s accuracy in- and out-of-sample (Figure 1) is consistent with comparisons of root mean square error (Figure 2). As expected, the RMSE for the out-of-sample period generally is larger than the RMSE 20 for the in-sample period. But much of this increase is associated with MIS 11, most of which occurs during the out- of-sample period (Figure 1). If we eliminate MIS 11 from the out-of-sample period, the RMSE of the in- and out-of- sample periods are similar (Figure 2). The outsized effect on the RMSE for the out-of-sample period is consistent with the ‘stage 11 paradox.’ The similarity between the model’s accuracy in- and out-of-sample (Figure 1) is consistent with comparisons of root mean square error (Figure 2). As expected, the RMSE for the out-of-sample period generally is larger than the RMSE 20 for the in-sample period. But much of this increase is associated with MIS 11, most of which occurs during the out- of-sample period (Figure 1). If we eliminate MIS 11 from the out-of-sample period, the RMSE of the in- and out-of- sample periods are similar (Figure 2). The outsized effect on the RMSE for the out-of-sample period is consistent with the ‘stage 11 paradox.’ Tests indicate that we cannot reject the null hypothesis that outliers are distributed randomly between the in- and out- 25 of-sample periods (Table 2). A test statistic χm(1) = 0.09 fails to reject (p > 0.76) the null hypothesis that as a group, outliers for the ten climate/physical variables are distributed randomly between the in- and out-of-sample periods. Conversely, a test statistic χm(1) = 52.5 rejects (p < 0.001) the null hypothesis that as a group, persisting errors for the ten climate/physical variables are distributed randomly between the in- and out-of-sample periods. 30 3.1.2 Comparisons Among Marine Isotope Stages Outliers and persisting errors are not distributed randomly among the nineteen marine isotope stages (Figure 3, Table 2). This result is generated in part by the ‘stage 11 paradox.’ If this stage is eliminated from consideration, we cannot reject the null hypothesis that outliers for variables other than methane are distributed randomly among the remaining 35 eighteen stages. Similarly, the RMSEs across variables are very similar in and out-of-sample when MIS 11 is excluded (Figure 2). 3.1.2 Comparisons Among Marine Isotope Stages Outliers and persisting errors are not distributed randomly among the nineteen marine isotope stages (Figure 3, Table 2). This result is generated in part by the ‘stage 11 paradox.’ If this stage is eliminated from consideration, we cannot reject the null hypothesis that outliers for variables other than methane are distributed randomly among the remaining 35 eighteen stages. Similarly, the RMSEs across variables are very similar in and out-of-sample when MIS 11 is excluded (Figure 2). Conversely, the number of persisting errors is not distributed randomly among the nineteen marine isotope stages, even if errors in MIS 11 are excluded (Table 2). https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 9 3.2 Causes for Model Failure Applying the p = 0.05 threshold to the tests that evaluate restrictions on Equation (7), sixteen of the one hundred tests reject the null hypothesis that lagged values for persisting errors (interacted with the non-zero dummy variable D) have no information about current values for the simulation errors on the left-hand side of Equation (7) beyond the 5 right-hand side variables that remain in Equation 7 (Table 3). For the eighty-one tests run on the nine endogenous variables (other than sea level), the null is rejected eleven times (Table 4). In both cases, the number of rejections observed is greater than the number expected due to repeated testing at p = 0.05, five and four rejections, respectively. Together, these results suggest that the test results reveal information about the statistical ordering of simulation errors. 4.3 Mechanisms for the Stage 11 Paradox MIS 11 also is the longest period of prolonged stable warm climate in the North Atlantic (Oppo et al 1998; McManus et al 1999; 2003) Finally many 25 4.1 Nonlinearties and/or threshold effects drive the timing and magnitude of glacial cycles A recent review of terminations states “Terminations clearly represent a strongly nonlinear response to regional changes in the seasonality of solar radiation (Past interglacials Working Group of Pages, 2016).” We test this statement 15 by using the CVAR to evaluate hypotheses about the importance of thresholds (e.g. Paillard, 1998; 2001; Ganopolski et al., 2016; Tzedakis, et al., 2017), nonlinearities (e.g. Tziperman et al., 2006), or governing equations that vary by phase of the glacial cycle. If any of these play an important role, the CVAR model, which does not include their effects, will not be able to simulate glacial cycles. The CVAR model is largely linear. Both long- and short-run relations among variables are linear. The only non-linear 20 relation is given by the fractional rate at which variables adjust to disequilibrium in the long-run relations (𝛼). But this nonlinearity is constrained by the fact that the fractional rate of adjustment is constant and applies during all phases of the glacial cycle. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 10 10 4.2 The Mid Brunhes Event 4.2 The Mid Brunhes Event The demarcation between the in- and out-of-sample period (391 kyr BP) falls close to the Mid-Brunhes event, MBE (Jansen et al., 1986). Compared to the in-sample period used to estimate KJ2013, the pre-MBE out-of-sample period has; (1) lower concentrations of CO2, (2) glacial cycles with a smaller amplitude, and (3) cooler but longer interglacial 5 periods (EPICA, et al., 2004; Luthi et al., 2008; Hoenisch et al., 2009). These three changes beg the question, do they represent a change in the dynamics that drive glacial cycles and/or a change in the drivers of glacial cycles. The latter is supported by Yin (2013), who concludes, “through a set of internal mechanisms insolation alone induces a systematic difference between the interglacials before and after the 430 kyr ago in some ocean processes that are critical for the carbon cycle.” Conversely, Tzedakis et al., (2009) argue ‘astronomical forcing alone cannot explain 10 the difference in interglacial intensity before and after the MBE.” Our model simulations contradict the latter, that the MBE represents a change in the dynamics that drive glacial cycles. As indicated in Figure 1, the single set of relations among orbital geometry and the climate system embodied in the CVAR model simulates the different characteristics of glacial cycles before and after the MBE. As such, the MBE is not a transition between regimes; rather there is something unique about the MBE in particular and MIS 11 in general 15 not a transition between regimes; rather there is something unique about the MBE in particular and MIS 11 in general. 15 4.3 Mechanisms for the Stage 11 Paradox Imbrie et al., (1993) describe ‘the stage 11 paradox’ as a significant mismatch between orbital position and changes in climate associated with MIS 11 in general and termination V (430 -415 kyr BP) in particular. The latter is defined 20 by the maximum in benthic 𝛿*+𝑂 of MIS 12 and the benthic 𝛿*+𝑂 plateau of MIS 11 (Broecker and van Donk, 1970). These periods are unique: Termination V is the longest of any during the previous half million years (Berger and Loutre, 1996; Droxler et al., 2003; Loutre and Berger, 2003; McManus et al., 2003; EPICA Community Members, 2004; Rohling et al., 2010; Liseicki and Raymo, 2005; Ruddiman, 2007). MIS 11 also is the longest period of prolonged, stable warm climate in the North Atlantic (Oppo et al., 1998; McManus et al., 1999; 2003). Finally, many 25 areas have air and sea surface temperatures that reach values consistent with interglacial periods even though large areas of the Earth’s surface are covered by ice (Ruddiman, 2007). Despite these large changes in climate, the changes in orbital geometry are small. Consistent with this seeming mismatch, the CVAR model does a poor job of simulating termination V in particular and MIS 11 in general. Figures 1-3 indicate that MIS 11 has more variables with persisting errors than any other 30 period, either in- or out-of- sample (as well as driving the higher RMSE out-of-sample). This indicates MIS 11 is a prolonged period during which the model is not able to use the four variables for orbital geometry to simulate climate, which is the definition of the ‘stage 11 paradox.’ Imbrie et al., (1993) describe ‘the stage 11 paradox’ as a significant mismatch between orbital position and changes in climate associated with MIS 11 in general and termination V (430 -415 kyr BP) in particular. The latter is defined 20 by the maximum in benthic 𝛿*+𝑂 of MIS 12 and the benthic 𝛿*+𝑂 plateau of MIS 11 (Broecker and van Donk, 1970). These periods are unique: Termination V is the longest of any during the previous half million years (Berger and Loutre, 1996; Droxler et al., 2003; Loutre and Berger, 2003; McManus et al., 2003; EPICA Community Members, 2004; Rohling et al., 2010; Liseicki and Raymo, 2005; Ruddiman, 2007). 4.3.1 Difficulties in orbital tuning 35 The CVAR’s model’s poor performance during MIS 11 could be caused by difficulties in orbital tuning. The insolation peak for MIS 11 occurs in the middle of the warm stage therefore, orbital tuning delays the interglacial peak in 𝛿*+𝑂 compared to other stages (Candy et al., 2014; Imbrie et al., 1984; Liseicki and Raymo, 2005). Furthermore, MIS 11 https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 11 contains fewer tie points that can be used to anchor the chronology (Desprat et al., 2005), which means that the orbitally tuned chronology of MIS 11 is less secure than other warm stages (Candy et al., 2014). As such, the model’s failure during this period may simply represent the poor quality of the chronology to which the simulation is compared. To evaluate whether the stage 11 paradox is an artifact of the poor quality of the chronology, we condition the model on some of the endogenous variables that are thought to play an important role in glacial cycles. Conditioning a model 5 on observed values for one or more endogenous variables always will improve performance (Oreskes et al., 1994), but the variable used to condition the model will have little effect on model performance if the model’s poor performance during MIS 11 is caused by the poor quality of the chronology because no endogenous variable will have more/less information about the poor chronology. Contrary to this expectation, model performance during stage 11 depends on the variable used to condition the model. Conditioning the model on observed values of CO2 or Na allows 10 the model to simulate more of the decline in Ice (and more accurately simulate other variables) throughout MIS 11, including termination V (Figure 4). Conversely, conditioning the model on observed values for SST, which is thought to play an important role in MIS 11 (see below), does not improve the model’s ability to simulate the interglacial in MIS 11. Although this failure may be explained by stronger latitudinal or meridional gradients in sea surface temperature (Kandiano et al., 2012), large variations in accuracy that depend on the endogenous variable used to 15 condition the model suggest that the model’s failure during MIS 11 is not caused solely by weaknesses in orbital tuning. 4.3.2 Mechanistic Explanations 20 The mechanisms and sequences that generate the ‘stage 11 paradox’ cannot be fully identified by the CVAR model because it greatly simplifies physical relations and it has a relatively coarse temporal resolution (1 kyr). Conversely, its ability to accurately simulate glacial cycles (except MIS 11) using orbital position alone allows the CVAR model to test competing hypotheses about the ‘stage 11 paradox’ by identifying exceptions to the model sequences that accurately simulate terminations other than termination V. In other words, the statistical ordering of simulation errors 25 allows us to identify what is unique about MIS 11 (and termination V) and whether these differences play an important role. Explanations for terminations in general - and stage 11 in particular - share several components. Many start with a change in meridonal overturning circulation and a bipolar seesaw that create a negative correlation between changes in hemispheric temperatures. Specifically, terminations may start with changes in orbital position that add freshwater 30 to the North Atlantic, this freshwater melt slows Atlantic meridonal overturning circulation (Elliot et al., 2002; McManus et al., 2004; Oppo et al., 1995; Vidal et al., 1997), and this slowdown creates a nearly simultaneous change in sea surface temperatures in the Southern Hemisphere via the bipolar seesaw (Barker et al., 2009; Broecker 1998; 1986; Schmittner et al., 2002; Stocker and Johnson, 2003). In addition to an opposite change in sea surface temperature, there is evidence that changes in buoyancy (Watson and Garabato, 2006), latitudinal shifts in the 35 Westerlies (Anderson et al., 2009; Ninnermann and Charles, 1997; Toggweiler et al., 2006), and/or a changes in sea ice (Stephens and Keeling, 2000) affect the flow of CO2 from the southern Ocean, which is an important reservoir for glacial/interglacial CO2 (Knox and McElroy, 1984; Sarmiento and Togeweiler, 1984; Seigenthaler and Wenk, 1984; Anderson et al., 2009; Skinner et al., 2013). https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 12 Uncertainties about this general schema include questions about the role of changes in sea surface temperature relative to the location of the Westerlies/sea ice and the role of CO2 from the Southern Ocean; does ventilation drive deglaciation or is it caused by the glaciation? 4.3.2 Mechanistic Explanations Riveiros et al., (2013) postulate that termination V is driven “primarily via meridonal heat transport anomalies that would have enhanced the incipient warming arising from relatively weak insolation forcing and only secondarily via CO2 release.” Conversely, Andersen et al., (2009) show that changes in 5 the position of the Westerlies are the main driver for the increased flow of CO2 to the atmosphere during the termination of the last ice age. Similarly, a shift by the Westerlies precedes the drop in atmospheric CO2 during MIS 5 (Govin et al., 2009). These competing hypothesis for terminations in general and stage 11 in particular can be tested by the statistical ordering of the model errors. If changes in sea surface temperature initiate Termination V, the model’s inability to 10 simulate termination V will ‘start’ with its inability to simulate SST. This inability will be indicated by simulation errors for SST that precede and have information about the simulation errors for other variables. Specifically, simulation errors for other variables, such as CO2, will not have prior information about the errors for SST and these errors will have prior information about the errors for the other variables, such as CO2. The statistical ordering of simulation errors indicates that the simulation errors for SST do not precede the model’s 15 inability to simulate MIS 11 and termination V. Errors for SST are preceded by the persisting errors for other variables (read across the SST row in Tables 3 and 4), such as CO2, and the persisting errors for SST do not have prior information about the simulation errors for any variables (read down the SST column) at . Using a threshold 𝑝≤0.10, there is some evidence that persisting errors for SST have information about Ice. Consistent with these results, conditioning the model on SST, which eliminates the simulation errors for SST, does not improve the model’s ability 20 to simulate Ice during MIS 11 relative to other potential causes for the stage 11 paradox (Figure 4). In toto, these results suggest that model failures do not ‘start with’ an inability to simulate sea surface temperature; rather the failure to simulate sea surface temperature is caused by the inability to simulate some other variable(s). 4.3.2 Mechanistic Explanations These CVAR 35 simulations also will be used to assess the early Anthropogenic hypothesis by evaluating the degree to which anthropogenic emissions of carbon dioxide and methane can account for outliers and persisting errors in Ice and other climate variables during the Holocene. 5 Conclusion 10 Our model is able to accurately simulate entire glacial cycles for an out-of-sample period that does not prescribe GHG forcing: the simulation is driven only by changes in orbital geometry. This ability suggests that the model can accurately hindcast climate using known climate parameters, which is the criterion proposed by Tzedakis et al., (2009) for understanding the current climate and where it is headed. Although satisfying this criterion has to be interpreted with caution because predictability is not necessarily informative about the quality of a model with respect to capturing 15 underlying causality (see e.g. Oreskes et al., 1994, or Clements and Hendry, 2005), the ability to hindcast climate suggests that our model could supplement the search for analogues for the Holocene (11,700 years before the present through the present), many of which focus on MIS 11 (Droxler et al., 2003; Tzedakis, 2010; Pol et al., 2011). Despite some similarities, our results suggest that such efforts are fraught with difficulty. Most importantly, the statistical model cannot use the four measures of orbital geometry to simulate the depth and length of the interglacial that is 20 associated with MIS 11. Conversely, the model is able to simulate many aspects of the current warm period (Figure 1 & 3): notable exceptions include peristing errors associated with Ice and SST (see below). This implies that any similarity in orbital geometry and feedback mechanisms (Imbrie et al., 1992; 1993, Ruddiman 2003; 2006) do not automatically translate into similar climates. As such, there probably are important differences between the Holocene and MIS 11. 25 4.3.2 Mechanistic Explanations As such, changes in sea surface temperature probably are not ultimately responsible for the ‘stage 11 paradox.’ p ≤0.05 Instead, the statistical ordering generated by Equation (7) highlights the importance of the model’s inability to simulate 25 atmospheric carbon dioxide. Reading across the CO2 row indicates that the simulation errors for other variables, including SST have no prior information about the simulation errors for CO2, which suggests that model failures ‘start with’ an inability to simulate carbon dioxide. Furthermore, these failures propagate through the system. Reading down the CO2 column indicates that the persisting errors for CO2 have information about the simulation errors for other variables including Ice and SST (Table 4). 30 Instead, the statistical ordering generated by Equation (7) highlights the importance of the model’s inability to simulate 25 atmospheric carbon dioxide. Reading across the CO2 row indicates that the simulation errors for other variables, including SST have no prior information about the simulation errors for CO2, which suggests that model failures ‘start with’ an inability to simulate carbon dioxide. Furthermore, these failures propagate through the system. Reading down the CO2 column indicates that the persisting errors for CO2 have information about the simulation errors for other variables including Ice and SST (Table 4). 30 https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 13 But the model’s inability to simulate MIS stage 11 may not start solely with an inability to simulate CO2. Persisting errors for Ice also are preceded by persisting errors for Ca and Fe (proxies for wind strength and aridity Section 2.1 and Supplemental Section I). And the persisting errors for Ca are preceded by the persisting errors for Na (a proxy for sea ice in the southern ocean Section 2.1 and Supplemental Section I) Although results cannot resolve the timing of the model’s inability to simulate wind (Ca, Fe) and sea ice (Na), their importance suggests that the model’s inability 5 to simulate the long interglacial of MIS 11 is generated in part by the model’s inability to simulate the location and strength of winds, the extent of sea ice, and/or the ventilation of CO2 from the Southern Ocean. 5 Conclusion 10 Our model is able to accurately simulate entire glacial cycles for an out-of-sample period that does not prescribe GHG forcing: the simulation is driven only by changes in orbital geometry. 4.3.2 Mechanistic Explanations This ability suggests that the model can accurately hindcast climate using known climate parameters, which is the criterion proposed by Tzedakis et al., (2009) for understanding the current climate and where it is headed. Although satisfying this criterion has to be interpreted with caution because predictability is not necessarily informative about the quality of a model with respect to capturing 15 underlying causality (see e.g. Oreskes et al., 1994, or Clements and Hendry, 2005), the ability to hindcast climate suggests that our model could supplement the search for analogues for the Holocene (11,700 years before the present through the present), many of which focus on MIS 11 (Droxler et al., 2003; Tzedakis, 2010; Pol et al., 2011). Despite some similarities, our results suggest that such efforts are fraught with difficulty. Most importantly, the statistical model cannot use the four measures of orbital geometry to simulate the depth and length of the interglacial that is 20 associated with MIS 11. Conversely, the model is able to simulate many aspects of the current warm period (Figure 1 & 3): notable exceptions include peristing errors associated with Ice and SST (see below). This implies that any similarity in orbital geometry and feedback mechanisms (Imbrie et al., 1992; 1993, Ruddiman 2003; 2006) do not automatically translate into similar climates. As such, there probably are important differences between the Holocene and MIS 11. 25 Ironically, the interglacials during MIS 11 and the Holocene may share an important similarity: an important role for carbon dioxide. The inability to simulate the interglacial in MIS 11 is likely caused by a poorly-modelled physical mechanism that raises atmospheric carbon dioxide. It is highly unlikely that this mechanism is related to human activity, even though MIS 11 contains the first evidence for the use of fire by people in Britain (Gowlett, 2005; Preece et al., 2006). Conversely, others argue that Holocene warming is amplified by anthropogenic emissions of carbon 30 dioxide and methane (Ruddiman 2003; 2005; 2007). Rather than trying to decide which aspects of the paleoclimate record ‘line up’ across marine isotope stages (e.g. Candy et al., 2014), future efforts will use the statistical model to identify the cause(s) for the current warming and how long it will last. Specifically, we will compile future values for orbital geometry and use them to simulate the model as a CVAR-based alternative to GCM-based simulations (see e.g. Ganopolski et al., 2016). Team list: The team includes Robert Kaufmann (RK) and Felix Pretis (FP). Team list: The team includes Robert Kaufmann (RK) and Felix Pretis (FP). Author contributions: This project was conceived by RK and FP. RK compiled the data from the statistical model and FP did the statistical analysis to identify impulses and steps. RK and FP write the manuscript, designed the tables, 10 and created the figures together. Author contributions: This project was conceived by RK and FP. RK compiled the data from the statistical model and FP did the statistical analysis to identify impulses and steps. RK and FP write the manuscript, designed the tables, 10 and created the figures together. Competing Interests: The authors have no financial or non-financial interests associated with the material in this manuscript. 15 Competing Interests: The authors have no financial or non-financial interests associated with the material in this manuscript. 15 15 Acknowledgements: We thank David F Hendry, Luke Jackson, and Katarina Juselius for helpful comments and suggestions. Financial support from the Robertson Foundation and British Academy is gratefully acknowledged." References Akaike, H.: 2nd International Symposium on Information Theory, P.N. Petrov and F. Csaki, Eds., Akadacemiai 20 Kiadaco, 267-281, 1997. Anderson, R.F., Ali, S., Bradtmiller, L.I., Nielsen, S.H.H., Fleisher, M.Q., Anderson, B.E., and Brickle, L.H.: Wind driven upwelling in the southern ocean and the deglacial rise in atmospheric CO2, Science, 323:1443-1448, 2009. Archer D., Winguth, A., Lea, D., and Mahowald N.: What caused the glacial/interglacial atmospheric pCO(2) Archer D., Winguth, A., Lea, D., and Mahowald N.: What caused the glacial/interglacial atmospheric pCO(2) cher D., Winguth, A., Lea, D., and Mahowald N.: What caused the glacial/interglacial atmospheric pCO(2) les? Rev. Geophys. 38(2), 159–189, 2000. cycles? Rev. Geophys. 38(2), 159–189, 2000. 25 Barker, S., Diz, P.,Vautravers, J., Pike, J., Knorr, G., Hall, I.R., and Broecker W.S., Interhemispheric Atlantic seesaw response during the last deglaciation, Nature 457:1097-1102, 2009. Basile, I., Grousset, F.E., Revel, M., Petit, J.R., Biscaye, P.E., and Barkov, N.I.: Patagonian origin of glacial dust deposited in East Antarctica (Vostok and Dome C) during glacial stages 2, 4 and 6, Earth and Planetary Science Letter, 146,573-589, 1997. 30 Berger, A., and Loutre, M. F.: Modelling the climate response to astronomical and CO2 forcings, C. R. Acad. Sci., Ser. IIa: Sci. Terre Planetes,323(1), 1–16, 1996. Broecker, W.S.: Paleocean circulation during the last deglaciation: a bipolar seesaw? Paleoceanography 13:119-121, 1998. Broecker, W.S., and vanDonk, J.: Insolation changes, ice volumes, and O-18 record in deep-sea cores, Reviews of 35 Geophysics and Space Physics: 8(1):169, 1970. Broecker, W.S., and vanDonk, J.: Insolation changes, ice volumes, and O-18 record in deep-sea cores, Reviews of 35 Geophysics and Space Physics: 8(1):169, 1970. Brovkin, V., Ganopolski, A., Archer, D., and Munhoven, G.: Glacial CO2 cycle as a succession of key physical and Broecker, W.S., and vanDonk, J.: Insolation changes, ice volumes, and O-18 record in deep-sea cores, Reviews of 35 Geophysics and Space Physics: 8(1):169, 1970. Brovkin, V., Ganopolski, A., Archer, D., and Munhoven, G.: Glacial CO2 cycle as a succession of key physical and biogeochemical processes. Climate of the Past, 8(1), 251-264, 2012. Candy I Schreve D C Sherriff J Tye G J : Marine isotope stage 11: paleoclimates paleoenvironments and its Geophysics and Space Physics: 8(1):169, 1970. Brovkin, V., Ganopolski, A., Archer, D., and Munhoven, G.: Glacial CO2 cycle as a succession of key physical and biogeochemical processes. Climate of the Past, 8(1), 251-264, 2012. 5 Conclusion 10 https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 14 Data and Code Availability: The data and computer code used in this analysis are available on OpenBU, which is FAIR-compliant, and can be accessed through a globally unique and eternally persistent identifier, Data and Code Availability: The data and computer code used in this analysis are available on OpenBU, which is FAIR-compliant, and can be accessed through a globally unique and eternally persistent identifier, https://hdl.handle.net/2144/40340 P. This dataset is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 License (http://creativecommons.org/licenses/by-sa/4.0). 5 Team list: The team includes Robert Kaufmann (RK) and Felix Pretis (FP). Author contributions: This project was conceived by RK and FP. RK compiled the data from the statistical model and FP did the statistical analysis to identify impulses and steps. RK and FP write the manuscript, designed the tables, 10 and created the figures together. Competing Interests: The authors have no financial or non-financial interests associated with the material in this manuscript. 15 Acknowledgements: We thank David F Hendry, Luke Jackson, and Katarina Juselius for helpful comments and suggestions. Financial support from the Robertson Foundation and British Academy is gratefully acknowledged." https://hdl.handle.net/2144/40340 P. This dataset is distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 License (http://creativecommons.org/licenses/by-sa/4.0). References Brovkin, V., Ganopolski, A., Archer, D., and Munhoven, G.: Glacial CO2 cycle as a succession of key physical and biogeochemical processes. 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Griffin and Co., London, 1929. 35 https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 20 Figure captions Fig. 1. The observed values for temperature (black line) and values simulated by the system model conditioned only on the four variables for solar insolation (red line). Thick portions of the red line represent time steps in which the simulation error is significantly different from zero (non-zero error). Red circles represent time steps when the 5 simulation error is an (innovational) outlier. The light gray area is the out-of-sample forecast period; MIS 11 is shaded dark gray. (b) same as above for carbon dioxide, (c) same as above for methane, (d) same as above for land ice, (e) same as above for Na, (f) same as above for SO4, (g) same as above for sea level, (h) same as above for SST1. Fig. 2. 1 Note that the series of SST exhibits non-zero simulation errors nearly throughout the sample, suggesting a non- zero bias throughout the observational record – simulated model values persistently exceed observations. References The root mean square simulation errors for all ten endogenous climate variables simulated by the CVAR system 10 model conditioned only on the four variables for solar insolation. RMSE for the in-sample period are shown as dark grey (left), out-of sample as grey (middle), and out-of sample excluding Marine Isotope Stage 11 as light grey (right). Fig. 3. The number of outliers (red spikes) and non-zero errors (darkly shaded) for each time step. Marine isotope stages are indicated by alternating areas of shading. 15 Fig. 4: The value of Ice conditioned on orbital geometry only (green line), CO2 (red line), non-sea-salt sodium (blue line), Ca (yellow line), and SST (orange line). Values from the proxy record are given by the black line. Figure captions Fig. 1. The observed values for temperature (black line) and values simulated by the system model conditioned only on the four variables for solar insolation (red line). Thick portions of the red line represent time steps in which the simulation error is significantly different from zero (non-zero error). Red circles represent time steps when the 5 simulation error is an (innovational) outlier. The light gray area is the out-of-sample forecast period; MIS 11 is shaded dark gray. (b) same as above for carbon dioxide, (c) same as above for methane, (d) same as above for land ice, (e) same as above for Na, (f) same as above for SO4, (g) same as above for sea level, (h) same as above for SST1. Fig. 4: The value of Ice conditioned on orbital geometry only (green line), CO2 (red line), non-sea-salt sodium (blue line), Ca (yellow line), and SST (orange line). Values from the proxy record are given by the black line. Fig. 4: The value of Ice conditioned on orbital geometry only (green line), CO2 (red line), non-sea-salt sodium (blue line), Ca (yellow line), and SST (orange line). Values from the proxy record are given by the black line. 1 Note that the series of SST exhibits non-zero simulation errors nearly throughout the sample, suggesting a non- zero bias throughout the observational record – simulated model values persistently exceed observations. Temp. CO2 CH4 Land Ice Na SO4 Sea Lev. SST Author(s) 2020. CC BY 4.0 License. Temp. CO2 CH4 Land Ice Na SO4 Sea Lev. SST ⃝ ( ) CO2 Temp. CH4 Land Ice Na SO4 Sea Lev. SST https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. In Out (no 11) Temp. 0.0 0.5 1.0 1.5 2.0 2.5 In Out (no 11) CO2 0 5 10 15 20 25 In Out (no 11) CH4 0 20 40 60 80 In Out (no 11) Ice 0.0 0.1 0.2 0.3 0.4 In Out (no 11) Fe 0 10 20 30 40 50 60 In Out (no 11) Na 0 50 100 150 In Out (no 11) SO4 0 50 100 150 200 In Out (no 11) Ca 0 50 100 150 In Out (no 11) Lev. Figure captions 0 10 20 30 40 In Out (no 11) SST 0.0 0.5 1.0 1.5 Out Out Out Out Out Out Out Out Out Out Figure 2 Figure 2 Figure 2 In Out (no 11) Temp. 0.0 0.5 1.0 1.5 2.0 2.5 In Out (no 11) CO2 0 5 10 15 20 25 In Out (no 11) CH4 0 20 40 60 80 In Out (no 11) Ice 0.0 0.1 0.2 0.3 0.4 In Out (no 11) Fe 0 10 20 30 40 50 60 Out Out Out Out Out In Out (no 11) Na 0 50 100 150 In Out (no 11) SO4 0 50 100 150 200 In Out (no 11) Ca 0 50 100 150 In Out (no 11) Lev. 0 10 20 30 40 In Out (no 11) SST 0.0 0.5 1.0 1.5 Out Out Out Out Out https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 0 1 2 3 4 5 6 0 1 801 751 701 651 601 551 501 451 401 351 301 251 201 151 101 51 1 Number of variables Thousand years before present In-Sample Period Out-of-Sample Period 19 17 15 13 11 9 7 5 3 1 Figure 3 0 1 2 3 4 5 6 0 1 801 751 701 651 601 551 501 451 401 351 301 251 201 151 101 51 1 Number of variables Thousand years before present In-Sample Period Out-of-Sample Period 19 17 15 13 11 9 7 5 3 1 Figure 3 Figure 3 Figure 3 Out-of-Sample Period https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 3 3.5 4 4.5 5 -432 -427 -422 -417 -412 -407 -402 -397 -392 -387 -382 -377 Ice volume (𝛿18 𝑂) Thousand years before present Figure 4 Figure 4 Ice volume (𝛿18 𝑂) https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Table 1: Time series included in the CVAR Variable Source Unit Time Scale Obs First Observation Temp Jouzel et al., (2007) ∆ avg. last 1 kyr EDC3 710 801kyr BP CO2 Lüthi et al., 2008 ppmv ECD3 517 798 kyr BP CH4 Loulergue et al., (2008) ppbv EDC3 1477 799 kyr BP Ice Lisiecki and Raymo, (2005) 180 LR04 390 801kyr BP Fe Wolff et al. (2006) µg m-2yr-1 EDC2 187 736 kyr BP Na Wolff et al. (2006) µg m-2yr-1 EDC2 195 739 kyr BP SO4 Wolff et al. (2006) µg m-2yr-1 EDC2 195 739 kyr BP Ca Wolff et al. (2006) µg m-2yr-1 EDC2 195 739 kyr BP Sea Level Siddal et al., (2003) Meters SPECMAP 125 466 kyr BP Sea Surface Temp Martinez-Garcia et al., (2009) Degrees C EDC3 121 801kyr BP Eccentricity Paillard et al., (1996) Dimensionless index _ 801 801kyr BP Obliquity Paillard et al., (1996) Degrees _ 801 801kyr BP Precession Paillard et al., (1996) Dimensionless index _ 801 801kyr BP Seasonal Insolation Paillard et al., (1996) W/m2 _ 801 801kyr BP € δ https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Table 2: Tests of simulation accuracy during various periods. In vs. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. out-of-sample Distribution among marine isotope stages Variable Outliers Persisting errors Outliers Persisting errors All stages Stage 11 excluded All stages Stage 11 excluded Temp 3.0+[0/3] 19.8** [34/6] 18.4 15.6 286.5** 53.8** CO2 3.0++[0/3] 2.5 [4/10] 15.8 13.2 57.9** 53.3** CH4 7.0**+[0/7] 49.3**[31/117] 46.5** 39.7** 535.9** 571.8** Ice 0.2 [3/2] 103.7**[4/116] 14.9 3.8 543.0** 506.5** Fe 1.4 [1/4] 91.8**[226/79] 19.1 12.6 325.4** 313.5** Na 4.2*+[6/1] 24.2**[172/107] 28.2 3.5 356.0** 342.2** SO4 7.9**+[7/0] 40.7**[36/0] 80.2** 3.8 398.2** 368.7** Ca 1.3 [3/1] 24.3 **[35/6] 22.9 3.00 332.3** 307.0** Sea Level 0.8 [0/4] 136.3**[73/69] 8.0 6.3 288.1** 275.4** SST 0.1 [4/5] 9.1 **[357/282] 20.0 11.6 101.7** 101.6** All 0.1 [24/30] 52.5**[972/793] 37.9** 3.5 561.4** 427.7** Value rejects the null hypothesis at p < .05 (*), p < 0.01 respectively (**). Blue indicates the out-of-sample simulation is more accurate than the in-sample simulation; red indicate the in-sample simulation is more accurate. Values in brackets indicate the number of outlier/persisting errors in the out-of-sample period relative to the number of outlier/nonzero mean errors in the in-sample period.. A large value implies that the in-sample simulation has significantly fewer outlier/persisting errors, which would make it more accurate than the out-of-sample simulation. Value rejects the null hypothesis at p < .05 ( ), p < 0.01 respectively ( ). Blue indicates the out-of-sample simulation is more accurate than the in-sample simulation; red indicate the in-sample simulation is more accurate. Values in brackets indicate the number of outlier/persisting errors in the out-of-sample period relative to the number of outlier/nonzero mean errors in the in-sample period.. A large value implies that the in-sample simulation has significantly fewer outlier/persisting errors, which would make it more accurate than the out-of-sample simulation. Value rejects the null hypothesis at p < .05 ( ), p < 0.01 respectively ( ). Blue indicates the out-of-sample simulation is more accurate than the in-sample simulation; red indicate the in-sample simulation is more accurate. Values in brackets indicate the number of outlier/persisting errors in the out-of-sample period relative to the number of outlier/nonzero mean errors in the in-sample period.. A large value implies that the in-sample simulation has significantly fewer outlier/persisting errors, which would make it more accurate than the out-of-sample simulation. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Table 3: p-values for test of significance on 𝜃" (equation 8) for the sample that includes all endogenous variables. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Red indicates rejection of the exclusion (p < 0.05) of lagged errors of other series, blue indicates rejection of the exclusion (p < 0.10) of lagged errors of other series, and green indicates rejection of the exclusion for the autoregressive lags ( p < 0.05). The red value of 0.023 in the second column of the first row indicates that the indicates that the simulation errors for CO2 have information about the simulation errors for temperature. Dep. Variable Eq. 8 Non zero simulation error excluded from equation 8 Temp CO2 CH4 Ice Fe Na SO4 Ca Level SST Temp 0.005 0.023 0.041 0.144 0.140 0.314 0.314 0.878 0.795 0.169 CO2 0.397 0.629 0.057 0.143 0.721 0.760 0.760 0.676 0.512 0.992 CH4 0.829 0.516 0.000 0.074 0.285 0.101 0.101 0.168 0.746 0.867 Ice 0.421 0.148 0.496 0.277 0.270 0.334 0.334 0.393 0.272 0.051 Fe 0.054 0.055 0.658 0.586 0.014 0.910 0.910 0.000 0.337 0.600 Na 0.013 0.442 0.064 0.917 0.007 0.875 0.875 0.752 0.476 0.622 SO4 0.234 0.283 0.111 0.705 0.301 0.902 0.902 0.042 0.957 0.158 Ca 0.044 0.842 0.884 0.902 0.032 0.475 0.475 0.006 0.965 0.106 Level 0.259 0.152 0.422 0.028 0.405 0.415 0.415 0.938 0.637 0.617 SST 0.015 0.036 0.368 0.052 0.949 0.119 0.119 0.271 0.969 0.000 https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. Table 4: p-values for test of significance on 𝜃" (equation 8) for the sample that includes variables other than sea-level. Red indicates rejection of the exclusion (p < 0.05) of lagged errors of other series, blue indicates rejection of the exclusion (p < 0.10) of lagged errors of other series, green indicates rejection of the exclusion for the autoregressive lags. Dependent variable Eq. https://doi.org/10.5194/cp-2020-58 Preprint. Discussion started: 11 May 2020 c⃝Author(s) 2020. CC BY 4.0 License. 8 Non zero simulation error excluded from equation 8 Temp CO2 CH4 Ice Fe Na SO4 Ca SST Temp 0.009 0.311 0.128 0.349 0.173 0.644 0.890 0.571 0.187 CO2 0.186 0.852 0.213 0.136 0.700 0.312 0.866 0.149 0.847 CH4 0.454 0.471 0.362 0.371 0.568 0.181 0.878 0.775 0.886 Ice 0.314 0.006 0.584 0.000 0.035 0.228 0.370 0.046 0.055 Fe 0.189 0.202 0.394 0.860 0.064 0.711 0.976 0.059 0.791 Na 0.212 0.668 0.632 0.881 0.272 0.812 0.066 0.687 0.932 SO4 0.166 0.226 0.039 0.881 0.736 0.460 0.000 0.025 0.246 Ca 0.094 0.954 0.766 0.955 0.376 0.024 0.000 0.113 0.204 SST 0.714 0.011 0.326 0.012 0.973 0.337 0.715 0.180 0.133 ependent i bl Non zero simulation error excluded from equation 8
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A malignus daganatok és az ischaemiás stroke kapcsolata.
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ÖSSZEFOGLALÓ KÖZLEMÉNY ÖSSZEFOGLALÓ KÖZLEMÉNY A malignus daganatok és az ischaemiás stroke kapcsolata I. A thrombosis kialakulása, átfedő kockázati tényezők Hajnóczky Nóra dr.1, 3 ■ Bereczki Dániel dr.1, 2 1Semmelweis Egyetem, Általános Orvostudományi Kar, Neurológiai Klinika, Budapest 2MTA-SE Neuroepidemiológiai Kutatócsoport – Eötvös Loránd Kutatási Hálózat, Budapest 3Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, USA A malignus daganat és a stroke egy-egy betegnél gyakran kombinálódik, sokszor egyidejűleg diagnosztizálják, vagy rövid idő telik el a két kórkép felismerése között. Az együttes megjelenés hátterében elsősorban a hasonló tradicioná­ lis rizikófaktorok állhatnak: az idősebb életkor, a magas vérnyomás, a hyperlipidaemia, a cukorbetegség, az elhízás és a dohányzás. Az átfedő kockázati tényezőkön túl a daganat által okozott hiperkoaguláció artériás és vénás thrombosis kialakulásához vezethet. A hiperkoaguláció hátterének kutatása főként a thrombocyták és a szöveti faktor aktiválására és a heparanáz fokozott expressziójára fókuszált, és felvetődött a neutrophil extracelluláris csapdák szerepe is. A da­ ganat által okozott hiperkoagulációhoz társuló cryptogen (tradicionális rizikófaktor nélküli) stroke-ban sokszor talál­ ható magasabb D-dimer-szint, és a CT/MRI-képeken gyakrabban látszanak multifokális, több ér ellátási területében megjelenő ischaemiás laesiók, melyek ritkábban fordulnak elő a tradicionális rizikófaktorokkal magyarázható stroke- okban. Az előzőkön kívül a daganatok kezelésére alkalmazott kemoterápia és sugárterápia is emeli a stroke kockáza­ tát. A malignus daganatokhoz társuló stroke-ok megelőzése érdekében további vizsgálatok szükségesek a daganat által okozott hiperkoaguláció és vascularis változások pontosabb megértéséhez. Orv Hetil. 2022; 163(1): 3–11. Kulcsszavak: tumorokhoz társuló stroke, daganat által okozott hiperkoaguláció, tumorokhoz társuló thrombosis, patomechanizmus, cryptogen stroke A daganatok és a stroke kapcsolatának történeti áttekintése A malignus tumorok és a stroke is az idősebb korcso­ portban fordul elő nagyobb gyakorisággal. A két kórkép egy-egy betegnél gyakran kombinálódik, ezért érdemes kapcsolatukat elemezni. Az összefüggés kétirányú lehet: egyrészt a malignus betegségek stroke-kockázati ténye­ zőként szerepelhetnek, például a tumorhoz társuló hi­ perkoagulabilitás révén, másrészt stroke után is gyakran jelentkezhet malignus betegség. Ilyen esetekben érde­ mes elemezni, hogy a két kórkép együttes előfordulá­ sának hátterében állhatnak-e csupán a közös kockázati tényezők, vagy pedig azokon túl egyéb okok is magya­ rázhatják a kapcsolatot. A probléma fontosságára utal­ hat, hogy míg egy tíz évet (1997–2006) átfogó, nagy esetszámú felmérés során csökkent a kórházba kerülő nem daganatos ischaemiás stroke-os betegek száma, ad­ dig a daganatos stroke-betegek kórházi felvételében nem volt szignifikáns változás [1]. A malignus daganatok és a stroke közötti kapcsolatot a másik irányban is érdemes vizsgálni – tehát az ischaemi­ ás stroke után jelentkező malignus tumorok előfordulá­ sát. Egy 2015-ös vizsgálatban akut ischaemiás stroke után 1282 beteg 4,3%-ában diagnosztizáltak malignus daganatot; a stroke és a daganat diagnózisa között eltelt medián időtartam 14 hónap volt [6]. p Jelen összefoglalónkban kitérünk (1) a vénás és az ar­ tériás thrombosisok kapcsolatára, (2) a daganatok által kiváltott hiperkoaguláció patofiziológiájára és (3) a ma­ lignus betegségek és a stroke közös rizikófaktoraira, és elemezzük (4) a malignus tumorok kezelésére alkalma­ zott kemoterápiához és sugárterápiához társuló stroke- kockázatot. Dolgozatunk II. részében elemezzük (5) a malignus tumorok után jelentkező stroke és a stroke után felismert malignus tumorok időbeli jellemzőit, (6) a daganatok mellett jelentkező akut stroke kezelési lehe­ tőségeit és a tumoros betegekben alkalmazható stroke- prevenció kérdéseit, valamint (7) kitérünk arra a kér­ désre, hogy milyen esetben indokolt tumor keresése cryptogen stroke-ban. A kapcsolat a malignus daganatok és a thromboticus érelzáródás kialakulása között már az 1800-as évek óta ismert. Armand Trousseau, a kor elismert kutatója vetet­ te fel először a kapcsolatot a hasi daganat és a migráló vénás thrombophlebitis kialakulása között [2]. Az ezt követő 150 évben tovább szélesedtek az ismeretek a da­ ganatok és a thrombosis kölcsönhatásairól, de a mai na­ pig sok kérdés maradt megválaszolatlanul. A Trousseau-tanulmány után kezdetben a thrombosis­ kutatás főleg a vénás thromboembolisatióval (VTE) fog­ lalkozott. A VTE-vizsgálatok döntően a mélyvénás ­thrombosisra (MVT) és a pulmonalis emboliára (PE) irányultak, amelyek együtt hétszer gyakrabban alakulnak ki malignus tumorban szenvedő betegekben, mint egész­ ségesekben [3]. A vizsgálatok a későbbiekben az MVT-n és a PE-n túl a központi idegrendszeri artériás thrombo­ ticus folyamatokra is irányultak. Rövidítések ASCO = (atherosclerosis, small-vessel disease, cardiac source, other cause) atherosclerosis, kisérbetegség, cardiogen eredet, egyéb ok; CI = (confidence interval) megbízhatósági tarto­ mány; CT = (computed tomography) komputertomográfia; DM = diabetes mellitus; DNS = dezoxiribonukleinsav; H3cit = citrullinált hiszton H3; HLD = hyperlipidaemia; HR = (hazard ratio) kockázati arány; MRI = (magnetic resonance imaging) mágnesesrezonancia-képalkotás; MVT = mélyvénás thrombo­ sis; NET = (neutrophil extracellular trap) neutrophil extracel­ luláris csapda; OR = (odds ratio) esélyhányados; PE = pulmo­ nalis embolia; RR = relatív rizikó; TF = (tissue factor) szöveti faktor; VTE = vénás thromboembolisatio Egy retrospektív összehasonlító vizsgálatban azt talál­ ták, hogy az artériás thrombosis (szívinfarktus és ischae­ miás stroke) szignifikánsan gyakoribb volt a daganatos betegekben, mint az illesztett daganatmentes kontroll­ csoportban [4]. Az ischaemiás stroke kockázata a daga­ nat felismerését követő első 6 hónap során tért el szigni­ fikáns mértékben a kontrolltól, de a követés második évének végére a különbség eltűnt [4]. Navi és mtsai tünetekkel járó artériás keringészavarban szenvedő betegek adatait elemezték [4], míg Graus és mtsai patológiai vizsgálata lehetőséget adott az agyi ér­ rendszeri betegségek gyakoriságának felmérésére a vér­ keringési zavar szempontjából tünetmentes daganatos betegekben is [5]. A vizsgálat során 3426, nem közpon­ ti idegrendszeri daganatos beteg 14,6%-ában (500 be­ tegben) találtak agyi érrendszeri betegséget (artériás és vénás eredetű laesiókat) [5]. Ezen 500 beteg mindössze csak felének (255/500) volt ismert korábbi tünetek alapján klinikai agyérbetegsége [5]. Ezek alapján ki­ mondható, hogy tumorokhoz társulva az agyban gyak­ ran alakulnak ki nem felismert keringési változások. A daganatok és a stroke kapcsolatának történeti áttekintése Ezenkívül fontos említe­ ni, hogy a malignus betegségekhez társuló vénás kerin­ gészavar agyinfarktust is okozhat: egyrészt paradox embolisatio révén MVT esetében nyitott foramen ovalé­ hoz társuló jobb-bal sönt mellett alakulhat ki az agyban artériás embolisatio, másrészt duralis sinus thrombosis I. Thrombosis development and shared risk factors Cancer and stroke have long been studied individually, but their detrimental forces together have also been a strong point of focus. The occurrence of both cancer and stroke in a patient is often a reflection of their similar risk factors (hypertension, hyperlipidemia, diabetes, obesity, and smoking), however, a subgroup of the cancer stroke population is believed to occur due to cancer-associated hypercoagulability. A deeper look into the cancer-associated hyperco­ agulable environment has indicated that thrombosis may be explained by cancer’s role in several factors, including activation of platelets and tissue factor, elevated expression of heparanase and influence on neutrophilic extracellular traps. When a cryptogenic stroke (stroke lacking the aforementioned risk factors) occurs due to the cancer-induced hypercoagulation state, patient serum D-dimer levels have been found elevated, and CT/MRI images of the brain have shown multivascular infarctions compared to stroke patients with traditional risk factors. Additionally, cancer treatment – chemotherapy and radiation – have also been found to increase the occurrence of cerebral vascular thrombosis. Further investigations are required to better understand cancer-associated vascular pathophysiologic changes and how to discern their unique strokes compared to strokes from other etiologies. With these insights, the prevalence of strokes in the cancer population could be decreased. Keywords: cancer-related stroke, cancer-associated hypercoagulability, cancer-associated thrombosis, pathomechan­ ism, cryptogenic stroke Hajnóczky N, Bereczki D. [The relationship between malignant tumors and ischemic stroke. I. Thrombosis develop­ ment and shared risk factors]. Orv Hetil. 2022; 163(1): 3–11. (Beérkezett: 2021. június 9.; elfogadva: 2021. augusztus 11.) 2022 ■ 163. évfolyam, 1. szám ■ 3–11. DOI: 10.1556/650.2022.32328 ■ © Szerző(k) 3 Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY vagy cerebralis vénás thrombosis is okozhat akut agyi vérkeringési zavart malignus kórképek mellett. 2022 ■ 163. évfolyam, 1. szám Az artériás occlusio lehetséges okai tumoros betegekben Prandoni hét tanulmány eredményeit foglalta össze, mindegyikben szignifikánsan gyakoribb volt az artériás cardiovascularis esemény a VTE-betegekben az egyes vizsgálatokban alkalmazott kontrollcsoporthoz képest [8]. Ezen vizsgálatok eredményei felvetették, hogy ha­ sonló mechanizmusok vagy rizikófaktorok állhatnak a vénás és az artériás thrombosis kialakulásának hátteré­ ben. Prandoni azt javasolja, hogy ismeretlen eredetű VTE esetén fontos vizsgálni, hogy van-e tünetmentes atherosclerosis [8]. Ha atherosclerosis is fennáll, akkor meg kell fontolni életmódbeli és gyógyszeres prevenció elkezdését [8]. A vizsgálatoknak négy fő limitációjuk volt: a kontrollcsoport nem egészséges személyekből állt; az adatbázis alapjául részben halotti bizonyítványok szolgáltak; a halotti bizonyítványokban a halál okaként többnyire cardiovascularis esemény szerepelt; végül, sok­ szor a szívroham diagnózisát használták halálokként a több társbetegséggel rendelkező betegekben [7]. A hiá­ nyosságok ellenére megalapozottnak tűnik a vénás és az  artériás thrombosis közötti kapcsolat feltételezése. Ismert atheroscleroticus betegek VTE-eseménye Ismert atheroscleroticus betegek VTE-eseménye A szakirodalomban az atheroscleroticus betegek VTE- hajlamára irányuló vizsgálatok eredményei nem mindig egybehangzóak. Reich és mtsai tünetmentes, de ultra­ hangvizsgálattal igazolt carotisplakkos betegekben nem találtak gyakoribb VTE-előfordulást a kontrollhoz ké­ pest (HR: 0,97, 95% CI: 0,72–1,29), viszont szignifi­ kánsan gyakoribb volt a VTE-esemény azon betegekben, akikben a követés során artériás cardiovascularis esemény (stroke vagy koszorúér-betegség) fordult elő (HR: 1,51, 95% CI: 1,01–2,25) [12]. Egy másik vizsgálatban szigni­ fikánsan gyakrabban alakult ki VTE a szívrohamon át­ esett és családi cardiovascularis előzményekkel rendelke­ ző páciensekben [13]. Sørensen és mtsai szerint stroke vagy szívroham után a betegek VTE-kockázata az első három hónapban bizonyult a legmagasabbnak [14]. Prandoni és mtsai 1996 és 2001 között 299 olyan, MVT miatt kezelt beteg adatait elemezték, akiknek nem volt tünetekkel járó atheroscleroticus betegségük [10]. A 299 betegből az MVT 153 esetben spontán jelentke­ zett, 146-nál pedig a háttérben álló ismert okok (például daganat, trauma, szülés) miatt következett be. Az MVT- betegeket korra és nemre illesztett 150 fős kontrollcso­ porthoz hasonlították. Mindkét csoportban elemezték az arteria carotisokban ultrahangvizsgálattal található tünetmentes elváltozásokat [10]. A 153 spontán MVT- beteg közül 72-ben, a másodlagos MVT-csoport 146 betege közül 40-ben, míg a 150 kontrollszemély közül 48-ban találtak legalább egy carotisplakkot [10]. A spon­ tán MVT-betegekben szignifikánsan gyakrabban fordult elő tünetmentes carotisplakk, mint (1) a másodlagos MVT-csoportban (OR: 2,3, 95% CI: 1,4–3,7) és mint (2) a kontrollcsoportban (OR: 1,8, 95% CI: 1,1–2,9) [10]. Nem volt szignifikáns különbség a másodlagos MVT-betegcsoportban a kontrollokhoz képest [10]. Többváltozós elemzésekben a következtetések nem mó­ dosultak. E vizsgálat alapján felvetődött, hogy hasonló vagy részben átfedő meghatározói lehetnek az athe­ rosclerosisnak és a vénás thrombosisnak. Az előzőek alapján elmondható, hogy VTE-betegek­ ben gyakrabban találtak atherosclerosist, viszont ismert atheroscleroticus betegekben nem volt egyértelműen gyakoribb a VTE. A fentiek alapján bizonyosnak tűnik ugyan, hogy létezik kapcsolat az artériás és a vénás ­thrombosis között, de annak pontos mechanizmusa még nem ismert. Az artériás és a vénás thrombosismechaniz­ musok (artériás: endotheliumsérülés, a vérlemezkék sti­ mulációja, a vér hemodinamikai változásai; vénás: a koa­ gulációs faktorok homeostasisváltozása, vénás pangás) részben átfedésben lehetnek egymással. A thrombosis kialakulása Napjainkban is úgy tekintünk a thrombosis kialakulására, ahogy Rudolf Virchow (1821–1902) leírta. A Virchow- triász három fontos tényezője az endothelkárosodás, a hemodinamikai változások és a hiperkoaguláció. Az egészséges endothelium, hemodinamika és koaguláció homeostasisa és harmonikus együttműködése szükséges 4 2022 ■ 163. évfolyam, 1. szám ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY ahhoz, hogy artériás vagy vénás thrombosis ne következ­ zen be. Franchini és Mannucci 15 vizsgálat elemzése során azt javasolták, hogy a klinikai gyakorlatban megfontolandó a teljes (vénás és artériás) thromboticus kockázatot figye­ lembe venni [11]. Az artériás és a vénás thrombosist korábban két külön entitásnak tartották. Az artériás thrombosis hátterében álló leggyakoribb ok az érfalkárosodás, a vérlemezkék aktiválódása és a hemodinamika megváltozása [7–9]. A vénás thrombosist elsősorban a koagulációs faktorok és a homeostasis változása váltja ki, például prokoaguláns és antifibrinolitikus hatású fehérjék aktiválódása révén [7, 8]. A neutrophil granulocyták daganat általi aktiválása A neutrophil granulocyták a leginkább a patogének ­eltüntetésében betöltött szerepükről ismertek. A neut­ rophil granulocyták az aktiválásuk során a granulumok­ ból és a sejtmagból származó alkotórészekből extra­ cellulárisan elhelyezkedő rostokat, neutrophil extracellu­ láris csapdákat (neutrophil extracellular trap, NET) hoz­ nak létre; ezek megkötik a fertőző mikroorganizmusokat, és meggátolják a terjedésüket [23]. A NET-nek az endo­ Hemodinamikai változások thelialis sejtek aktiválása révén szerepe van mind az athe­ roscleroticus plakkok, mind a thrombosis képződésében [24]. A NET-ek a macrophagok aktiválása révén tovább fokozzák az atherosclerosist a gyulladásos citokinek szint­ jének emelésével és az endothelsejtekhez történő immun­ sejtkötődés fokozásával [24]. A daganatos betegekben a tumor által szekretált granulocytakolónia-stimuláló fak­ torok emelik a neutrophilgranulocyta-számot, és a neut­ rophilek stimulálásával növelik a NET mennyiségét [25]. Intracranialis tumorokban a hemodinamikai változáso­ kat a tumor által a vérerekre gyakorolt nyomás idézi elő, ami a tumor növekedése során egyre fokozódik. A malig­ nus daganatos betegek körülbelül 25%-ában képződik agyi áttét. Az intracranialis primer tumorok és az agyi áttétek a növekedésük során nyomást gyakorolhatnak az agyi erekre, ami a normális véráramlás csökkenését okoz­ va thrombosis kialakulásához vezethet [17]. Az intracra­ nialis tumor közvetlen hatása mellett a perifokális ödéma is nyomást gyakorolhat a környező struktúrákra vagy erekre, ami szintén a lokális keringés csökkenéséhez vagy megszűnéséhez vezethet [17]. 2012-ben mutatták ki először állatkísérletekben a NET jelenlétét a kísérletesen létrehozott atheroscleroti­ cus plakkokban [26]. A NET thrombosisban és athe­ rosclerosisban betöltött lehetséges szerepének vizsgálata során a plazma-NET-szintet tükröző markerek (például extracelluláris DNS, citrullinált hiszton H3 [H3cit] és mieloperoxidáz-DNS) szintjét mérik. Stroke-betegek­ ben a stroke súlyossága kapcsolatban volt a plazma extra­ celluláris DNS-szinttel [27] és a nucleosoma- és a H3cit- arányokkal [27–29]. Kapcsolatot találtak a markerek szintje és a kórházi halálozás mértéke között is [27]. Ezek alapján azt feltételezzük, hogy a plazma-NET-alko­ tóelemek mennyisége tükrözheti a stroke súlyosságát. Thrombectomiát követően a kivett thrombusban a H3cit mindig jelen volt, amiből arra következtettek, hogy a NET-nek mindig van szerepe az ischaemiás stroke-ot okozó thrombus kialakulásában [30]. A NET mennyisége kétszer nagyobb volt a cardiogen eredetű thrombusokban, és a H3cit-szint mindig magasabb volt az 1 napnál idősebb thrombusokban [30]. A neutrophil sejtek számából is következtetni lehet a thrombus korá­ ra: magasabb volt a neutrophilszám az 1 napnál idősebb thrombusban. A thrombus eredetét (szív- vagy egyéb eredetű) jelzi annak összetétele: a magasabb fehérvér­ sejtarány a cardiogen eredet mellett szólt, és ezen esetek­ ben a thrombectomia kevésbé volt eredményes [31]. Daganat által szekretált citokinek A daganat által szekretált citokinek hatására a transz­ membrán protein TF aktiválja az extrinszik koagulációs folyamatot, ami lokális thrombosisképződéshez vezethet [16]. A heparanáz fokozott expressziója daganatokban A heparanáz egy heparin-szulfát endo-D-glükuronidáz, amely jelen van minden emlőssejtben, és fontos szerepe van az extracelluláris mátrix lebontásában és felépíté­ sében. A heparanáz szerepét a tumorsejtekben már az 1980-as évek óta vizsgálják, és ismert a kulcsszerepe a metastasisok kialakulásában [18, 19]. Fontos nonenzi­ matikus hatása van a koaguláció folyamatára: szabályozza a szöveti faktor (tissue factor, TF) expresszióját, aminek révén emelni tudja az aktivált X. faktor (Xa) mennyisé­ gét, és így aktiválja a véralvadás folyamatát [20, 21]. Ezenkívül a heparanáz befolyásolja a ’tissue factor path­ way inhibitor’ expresszióját és extracelluláris szekrécióját az emberi köldökzsinórér endothelialis sejtjeiben és em­ beri tumorsejtekben is, ilyen módon is aktiválva a vér­ alvadást [22]. Egy másik vizsgálat szerint a heparanáz fokozottan expresszálódik a mell-, a vastagbél- és a máj­ daganatokban [19]. A fentiek alapján feltételezhető, hogy a daganatok a heparanáz nonenzimatikus szerepé­ nek befolyásolásával hatnak a véralvadásra. Egy újabb vizsgálat igazolta a NET-ek szerepét meta­ stasisokban is. A plazma-NET DNS-szintje magasabb volt elsődleges emlő- és gastrointestinalis daganatok májmetastasisai esetén [32]. Felvetődött, hogy a plazma- NET szintje előre jelezheti ezen primer daganatokban a májmetastasisok kialakulására való hajlamot már a korai fázisokban is [32]. A NET-eknek tehát az atherosclero­ sisban, a thrombusképződésben és a metastasisok megje­ lenésében egyaránt leírt szerepe miatt elképzelhető, hogy a NET-eknek szerepük lehet a metastaticus tumo­ rok mellett jelentkező ischaemiás stroke-ok etiológiájá­ ban is. A malignus tumorok mellett kialakuló ischaemiás stroke patomechanizmusait az 1. ábra szemlélteti. Endotheliumsérülés és vérlemezke-aktiválás Artériás thrombosis a leggyakrabban endothelsérülésből következik [15]. Ha az atheroscleroticus plakk egy érben megreped, akkor a vérlemezke-aktiválódás thrombosist okozhat [15]. Daganatos betegekben azonban az artéri­ ás thrombosis kialakulásához plakkruptura nem szüksé­ ges, mivel a tumorsejtek olyan nekrotizáló faktorokat és interleukint is szekretálhatnak, melyek az erek endothe­ lialis felszínén gyulladásos folyamatot indítanak, throm­ bosist kiváltva [6]. Ezenkívül a daganatok direkt és indi­ rekt módon aktiválhatják a vérlemezkéket. A vérlemezkék direkt aktiválásának pontos mechanizmusa még nem is­ mert [16]. Indirekt módon történő aktiválás során a da­ ganatok szekretálhatnak egy olyan cisztein-proteázt (malignus tumorhoz társuló prokoagulánst), amely a vérlemezkék aktiválását és adhézióját okozza [15]. 5 2022 ■ 163. évfolyam, 1. szám ORVOSI HETILAP ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY 2022 ■ 163. évfolyam, 1. szám A malignus tumorok és az ischaemiás stroke rizikófaktorai A malignus tumorok és az ischaemiás stroke hátterében közös rizikófaktorok is állnak: mind a daganat, mind az agyinfarktus nagyobb valószínűséggel alakul ki olyan be­ tegekben, akiknek magas a vérnyomásuk, akik elhízot­ tak, hyperlipidaemiásak (HLD), akik dohányoznak, és 2022 ■ 163. évfolyam, 1. szám 6 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Hiperkoagulabilitás Endothelkárosodás Hemodinamikai változások Vérlemezkék aktiválódása és adhéziója Nekrotizáló faktorok Interleukinek Gyulladásos folyamat az endothelialis felszínen Thrombosis, érelzáródás Cisztein-proteáz (malignus tumorhoz társuló prokoaguláns) Intracranialis tumor Perifokális ödéma Intracranialis erek kompressziója Heparanáz Xa-szint-növekedés TF-expresszió TFPI-expresszió Keringő transzmembrán protein szöveti faktor NET Keringő tumorsejtek VIIa MALIGNUS TUMOR STROKE 1. ábra A malignus tumorok mellett kialakuló ischaemiás stroke patomechanizmusai VIIa = aktivált VII. véralvadási faktor; NET = neutrophil extracelluláris csapda; TF = szöveti faktor; TFPI = tissue factor pathway inhibitor; Xa = akti­ vált X. véralvadási faktor ÖSSZEFOGLALÓ KÖZLEMÉNY MALIGNUS TUMOR Intracranialis tumor Keringő transzmembrán protein szöveti faktor Heparanáz Interleukinek Cisztein-proteáz (malignus tumorhoz társuló prokoaguláns) Perifokális ödéma TFPI-expresszió Intracranialis erek kompressziója Xa-szint-növekedés Vérlemezkék aktiválódása és adhéziója Hemodinamikai változások 1. ábra A malignus tumorok mellett kialakuló ischaemiás stroke patomechanizmusai VIIa = aktivált VII. véralvadási faktor; NET = neutrophil extracelluláris csapda; TF = szöveti faktor; TFPI = tissue factor pathway inhibitor; Xa = akti­ vált X. véralvadási faktor VIIa = aktivált VII. véralvadási faktor; NET = neutrophil extracelluláris csapda; TF = szöveti faktor; TFPI = tissue factor pathway inhibitor; Xa = akti­ vált X. véralvadási faktor akiknek nem jól kezelt cukorbetegségük (DM) van. Ezért nehéz eldönteni, hogy a daganat és a stroke együt­ tes kialakulásának hátterében az idősebb életkor és a több közös kockázati betegség jelenléte felelős, vagy pe­ dig a daganatok és a stroke ennél közvetlenebb egymásra hatása áll a háttérben [33, 34]. Egy dél-koreai tanulmányban 161 olyan, malignus da­ ganatban szenvedő beteget vizsgáltak, akinek később stroke-ja jelentkezett [33]. A betegeket két csoportra osztották: az első csoportba a tradicionális rizikóténye­ zőkkel rendelkezők (97/161), míg a másodikba a cryp­ togen stroke betegek (fiatal, egyéb ismert betegség nél­ küliek) kerültek (64/161) [33]. A két csoportban összehasonlították a klinikai tüneteket, a diffúziósúlyo­ zott agy-MRI-képeket és a D-dimer (amely egy fib­ rindegradációs termék) értékét [33]. Átfedő rizikófaktorok Már korábban felvetődött a kérdés, hogy a daganat és a stroke együttes megjelenésének hátterében átfedő, ha­ sonló tradicionális rizikófaktorok (magas vérnyomás, HLD, elhízás, dohányzás, DM) állnak-e, vagy pedig köz­ vetlen kapcsolat van a két kórképcsoport között. Stroke és daganat együttes fennállása mellett 100 akut stroke- beteg 60%-ában magas vérnyomás, 31%-ában atheroscle­ rosis, 30%-ában DM, 26%-ában HLD fordult elő, és 45%- uk dohányzott [35]. A betegek 79%-ában voltak jelen tradicionális rizikófaktorok [35]. Ebből a visszatekintő vizsgálatból úgy tűnik, hogy daganatos betegekben dön­ tően a hagyományos rizikófaktorok tehetők felelőssé a stroke kialakulásáért, és ezért fontos ­megvizsgálni a stroke gyakoriságát olyan daganatos betegekben is, akiknek nin­ csenek tradicionális rizikófakto­raik. Egy másik vizsgálatban transcranialis ultrahanggal vizsgálták a cryptogen stroke csoportját, és szignifikáns kapcsolatot találtak az arteria cerebri mediában detektált emboliás szignál gyakorisága és a D-dimer-érték között [36]. Seok és mtsai vizsgálatának egyik fontos limitációja az egészséges kontrollcsoport hiánya volt [36]. Egy Németországban végzett retrospektív vizsgálat­ ban egy 140 fős, daganat mellett jelentkező stroke-os, valamint egy 140 fős, daganat nélküli (kontroll) stroke- os betegcsoportot hasonlítottak össze [34]. Mindkét csoportot továbbosztották az ASCO-pontszám [37] szerint: (1) tradicionális stroke etiológiájú és (2) crypto­ gen: nem ismert stroke etiológiájú csoportba [34]. A tel­ jes daganatos csoportban (tradicionális és cryptogen stroke együtt) gyakoribb volt a cryptogen típusú stroke (67/140), a többszörös agyinfarktusos és a magasabb A malignus tumorok és az ischaemiás stroke rizikófaktorai A cryptogen stroke miatt kezelt betegekben az agy-MRI-laesiók multivascu­ laris eloszlást mutattak (OR: 11,2, 95% CI: 3,74–33,3), és a betegeknek magasabb D-dimer-szintjük volt, mint a tradicionális rizikófaktorokkal rendelkező csoportnak (OR: 10,6, 95% CI: 3,29–33,8) [33]. Malignus tumorhoz társuló cryptogen stroke Az átfedő rizikófaktorok miatt több vizsgálatban össze­ hasonlították a tradicionális rizikófaktorokkal rendelke­ zők csoportját azokkal, akikben ezek nem voltak jelen (cryptogen stroke csoport). 7 2022 ■ 163. évfolyam, 1. szám ORVOSI HETILAP ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY A kemoterápia kapcsolata a vénás és az artériás thrombosissal D-dimer-szinttel rendelkező beteg, mint a kontroll- ­(daganat nélküli) csoportban (38/140, p<0,001) [34]. A daganatmentes csoportban gyakoribb volt a magas vérnyomás (p<0,05) és a HLD (p<0,01), és több tradici­ onális etiológiájú stroke-os volt (102/140), mint a da­ ganatos betegcsoportban (73/140) [34]. A daganatos stroke-os csoporton belül a cryptogen stroke-os alcso­ portban gyakoribb volt az MVT és a tüdőembolia, és magasabb volt a D-dimer-érték, mint a daganatos, tradi­ cionális etiológiájú stroke-os alcsoportban (p<0,01). A daganatok közül a tüdő- és hasnyálmirigy-daganat volt a leggyakoribb [34]. Az eredmény alapján elmondható, hogy a cryptogen stroke gyakoribb a daganatos betegek­ ben, mint a nem daganatos betegekben, és ez valószínű­ leg a paraneoplasiás hiperkoaguláció miatt van így [34]. Korábban kemoterápiás kezelést követően elsősorban a VTE előfordulását vizsgálták. Egy populációalapú eset- kontroll vizsgálatban 625 VTE-beteget és 625 VTE- mentes kontrollszemélyt hasonlítottak össze [38]. A kontrollcsoporthoz képest a VTE esélye a kemoterápiá­ val kezelt aktív malignus daganatos betegek esetében több mint hatszor (OR: 6,5, 95% CI: 2,1–20,2), a daga­ natos, de kemoterápiával nem kezeltek esetében négy­ szer nagyobb volt (OR: 4,1, 95% CI: 1,9–8,5) [38]. A  kemoterápia következményeire irányuló kutatások korlátja, hogy a statisztikák nagyrészt orvosi rekordok visszatekintő elemzésével készültek. Egy másik vizsgálat­ ban emlőrákos nőbetegek között a VTE placebokezelés mellett 0,2%-ban, tamoxifénnel kezelteknél 0,9%-ban és kemoterápiával kezelteknél 4,2%-ban fordult elő [39]. Az ANC Study Group vizsgálatában 4466 beteg közül 141 meghalt az első 4 kemoterápiás ciklus során. E 141 esetben 13 betegnél thrombosis (8 artériás és 5 vénás) volt a halál oka [40]. A malignus tumorokhoz társuló stroke etiológiájával kapcsolatos vizsgálatokat az 1. táblázatban foglaljuk ös�­ sze [33, 34, 36]. CI = 95%-os konfidenciaintervallum; HLD = hyperlipidaemia; MRI = mágnesesrezonancia-képalkotás; MVT = mélyvénás thrombosis; OR = esélyhányados; PE = pulmonalis embolia 2022 ■ 163. évfolyam, 1. szám CI = 95%-os konfidenciaintervallum; HLD = hyperlipidaemia; MRI = mágnesesrezonancia-képalkotás; MVT = mélyvénás thrombosis; OR él há d PE l li b li Kemoterápia és sugárterápia mint stroke- rizikófaktor Egyváltozós elemzésben a kemoterápiával kezelt csoportban közel kétszer nagyobb volt a stroke kockázata (HR: 1,84, 95% CI: 1,23–2,75), de a különbség eltűnt, ha többváltozós elemzésben fi­ gyelembe vették a beteg állapotának súlyosságát [41]. betegcsoportban általában nincsenek tradicionális stroke-rizikófaktorok, és a sugárterápia késői következ­ ményét nem befolyásolják az egyéb hagyományos kocká­ zati tényezők. Felnőttekben Burch és mtsai tanulmányában 83 beteg vizsgálata alapján a gliomák (72%) és a meningeomák (13%) voltak a leggyakoribb agyi daganatok, és a stroke- ok egyrészt a sugárkezelés vascularis szövődményeként (36%), illetve műtétek során (19%) következtek be, vala­ mint gyakorinak találták a stroke ismétlődését [51]. Egy 17 vizsgálatot összegző áttekintésben (N = 12 917) bevacizumabkezelés mellett több mint három­ szoros stroke-kockázatot találtak (RR: 3,22, 95% CI: 1,71–6,07) [42]. A kockázat arányos volt a bevacizumab dózisával [42], és függött a tumor típusától is: a legin­ kább a metastaticus colorectalis daganatokban fordult elő, a többi tumortípusban nem találtak szignifikáns koc­ kázatot [42]. A malignus daganatok és a stroke kapcsolatát vizsgáló összefoglaló áttekintésünk I. részében a malignus daga­ natokhoz társuló stroke patofiziológiáját és a két beteg­ ségcsoport átfedő kockázati tényezőit tekintettük át. A  dolgozat II. részében elemezzük a daganattípusok figye­lembevételével a malignus tumorok után jelentkező stroke és a stroke után felismert malignus tumorok idő­ beli jellegzetességeit; kitérünk a daganatok mellett je­ lentkező stroke akut kezelésére és a stroke-prevenció kérdéseire; és áttekintjük, hogy cryptogen stroke-ok ese­ tén mikor indokolt a tumorkeresés. Több vizsgálatban kapcsolatot találtak egyes citoszta­ tikumok alkalmazása és a stroke kialakulása között, pél­ dául ciszplatinkezelés során nem kissejtes tüdő- és ovári­ umtumorban, metotrexát adása mellett gyermekkori agyi tumorban, antiösztrogén (tamoxifén, raloxifén) al­ kalmazása mellett emlődaganatban és antiandrogén ké­ szítmények mellett prosztatadaganatban [43]. Ezen kap­ csolatokat részletesen összefoglalták [44, 45]. Tüneti morfinkezelés mellett is leírtak emelkedett stroke-kocká­ zatot prosztatadaganatos betegekben [46], ezért nem egyértelmű, hogy a stroke-kockázatért a kemoterápia vagy pedig a daganathoz társuló hiperkoagulációs hatás – esetleg mindkettő – felelős. Következtetés – Az artériás és a vénás thrombosismechanizmusok (ar­ tériás: endotheliumsérülés, a vérlemezkék stimuláció­ ja, a vér hemodinamikai változásai; vénás: a koaguláci­ ós faktorok homeostasisváltozása, vénás pangás) részben átfedésben lehetnek egymással. Kemoterápia és sugárterápia mint stroke- rizikófaktor A daganat hatásán túl a daganat kezelésének következté­ ben, azaz a kemoterápia és a radioterápia szövődménye­ ként is kialakulhat artériás thrombosis és/vagy VTE [35]. Kitano és mtsai tanulmányában 19 006, daganatos be­ teg vizsgálata során a kemoterápiás kezelésben részesülő 5887 betegből 44-nél (0,75%) jelentkezett stroke, míg a kemoterápiás kezelés nélküli csoportban 51 esetben ala­ 1. táblázat A malignus tumorokhoz társuló, tradicionális rizikófaktorokkal rendelkező és azokkal nem rendelkező (cryptogen) stroke-okat összehasonlító vizsgá­ latok összefoglalása [33, 34, 36] Szerzők és évszám Vizsgálati csoport Kontrollcsoport Vizsgált tényezők Eredmények Kim et al. 2010 [33] Tradicionális rizikótényezők nélkül (cryptogen stroke, N = 64) Tradicionális rizikótényezőkkel (n = 97) 1. Diffúziósúlyozott agyi MRI 2. Szérum-D-dimer-szint 1. Cryptogen: gyakoribb a multivascularis lokalizáció (OR: 11,2, 95% CI: 3,74–33,3) 2. Cryptogen: magasabb D-dimer- szint (OR: 10,6, 95% CI: 3,29–33,8) Seok et al. 2010 [36] Tradicionális rizikótényezők nélkül (cryptogen stroke, N = 38) Tradicionális rizikótényezőkkel (n = 36) 1. Emboliásszignál-gyako­ riság transcranialis Doppler-vizsgálattal 2. Szérum-D-dimer-szint Szignifikáns kapcsolat az embolia- szignál-gyakoriság és a szérum-D- dimer-érték között Schwarzbach et al. 2012 [34] A. Daganat mellett jelentkező stroke, tradicionális rizikófaktorok nélkül (cryptogen stroke, N = 67) B. Daganatmentes stroke-os, tradicionális rizikófaktorok nélkül (cryptogen stroke, N = 38) C. Daganat mellett jelentkező stroke, tradicionális rizikófaktorokkal (n = 73) D. Daganatmentes stroke-os, tradicionális rizikófaktorokkal (n = 102) 1. Agyinfarktusok száma 2. Szérum-D-dimer-szint 3. Magas vérnyomás, HLD 4. MVT/tüdőembolia 1. A daganatmentes csoportban gyakoribb volt a magas vérnyomás (p<0,05) és a HLD (p<0,01), és több volt a tradicionális etiológiájú stroke-os, mint a teljes daganatos csoportban 2. A teljes daganatos csoportban gyakoribb volt a cryptogen stroke, a többszörös agyinfarktusos és a magasabb D-dimer-szinttel rendelkező beteg, mint a daganatmentes csoportban 3. A daganatos stroke-os csoporton belül a cryptogen alcsoportban gyakoribb volt az MVT és a PE, és magasabb volt a D-dimer-érték (p<0,01) CI = 95%-os konfidenciaintervallum; HLD = hyperlipidaemia; MRI = mágnesesrezonancia-képalkotás; MVT = mélyvénás thrombosis; OR = esélyhányados; PE = pulmonalis embolia orokhoz társuló, tradicionális rizikófaktorokkal rendelkező és azokkal nem rendelkező (cryptogen) stroke-okat összehasonlító vizsgá­ ása [33, 34, 36] 2022 ■ 163. évfolyam, 1. szám 8 ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY kult ki stroke (0,39%) [41]. A fej- és nyakrégióra alkalmazott radioterápia A fejre és nyakra irányuló radioterápia vasculopathiát okozhat a közepes és nagy átmérőjű nyaki erekben (pél­ dául arteria carotis), felgyorsult atherosclerosis formájá­ ban [47], aminek következtében carotisszűkület és stroke jelenhet meg. Az így jelentkező ischaemiás stroke a plakkokból eredő embolisatio következménye lehet, vagy a carotisok szűkülete okozhat ischaemiát. Egy ös�­ szefoglaló áttekintés szerint a fejre és nyakra irányuló ra­ dioterápia legalább kétszeresére növeli az ischaemiás agyi vérkeringési zavar (ischaemiás stroke és tranziens ischae­ miás attak) kockázatát 2 éves vagy hosszabb követés so­ rán [48]. Bowers és mtsai a sugárterápiát követően leg­ alább 5 évvel vizsgáltak sugárterápiában részesült és daganatmentessé vált gyerekeket. Az agydaganatban nem szenvedő testvéreikhez hasonlítva a gyermekkori sugárterápiában részesülők hosszú távú stroke-kockázata jelentősen nagyobb volt (RR: 37,2; 95% CI: 17,5–79,1; P<0,0001) [49]. A sugárterápiával nem kezelt és daga­ natmentessé vált agytumoros betegekhez hasonlítva a legalább 50 Gy sugárdózisban részesült, daganatmentes­ sé vált agytumoros betegekben 3,3-szer gyakoribb volt a stroke [49]. Keene és mtsai besugárzással kezelt 244, agy­ daganatos gyermek (medián életkor 8,9 év) 4,5%-ában (11/244) találtak cerebrovascularis eseményt a sugárke­ zelést követő 8 éven belül [50]. A 11 betegből 6 esetben nagyérszűkület igazolódott [50]. A sugárkezelés követ­ kezményének vizsgálata agydaganatos gyermekekben a későbbi stroke kialakulására azért fontos, mert ebben a – A tradicionális rizikófaktorok – mint a magas vérnyo­ más, a HLD, a dohányzás, a cukorbetegség – predisz­ ponálják a betegeket mind a malignus daganatok, mind az ischaemiás stroke megjelenésére. – Ismeretlen eredetű (cryptogen) ischaemiás stroke-ok hátterében a daganat által okozott hiperkoaguláció is állhat. – Cryptogen stroke-ban gyakran található magasabb D- dimer-szint és a CT/MRI-képeken multifokális ischae­ mia; ezek felvethetik a daganat által okozott hiperko­ aguláció lehetőségét. – A malignus daganatok kezelésére adott kemoterápia és sugárterápia is okozhat artériás agyi thrombosist. – A malignus daganatok és a stroke diagnózisának idő­ rendjére és a tumorokhoz társuló stroke kezelésére a kísérő közleményben térünk ki. Anyagi támogatás: A kutatás a Fulbright-ösztöndíj tá­ mogatásával valósult meg. Anyagi támogatás: A kutatás a Fulbright-ösztöndíj tá­ mogatásával valósult meg. Anyagi támogatás: A kutatás a Fulbright-ösztöndíj tá­ mogatásával valósult meg. Szerzői munkamegosztás: H. N.: A koncepció, irodalom­ kutatás, a kézirat megfogalmazása és végleges formába hozása. B. D.: A koncepció, a kézirat egymást követő verzióinak kritikus véleményezése. A cikk végleges válto­ zatát mindkét szerző elolvasta és jóváhagyta. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. 9 2022 ■ 163. évfolyam, 1. Anyagi támogatás: A kutatás a Fulbright-ösztöndíj tá­ mogatásával valósult meg. Érdekeltségek: A szerzőknek nincsenek érdekeltségeik. Irodalom Irodalom [24]  Qi H, Yang S, Zhang L. Neutrophil extracellular traps and en­ dothelial dysfunction in atherosclerosis and thrombosis. Front Immunol. 2017; 8: 928. [1]  Sanossian N, Djabiras C, Mack WJ, et al. Trends in cancer diag­ noses among inpatients hospitalized with stroke. J Stroke Cere­ brovasc Dis. 2013; 22: 1146–1150. [2]  Trousseau A. 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A fej- és nyakrégióra alkalmazott radioterápia szám ORVOSI HETILAP ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY [23]  Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extra­ cellular traps kill bacteria. Science 2004; 303: 1532–1535. Irodalom J Neurol Transl Neurosci. 2014; 2: 1039. [38]  Heit JA, Silverstein MD, Mohr DN, et al. Risk factors for deep vein thrombosis and pulmonary embolism: a population-based case-control study. Arch Intern Med. 2000; 160: 809–815. [18]  Parish CR, Coombe DR, Jakobsen KB, et al. Evidence that sul­ phated polysaccharides inhibit tumour metastasis by blocking tumour-cell-derived heparanases. Int J Cancer 1987; 40: 511– 518. [39]  Lee AY, Levine MN. Venous thromboembolism and cancer: risks and outcomes. Circulation 2003; 107(23 Suppl 1): I17–I21. [19]  Vlodavsky I, Friedmann Y, Elkin M, et al. 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[22]  Nadir Y, Brenner B, Gingis-Velitski S, et al. Heparanase induces tissue factor pathway inhibitor expression and extracellular ac­ cumulation in endothelial and tumor cells. Thromb Haemost. 2008; 99: 133–141. [43]  Azoulay L, Yin H, Benayoun S, et al. Androgen-deprivation therapy and the risk of stroke in patients with prostate cancer. Eur Urol. 2011; 60: 1244–1250. 10 2022 ■ 163. évfolyam, 1. szám ORVOSI HETILAP ORVOSI HETILAP Brought to you by Library and Information Centre of the Hungarian Academy of Sciences MTA | Unauthenticated | Downloaded 02/21/23 08:32 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY [44]  Grisold W, Oberndorfer S, Struhal W. Stroke and cancer: a re­ view. Acta Neurol Scand. 2009; 119: 1–16. [49]  Bowers DC, Liu Y, Leisenring W, et al. Late-occurring stroke among long-term survivors of childhood leukemia and brain tu­ mors: a report from the Childhood Cancer Survivor Study. J Clin Oncol. 2006; 24: 5277–5282. Irodalom [45]  Dardiotis E, Aloizou AM, Markoula S, et al. Cancer-associated stroke: pathophysiology, detection and management. (Review.) Int J Oncol. 2019; 54: 779–796. [50]  Keene DL, Johnston DL, Grimard L, et al. Vascular complica­ tions of cranial radiation. Childs Nerv Syst. 2006; 22: 547–555. [46]  Lee CW, Muo CH, Liang JA, et al. Association of intensive mor­ phine treatment and increased stroke incidence in prostate can­ cer patients: a population-based nested case-control study. Jpn J Clin Oncol. 2013; 43: 776–781. [51]  Burch JE, Parikh NS, Kamel H, et al. Abstract TMP51: Ischemic stroke in patient with primary brain tumors: mechanisms and risk of recurrence. Stroke 2017; 48: ATMP51 [47]  Loftus CM, Biller J, Hart MN, et al. Management of radiation- induced accelerated carotid atherosclerosis. Arch Neurol. 1987; 44: 711–714. (Bereczki Dániel dr., Budapest, Balassa u. 6., 1083 e-mail: bereczki.daniel@med.semmelweis-univ.hu) [48]  Plummer C, Henderson RD, O’Sullivan JD, et al. Ischemic stroke and transient ischemic attack after head and neck radio­ therapy: a review. Stroke 2011; 42: 2410–2418. A cikk a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) feltételei szerint publikált Open Access közlemény, melynek szellemében a cikk bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető, feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. (SID_1) (Bereczki Dániel dr., Budapest, Balassa u. 6., 1083 e-mail: bereczki.daniel@med.semmelweis-univ.hu) PÁLYÁZAT PÁLYÁZAT A Prof. Dr. Romics László Akadémikus Emlékére Alapítvány pályázatot hirdet Magyarországon dolgozó, magyar állampolgárságú, 40 éven aluli orvosok és orvosbiológiai kutatással foglalkozó személyek számára. A nyertes pályázó(k) között 500 000 Ft alapítványi adomány, illetve különdíjak kerülnek kiosztásra. A pályázat célja: a klinikai gyógyítás, vagy orvosi tudományos kutatás területén dolgozók kiemelkedő tudományos tevékenységének elismerése. Előnyt élveznek azok a pályázók, akik az Alapítvány névadójának munkásságát folytatva cardiovascularis és anyag­ csere-betegségek területéről nyújtanak be pályázatot. A pályázat benyújtásának határideje: 2022. február 28. (elbírálásának határideje: 2022. április 30.) A pályázatot a palyazat@romicsalapitvany.hu e-mail címre elektronikus aláírással ellátva (ügyfélkapuval létrehozott AVDH aláírás is megfelelő), PDF formátumban kell benyújtani. A pályázatot természetes személy, saját nevében, magyar nyelven nyújthatja be, a pályázati anyag ábrák nélkül maximum 15.000 leütés (karakter) terjedelmű lehet. A pdf fájl mérete nem haladhatja meg a 25 MB-ot. A pályázathoz a fentiekhez azonos módon, külön pdf formátumú fájlban mellékelni kell rövid szakmai életrajzot, a születési idő, lakcím és telefon elérhetőségek megjelölésével. A szakmai önéletrajz végén nyilatkozni kell, hogy a pályázó a közölt személyes adatoknak a Romics Alapítvány által történő kezeléséhez hozzájárul, tudomásul veszi, hogy a Kuratórium minden tagja megismerheti adatait és pályázatát. A pályázatot papíron kinyomtatott formában nem kell megküldeni. ány adatairól, működéséről az alapítvány honlapján – www.romicsalapitvany.hu – található információ. 2022 ■ 163. évfolyam, 1. szám 11 ORVOSI HETILAP
https://openalex.org/W2074040599
https://europepmc.org/articles/pmc3213092?pdf=render
English
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The 'help' question doesn't help when screening for major depression: external validation of the three-question screening test for primary care patients managed for physical complaints
BMC medicine
2,011
cc-by
6,416
Abstract Background: Major depression, although frequent in primary care, is commonly hidden behind multiple physical complaints that are often the first and only reason for patient consultation. Major depression can be screened by two validated questions that are easier to use in primary care than the full Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria. A third question, called the ‘help’ question, improves the specificity without apparently decreasing the sensitivity of this screening procedure. We validated the abbreviated screening procedure for major depression with and without the ‘help’ question in primary care patients managed for a physical complaint. Methods: This diagnostic accuracy study used data from the SODA (for ‘SOmatisation Depression Anxiety’) cohort study conducted by 24 general practitioners (GPs) in western Switzerland that included patients over 18 years of age with at least a single physical complaint at index consultation. Major depression was identified with the full Patient Health Questionnaire. GPs were asked to screen patients for major depression with the three screening questions 1 year after inclusion. Results: Of 937 patients with at least a single physical complaint, 751 were eligible 1 year after index consultation. Major depression was diagnosed in 69/724 (9.5%) patients. The sensitivity and specificity of the two-question method alone were 91.3% (95% CI 81.4 to 96.4) and 65.0% (95% CI 61.2 to 68.6), respectively. Adding the ‘help’ question decreased the sensitivity (59.4%; 95% CI 47.0 to 70.9) but improved the specificity (88.2%; 95% CI 85.4 to 90.5) of the three-question method. Conclusions: The use of two screening questions for major depression was associated with high sensitivity and low specificity in primary care patients presenting a physical complaint. Adding the ‘help’ question improved the specificity but clearly decreased the sensitivity; when using the ‘help’ question, four out of ten patients with depression will be missed, compared to only one out of ten with the two-question method. Therefore, the ‘help’ question is not useful as a screening question, but may help discussing management strategies. The ‘help’ question doesn’t help when screening for major depression: external validation of the three-question screening test for primary care patients managed for physical complaints The ‘help’ question doesn’t help when screening for major depression: external validation of the three-question screening test for primary care patients managed for physical complaints Patrick Lombardo1, Paul Vaucher1, Nader Haftgoli1, Bernard Burnand2, Bernard Favrat3, François Verdon1, Thomas Bischoff1 and Lilli Herzig1* © 2011 Lombardo 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. Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Lombardo et al. BMC Medicine 2011, 9:114 RESEARCH ARTICLE Open Access * Correspondence: lilli.herzig@hin.ch 1Institute of General Medicine, University of Lausanne, Lausanne, Switzerland Full list of author information is available at the end of the article Background hidden behind multiple and sometimes unexplained phy- sical complaints that are often the first and only reason for patients to request consultation [8-12]. Detecting mental disorders in the presence of such complaints is thus an important challenge for general practitioners (GPs) [13]. To help GPs detect major depression, a screening tool containing two questions has been derived from the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria and validated Major depression is found in 3.9% of the general popula- tion in Europe [1] and a prevalence of 5% to 14% has been reported in primary care patients [2-6]. In a more recent meta-analysis the rate of depression was even of 17% to 19% [7]. However, major depression is commonly * Correspondence: lilli.herzig@hin.ch 1Institute of General Medicine, University of Lausanne, Lausanne, Switzerland Full list of author information is available at the end of the article Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Page 2 of 9 Page 2 of 9 enrolled (MD trainees see fewer patients) nevertheless more patients could not be included, mainly due to lan- guage barriers. GPs completed a case report form for each patient. Each patient received a self-administered ques- tionnaire that was either to be completed in the waiting room or returned by mail in the next few days. Patients were followed-up by their GPs as needed according to usual practice. The 1-year follow-up consultation took place during a scheduled visit 9-15 months after the index consultation. Patients who did not consult their physicians spontaneously during the 1-year follow-up were invited by phone to plan a visit within the next 3 months. Data col- lected during the follow-up consultation allowed the assessment of the accuracy of the screening questions in detecting major depression. [14]. These questions are simple, respectful, easy to inte- grate into the consultation, and require less time than the full DSM-IV criteria. Arroll et al. [15,16] suggested the addition of a third question called the ‘help’ question, in which the patient is asked whether they would like help regarding the issues raised by the first two screening questions. This new screening tool was reported to result in increased specificity (from 67% to 89%) not accompa- nied by decreased sensitivity (from 97% to 96%). Methods This diagnostic accuracy study was nested within a larger cohort study on the occurrence and correlations of depres- sion, anxiety, and somatoform disorders (the SODA (for ‘SOmatisation Depression Anxiety’) cohort study [17]) in primary care patients with physical complaints who were followed over 1 year. Data were collected in western French-speaking Switzerland by 21 GPs in private practice and 3 medical doctor (MD) trainees from 1 academic pri- mary care centre from November 2004 to March 2007. This study protocol was approved by the State Ethics Committee of the Canton of Vaud (Prot.100/04). Background In gen- eral, the addition of a mandatory qualifying question to a screening tool usually decreases the sensitivity and increases the specificity of the test, unless the added question is perfectly discriminatory. Since most primary care patients are usually followed by their GP for many years, we conducted a novel investiga- tion into the utility of these screening procedures over time. We examined the contribution to diagnosis of the two screening questions and the additional ‘help’ question in patients previously seen by a GP for a physical com- plaint (index consultation) and followed-up for a year. The accuracies of the two-question and three-question screen- ing methods were explored across subgroups defined by age, gender, education level, migration status, presence of other mental disorders (anxiety, somatoform disorder, alcohol abuse), and presentation of major or minor depression at the time of index consultation. The participating primary care physicians were all trained in family practice or general internal medicine and worked in primary care settings. These physicians were trained in the use of the three screening questions for major depression. GPs were allowed to investigate depres- sion only after they asked the three screening questions. Physicians were blinded to the reference standard results of both the initial and follow-up consultations, but were not necessarily blinded to the patient’s depression status. Questionnaires h d During the index and follow-up consultations, GPs read out the two screening questions for major depression: ‘During the past month have you often been bothered by feeling down, depressed, or hopeless?’ and ‘During the past month have you often been bothered by little interest or pleasure in doing things?’. Patients responding posi- tively to either of these questions were asked the ‘help’ question: ‘Is this something with which you would like help?’ with three possible responses: ‘no’, ‘yes, but not today’, or ‘yes’. These three screening questions were translated from English to French and then reverse trans- lated. Patients responding positively to either of the first two questions were considered ‘positive’ for the two screening questions. Patients who responded positively to either of the two questions and to the ‘help’ question (’yes’ or ‘yes, but not today’) were considered ‘positive’ for the three screening questions. All other patients were consid- ered ‘negative’. Results Between November 2004 and July 2005, 937 patients were included in the present study. At 1 year after inclu- sion, 751 patients agreed to be questioned (Figure 1). A total of 12 patients did not answer all PHQ questions, making it impossible to know whether they were suffer- ing from depression, and the physician did not report the results of 3 screening questions for 15 other patients. Thus, 724 patients were included in the analysis. The included patients were similar to those excluded regard- ing gender (63.3% of women in the group included vs 62.4%), age of 65 years or over (29.8% vs 25.3%), educa- tion level (79.9% vs 79.8%), and presence of major depression at the index consultation (11.3% vs 14.0%). Most patients (91.3%) were recruited from private prac- tices, with the number of patients from each practice ran- ging from 6 to 58. Patients were mainly women (63.3%) and had a mean age of 54.7 years (SD 17 years). The most frequent diagnoses for the main physical complaint were musculoskeletal (29.9%) or digestive (8.4%). In 94 patients (13%), a mental disorder was considered to be related to the initial physical complaint. During the year of follow-up, 83.1% of patients visited their GP at least once, and 40.4% received psychotherapeutic care from their GP. Psychotropic drugs were used by 34.2% of the patients and 8.1% were referred to either a psychiatrist or a psychologist. At 1 year after the index consultation the prevalence of major depression was of 9.5%. Anxiety, somatoform disorder, alcohol abuse and expo- sure to psychosocial stressors were assessed with PHQ questions. Patients were considered to be exposed to a psychosocial stressor if they reported being bothered a lot by at least one of the ten stressors assessed with question 12 of the full PHQ [18] (1, health; 2, weight or appearance; 3, having little or no sexual desire or pleasure during sex; 4, difficulties with husband/wife, partner/lover or boy- friend/girlfriend; 5, the stress of taking care of children, parents or other family members; 6, stress at work or out- side of the home or at school; 7, financial problems or worries; 8, having no one to turn to when having a pro- blem; 9, something bad that happened recently; 10, think- ing or dreaming about something terrible that happened in the past). Sociodemographic questions included age, gender, and nationality (dichotomised into Swiss or non- Swiss). Patients and follow-up This study, conducted 1 year after the index consultation, included consenting patients aged 18 years and over who presented with at least 1 physical complaint during the index consultation at 1 of 22 recruiting centres. Patients with vital emergencies, dementia, intellectual deficiency, inability to understand French, or acute psychiatric dis- eases that prevented the patient from answering appropri- ately were excluded. The GPs included one patient per each half-day of consultation. To minimise selection bias, patients eligible for inclusion were selected by each GP using a pre-established, daily, randomised rank order list, thus defining each eligible patient for every half-day. In the academic primary care centre all eligible patients were After the consultation, the patients independently com- pleted the reference standard questionnaire (full Patient Health Questionnaire (PHQ)) [3,18,19], a validated French version of the self-reported Primary Care Evaluation of Mental Disorders (PRIME-MD) [20] questionnaire. This questionnaire was designed to detect mental disorders in primary care practice, including depression, anxiety, alco- hol abuse, and eating and somatoform disorders. To clas- sify whether patients had major depression, we used nine questions corresponding to DSM-IV criteria (questions 2a Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Page 3 of 9 compared between patients included and those excluded from the analysis to assess potential selection bias. to 2i) [18]. Patients who responded positively to at least one of the first two screening questions and to five or more of the nine questions were considered to have major depressive syndrome. Minor depression was considered present when three or four of the nine questions were answered positively and at least one of the two core questions. Results Professional education included eight categories summarised in a dichotomised variable: presence or absence of fully achieved training beyond compulsory school. Questionnaires were sent to the data centre, and all vari- ables were double entered and checked. A researcher, blinded to index consultation results, determined which patients presented PHQ criteria for major depression. The depression screening test administered by GPs was completed on the same day as the reference test (PHQ) by 59.3% and within 1 week by additional 25% of patients. Physicians did not report any adverse effects of using the three screening questions. GPs did not report an answer to the ‘help’ question in five patients (0.7%). Statistical methods The sample size necessary to obtain a 10%-wide interval around a 70% expected sensitivity (a = 0.05) was calcu- lated, assuming a 10% prevalence of major depression. The expectation of 20% loss to follow-up led to a total of 947 patients required for inclusion, a figure that was rounded to 1,000 patients. The sensitivity and specificity of the two screening questions were 91.3% (95% CI 81.4 to 96.4) and 65.0% (95% CI 61.2 to 68.6), respectively (Table 1). Adding the ‘help’ question improved the specificity to 88.2% (95% CI 85.4 to 90.5), but the sensitivity decreased to 59.4% (95% CI 47.0 to 70.9). In fact, 118 (40.4%) of the patients initi- ally screened positive for depression (N 292) were willing to accept help (Figure 2). Considering the patients who were not already being treated for major depression only, the sensitivity and the specificity of the two-question method are, respectively, 84.6% (95% CI 54.6 to 98.1) and 76.8% (95% CI 72.0 to 81.2). For the three-question method the sensitivity decreased to 46.2% (95% CI 19.2 to 74.9) and the specificity increased to 94.5% (95% CI 91.5 to 96.7). Sensitivity, specificity, positive and negative likelihoods, and predictive values were calculated, with their respective 95% confidence intervals (95% CIs), to determine screen- ing test accuracy. Sensitivity, specificity, and 95% CIs were also calculated for subpopulations stratified by age, gender, nationality, education level, anxiety, somatoform disorder, depression status at the index consultation, and exposure to a psychosocial stressor. Although these variables were predefined before analysis, this study was not sufficiently powerful to detect significant clinical differences between subgroups. The effects of these factors on the screening method were estimated by likelihood ratio test comparing logistic regression models with or without an interaction term. Characteristics of the patients (age, gender, level of education, and depression at index consultation) were We next explored the sensitivity and specificity of both screening instruments in various patient subpopulations Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Page 4 of 9 (Table 2). Discussion In primary care patients well known by their GPs, the two-question screening method for major depression dis- played high sensitivity (91%) and low specificity (65%). As suspected, adding the ‘help’ question led to a decreased sensitivity (59%) but a higher specificity (88%). We also observed a lower specificity for the two-question and three-question methods in subpopulations with other psychiatric conditions (such as generalised anxiety) and in patients who had exhibited major depression 1 year previously. y g j p p Introduction of the third ‘help’ question was a very interesting and logical proposition, and should have facilitated the diagnosis of major depression. When we added the ‘help’ question to the screening method, how- ever, our observations were substantially different from those of Arroll et al., [15] who reported increased specifi- city (89%) but identical sensitivity (96%). As an important number of their patients with major depression responded ‘no’ to the ‘help’ question, it is not clear why the sensitivity remained identical. In a second study, Goodyear et al. [16] validated the two-question and three-question methods using the PHQ-9 as a reference standard for major depression. Although the two-ques- tion method was associated with a sensitivity of 98% and a specificity of 73%, and the specificity of the three-ques- tion method questions was reported to be 99%, the sensi- tivity of the three-question method was not provided. A recent publication by the same authors determines a sensitivity of 99.2% and a specificity of 70.4% for the two- question method, whereas the sensitivity decreased to 87.1% and the specificity increased to 94.8% for the three-question method [26]. The strengths of our study are its large sample size, the number and diversity of the participating GPs, and the use of standardised, validated measures for mental disor- ders. Furthermore, the random selection of patients and their recruitment from a large number of GPs in various settings decreased the risk of selection bias. We therefore believe that our observations are relevant for most patients with physical complaints in primary care in developed countries. However, our study is limited because the two screening questions for major depression were similar to those of the PHQ-9, our reference stan- dard. Therefore, the sensitivity of the screening method is expected to be very high. Statistical methods BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Table 1 Sensitivity, specificity, positive/negative predictive values, positive/negative likelihood ratios for major depression Parameter Two screening questions % (95% CI) Three screening questions % (95% CI) Sensitivity 91.3% (81.4 to 96.4) 59.4% (47.0 to 70.9) Specificity 65.0% (61.2 to 68.6) 88.2% (85.4 to 90.5) Positive predictive value 21.6% (17.1 to 26.8) 34.7% (26.4 to 44.1) Negative predictive value 98.6% (96.8 to 99.4) 95.3% (93.3 to 96.8) Positive likelihood ratio 2.6 (2.3 to 3.0) 5.0 (3.8 to 6.7) Negative likelihood ratio 0.1 (0.06 to 0.28) 0.5 (0.3 to 0.6) Table 1 Sensitivity, specificity, positive/negative predictive values, positive/negative likelihood ratios for major depression sitivity, specificity, positive/negative predictive values, positive/negative likelihood ratios for majo ositive/negative predictive values, positive/negative likelihood ratios for major stressor during the 4 previous weeks, or who were diag- nosed with either anxiety or somatoform disorder were more likely to answer positively to each screening instru- ment without being diagnosed as having depression, as indicated by a lower specificity (Table 2). specificities (57% and 67%, respectively). Löwe et al. [25] evaluated the two screening questions in outpatients and obtained similar results with a dichotomous answer (yes/ no). Furthermore, the two-question method was able to detect changes in a patient’s state of depression. Here we report observations similar to those of Arroll et al. [24] regarding screening for major depression with two ques- tions. The high sensitivity of these questions allows GPs to securely rule out negative patients, but the relatively low specificity requires further investigations to confi- dently diagnose major depression in positive cases [14]. Statistical methods The sensitivity of the two-question method was high and consistent through the entire population ran- specificity of both screening instruments exhibited important disparities across patients with various mental Eligible patients N=1020 Refused to participate N=83 Consent to participate N=937 Follow-up patients at one year N=751 Patients with data completed enough for our analysis N=724 Lost at follow-up N=186 • 10 patients died • 44 of them were unreachable • 31 CRFs were not completed by physicians without any given explanation • 17 refused to answer questions at one year • 84 Questionnaire was not returned by the patient Missing data N=27 • 1 Missing questionnaire from the GP • 12 patients with missing data for the diagnostic of depresion • 14 no response were given about the screening questions Figure 1 Flowchart of eligible patients. Eligible patients N=1020 Refused to participate N=83 Consent to participate N=937 Lost at follow-up N=186 • 10 patients died • 44 of them were unreachable • 31 CRFs were not completed by physicians without any given explanation • 17 refused to answer questions at one year • 84 Questionnaire was not returned by the patient Follow-up patients at one year N=751 Patients with data completed enough for our analysis N=724 specificity of both screening instruments exhibited important disparities across patients with various mental states. Patients who suffered from depression at the index consultation, who were exposed to a psychosocial (Table 2). The sensitivity of the two-question method was high and consistent through the entire population, ran- ging from 80% (95% CI 51.3 to 94.6) in patients older than 65 years to 100% (95% CI 83.4 to 100) in men. The Page 5 of 9 Page 5 of 9 Lombardo et al. Discussion Finally, the PHQ-9 may not be the best reference standard for major depression for the following three reasons: (1) it is self-report, (2) it doesn’t apply exclusion criteria, and (3) it doesn’t apply clinical significance criteria. Thus PHQ-9 can only be interpreted as a proxy of DSM-IV [21,22]. Therefore a standardised visit to a psychiatrist would have been preferred. An independent study by Baker-Glenn et al. [27] observed a sensitivity of 23.7% and specificity 97.8% in patients attending chemotherapy with the three-question method. We therefore believe Arrol et al.’s [15] results to be misleading. These findings support the latest NICE [28] guidelines that recommend only the use of the two screening questions. Whooley et al. [23] and Arroll et al. [24] first intro- duced the two-question screening method and reported high sensitivities (96% and 97%, respectively) and low Page 6 of 9 Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Our analysis indicates that although the three-ques- tion method has high negative predictive value, the high false negative rate implies that as many as four patients out of ten (28/69) with major depression would not be correctly diagnosed with this method. In comparison, less than one out of ten patients (6/69) with major depression will not be diagnosed when using the two- question method. It is therefore not helpful to include Patients with physical complaint N=724 « No » to both screening questions « Yes » to at least one of the screening questions Negative to the two-screening questions n= 432 Positive to the two-screening questions n= 292 n % Major depression 6 1.4% Minor depression 10 2.3% No depression 416 96.3% Negative to the three-screening questions n= 606 Negative to the Help question n= 174 Positive to the three-screening questions n= 118 « Yes » to the help question « No » to the help question n % Major depression 28 4.6% Minor depression 41 6.8% No depression 537 88.6% n % Major depression 63 21.6% Minor depression 47 16.1% No depression 182 62.3% n % Major depression 41 34.7% Minor depression 16 13.6% No depression 61 51.7% Figure 2 Flowchart of screening. Patients with physical complaint N=724 Our analysis indicates that although the three-ques- tion method has high negative predictive value, the high false negative rate implies that as many as four patients out of ten (28/69) with major depression would not be correctly diagnosed with this method. Discussion the third ‘help’ question to rule out major depression in patients well known by their GPs. But as Kroenke [29] suggests, ‘screening for depression is not enough’. Patients identified with depression have to be treated. Therefore the ‘help’ question remains clinically relevant, even if more than half of patients with major depression did not ask for help. But within the context of the con- sultation, the ‘help’ question enables a continuing dis- cussion about mood disorders and allows evaluation of the appropriateness of a psychiatric treatment and refer- ral. Baker-Glenn et al. conclude, as we do, that the ‘help’ question may highlight patients willing to accept support [27]. This also underlines GPs’ role in investi- gating and answering patient expectations for their psy- chological distress as described by Walters showing that patients with milder symptoms usually prefer simple human contact, and informal resource rather than for- mal interventions or medication [30]. While all these questions may help GPs screen for major depression in their patients, this tool should not replace clinical the third ‘help’ question to rule out major depression in patients well known by their GPs. But as Kroenke [29] suggests, ‘screening for depression is not enough’. Patients identified with depression have to be treated. Therefore the ‘help’ question remains clinically relevant, even if more than half of patients with major depression did not ask for help. But within the context of the con- sultation, the ‘help’ question enables a continuing dis- cussion about mood disorders and allows evaluation of the appropriateness of a psychiatric treatment and refer- ral. Baker-Glenn et al. conclude, as we do, that the ‘help’ question may highlight patients willing to accept support [27]. This also underlines GPs’ role in investi- gating and answering patient expectations for their psy- chological distress as described by Walters showing that patients with milder symptoms usually prefer simple human contact, and informal resource rather than for- mal interventions or medication [30]. While all these questions may help GPs screen for major depression in their patients, this tool should not replace clinical Our observations suggest that the sensitivity of the two screening questions is consistent across various patient subpopulations guaranteeing a low number of false nega- tives regardless of patient characteristics. However, as the specificity differs across patients, GPs may frequently and falsely diagnose major depression in patients who present other mental disorders. Discussion In comparison, less than one out of ten patients (6/69) with major depression will not be diagnosed when using the two- question method. It is therefore not helpful to include correctly diagnosed with this method. In comparison, less than one out of ten patients (6/69) with major depression will not be diagnosed when using the two- question method. It is therefore not helpful to include Page 7 of 9 Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Page 7 of 9 Table 2 Stratified specificity of screening questions for major depression Characteristic Prevalence of depression, % (95% CI) Specificity, % (95% CI) Two screening questions Three screening questions Overall 9.5% (7.6 to 12.0) 65.0% (61.2 to 68.6) 88.2% (85.4 to 90.5) Gender Male 9.4% (6.3 to 13.7) 69.2% (63.0 to 75.0) 90.0% (85.4 to 93.4) Female 9.6% (7.1 to 12.8) 62.5% (57.7 to 67.2) 87.2% (83.5 to 90.2) Age < 65 years 10.9% (8.4 to 14.1) 66.1% (61.4 to 70.5) 87.4% (83.8 to 90.3) ≥65 years 6.4% (3.8 to 10.6) 62.8% (56.0 to 69.2) 89.9% (84.9 to 93.4) Nationality Swiss 8.0% (6.0 to 10.6) 67.6% (63.3 to 71.5) 89.1% (86.0 to 91.5) Not Swiss 14.8% (9.4 to 22.5) 53.2% (43.4 to 62.7) 84.4% (75.9 to 90.4) Education level Professional training 9.4% (71.5 to 12.3) 66.8% (62.4 to 70.9) 88.3% (85.1 to 91.0) No professional training 9.0% (5.0 to 15.1) 60.6% (51.7 to 68.8) 88.6% (81.7 to 93.3) Psychosocial stressors ≥1 major stressor 21.5% (16.9 to 26.9) 44.3% (37.6 to 51.1) 77.6% (71.4 to 82.8) No major stressor 2.0% (1.0 to 4.0) 76.0% (71.7 to 79.9) 93.7% (90.9 to 95.7) Mood disorders 1 year previously Major depression 39.0% (28.6 to 50.4) 34.0% (21.6 to 48.8) 62.0% (47.1 to 75.0) Minor depression 15.0% (7.5 to 27.1) 43.1% (29.6 to 57.7) 84.3% (70.8 to 92.5) No depression 39.1% (25.2 to 59.5) 70.1% (66.0 to 73.9) 91.1% (88.3 to 93.3) Anxiety Anxiety syndrome 60.0% (45.2 to 73.2) 5% (0.2 to 26.9) 40.0% (20.0 to 63.6) No anxiety 57.1% (41.2 to 78.3) 67.1% (63.3 to 70.8) 89.8% (87.1 to 92.0) Somatoform disorder ≥3 symptoms 31.7% (22.1 to 43.0) 46.4% (33.2 to 60.1) 67.9% (53.9 to 79.4) < 3 symptoms 6.7% (5.0 to 9.0) 67.7% (62.7 to 70.4) 90.1% (87.4 to 92.3) Table 2 Stratified specificity of screening questions for major depression judgment; indeed, GPs seldom rely on questionnaires alone [31,32]. Discussion Additional studies are necessary to quantify the actual benefits of screening mental disorders in primary care with the two-question and three-question screening methods. Authors’ contributions LH, BF, FV, and BB participated in the conception and design of the study. NH, LH, FV, and TB collected data. NH monitored data collection and completed missing data, PL, PV, LH, and BF planned and analysed the data, PL, PV, BF, and LH participated in data interpretation, drafting, and revising the manuscript. NH, BB, BF, FV, and TB reviewed the manuscript. LH is the guarantor of the paper. All authors read and approved the final manuscript. 16. Goodyear-Smith F, Arroll B, Coupe N: Asking for help is helpful: validation of a brief lifestyle and mood assessment tool in primary health care. Ann Fam Med 2009, 7:239-244. 17. Haftgoli N, Favrat B, Verdon F, Vaucher P, Bischoff T, Burnand B, Herzig L: Patients presenting with somatic complaints in general practice: depression, anxiety and somatoform disorders are frequent and associated with psychosocial stressors. BMC Fam Pract 2010, 11:67. Conclusions The two-question screening method for major depression exhibited a high sensitivity and a low specificity when applied to well known primary care patients with a physi- cal complaint. Adding the ‘help’ question improved the specificity of the test, but clearly decreased its sensitivity: four out of ten patients will thus be missed with the Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Page 8 of 9 Page 8 of 9 6. Norton J, De Roquefeuil G, Boulenger JP, Ritchie K, Mann A, Tylee A: Use of the PRIME-MD Patient Health Questionnaire for estimating the prevalence of psychiatric disorders in French primary care: comparison with family practitioner estimates and relationship to psychotropic medication use. Gen Hosp Psychiatry 2007, 29:285-293. three-question method, compared to only one out of ten with the two-question method. Although the ‘help’ ques- tion is not useful as a screening question in this patient group, it may facilitate discussion about mood disorders and its management. 7. Mitchell AJ, Vaze A, Rao S: Clinical diagnosis of depression in primary care: a meta-analysis. Lancet 2009, 374:609-619. 8. Kroenke K, Jackson JL, Chamberlin J: Depressive and anxiety disorders in patients presenting with physical complaints: clinical predictors and outcome. Am J Med 1997, 103:339-347. Acknowledgements W h k ll h i i We thank all physicians who participated in the present study: Dr C Bonnard, Dr M Bonnard, Dr J-P Bussien, Dr C Chapuis, Dr G Conne, Dr M Dafflon, Dr M Danese, Dr M De Vevey, Dr C Dvorak, Dr M Junod, Dr G Lorenz, Dr A Michaud, Dr N Mühlemann, Dr F Pilet, Dr P-A Schmied, Dr A Schwob, Dr J-P Studer, Dr M Wenner, and Dr K Würzner. We also thank Françoise Secretan and Dr B Chiarini for data management. 9. Haug TT, Mykletun A, Dahl AA: The association between anxiety, depression, and somatic symptoms in a large population: the HUNT-II study. Psychosom Med 2004, 66:845-851. 10. Simon GE, VonKorff M, Piccinelli M, Fullerton C, Ormel J: An international study of the relation between somatic symptoms and depression. N Engl J Med 1999, 341:1329-1335. This study was supported and financed by the Department of Ambulatory Care and Community Medicine, University of Lausanne, Switzerland and by a grant from the Swiss Academy of Medical Sciences (Projekt RRMA 2/04). The sponsors were not involved in data collection and analysis, or in writing or editing the manuscript. 11. Kroenke K, Spitzer RL, Williams JB, Linzer M, Hahn SR, de Gruy FV, Brody D: Physical symptoms in primary care. Predictors of psychiatric disorders and functional impairment. Arch Fam Med 1994, 3:774-779. 12. Ohayon MM, Schatzberg AF: Using chronic pain to predict depressive morbidity in the general population. Arch Gen Psychiatry 2003, 60:39-47. 13. Simon GE, Goldberg SD, Tiemens BG, Ustun TB: Outcomes of recognized and unrecognized depression in an international primary care study. Gen Hosp Psychiatry 1999, 21(2):97-105. Competing interests All authors had full access to all data (including statistical reports and tables) and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors declare that they have no competing interests. y 18. Deployment Health Clinical Center: Full Patient Health Questionnaire (English).[http://www.pdhealth.mil/guidelines/downloads/appendix2.pdf]. 19. Kroenke K, Spitzer RL, Williams JB: The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med 2001, 16:606-613. ll k d h d Received: 22 May 2011 Accepted: 18 October 2011 Published: 18 October 2011 Received: 22 May 2011 Accepted: 18 October 2011 Published: 18 October 2011 20. Spitzer RL, Williams JB, Kroenke K, Linzer M, de Gruy FV, Hahn SR, Brody D, Johnson JG: Utility of a new procedure for diagnosing mental disorders in primary care. The PRIME-MD 1000 study. JAMA 1994, 272:1749-1756. y y 21. Wittkampf KA, Naeije L, Schene AH, Huyser J, van Weert HC: Diagnostic accuracy of the mood module of the Patient Health Questionnaire: a systematic review. Gen Hosp Psychiatry 2007, 29:388-395. Author details 1I tit t f G 1Institute of General Medicine, University of Lausanne, Lausanne, Switzerland. 2Clinical Epidemiology Centre, Institute of Social and Preventive Medicine, University of Lausanne, Lausanne, Switzerland. 3Department of Ambulatory Care and Community Medicine, University of Lausanne, Lausanne, Switzerland. 1Institute of General Medicine, University of Lausanne, Lausanne, Switzerland. 2Clinical Epidemiology Centre, Institute of Social and Preventive Medicine, University of Lausanne, Lausanne, Switzerland. 3Department of Ambulatory Care and Community Medicine, University of Lausanne, Lausanne, Switzerland. 1Institute of General Medicine, University of Lausanne, Lausanne, Switzerland. 2Clinical Epidemiology Centre, Institute of Social and Preventive Medicine, University of Lausanne Lausanne Switzerland 3Department of Ambulatory 14. Mitchell AJ, Coyne JC: Do ultra-short screening instruments accurately detect depression in primary care? A pooled analysis and meta-analysis of 22 studies. Br J Gen Pract 2007, 57:144-151. 15. Arroll B, Goodyear-Smith F, Kerse N, Fishman T, Gunn J: Effect of the addition of a “help” question to two screening questions on specificity for diagnosis of depression in general practice: diagnostic validity study. BMJ 2005, 331:884. References 1 Al J 1. Alonso J, Angermeyer MC, Bernert S, Bruffaerts R, Brugha TS, Bryson H, de Girolamo G, Graaf R, Demyttenaere K, Gasquet I, Haro JM, Katz SJ, Kessler RC, Kovess V, Lépine JP, Ormel J, Polidori G, Russo LJ, Vilagut G, Almansa J, Arbabzadeh-Bouchez S, Autonell J, Bernal M, Buist- Bouwman MA, Codony M, Domingo-Salvany A, Ferrer M, Joo SS, Martínez- Alonso M, et al: 12-Month comorbidity patterns and associated factors in Europe: results from the European Study of the Epidemiology of Mental Disorders (ESEMeD) project. Acta Psychiatr Scand Suppl 2004, 420:28-37. 22. Wittkampf K, van Ravesteijn H, Baas K, van de Hoogen H, Schene A, Bindels P, Lucassen P, van de Lisdonk E, van Weert H: The accuracy of Patient Health Questionnaire-9 in detecting depression and measuring depression severity in high-risk groups in primary care. Gen Hosp Psychiatry 2009, 31:451-459. 23. Whooley MA, Avins AL, Miranda J, Browner WS: Case-finding instruments for depression. Two questions are as good as many. J Gen Intern Med 1997, 12:439-445. 2. Serrano-Blanco A, Palao DJ, Luciano JV, Pinto-Meza A, Lujan L, Fernandez A, Roura P, Bertsch J, Mercader M, Haro JM: Prevalence of mental disorders in primary care: results from the diagnosis and treatment of mental disorders in primary care study (DASMAP). Soc Psychiatry Psychiatr Epidemiol 2010, 45:201-210. 24. Arroll B, Khin N, Kerse N: Screening for depression in primary care with two verbally asked questions: cross sectional study. BMJ 2003, 327:1144-1146. 25. Lowe B, Kroenke K, Grafe K: Detecting and monitoring depression with a two-item questionnaire (PHQ-2). J Psychosom Res 2005, 58:163-171. 3. Spitzer RL, Kroenke K, Williams JB: Validation and utility of a self-report version of PRIME-MD: the PHQ primary care study. Primary Care Evaluation of Mental Disorders. Patient Health Questionnaire. JAMA 1999, 282:1737-1744. 3. Spitzer RL, Kroenke K, Williams JB: Validation and utility of a self-report version of PRIME-MD: the PHQ primary care study. Primary Care Evaluation of Mental Disorders. Patient Health Questionnaire. JAMA 1999, 282:1737-1744. 26. Mohd-Sidik S, Arroll B, Goodyear-Smith F, Zain AM: Screening for depression with a brief questionnaire in a primary care setting: validation of the two questions with help question (Malay version). Int J Psychiatry Med 2011, 41:143-154. 4. Ansseau M, Dierick M, Buntinkx F, Cnockaert P, De Smedt J, Van Den Haute M, Vander Mijnsbrugge D: High prevalence of mental disorders in primary care. J Affect Disord 2004, 78:49-55. 4. References 1 Al J Ansseau M, Dierick M, Buntinkx F, Cnockaert P, De Smedt J, Van Den Haute M, Vander Mijnsbrugge D: High prevalence of mental disorders in primary care. J Affect Disord 2004, 78:49-55. 27. Baker-Glenn EA, Park B, Granger L, Symonds P, Mitchell AJ: Desire for psychological support in cancer patients with depression or distress: validation of a simple help question. Psychooncology 2011, 20:525-531. 5. Ansseau M, Fischler B, Dierick M, Mignon A, Leyman S: Prevalence and impact of generalized anxiety disorder and major depression in primary care in Belgium and Luxemburg: the GADIS study. Eur Psychiatry 2005, 20:229-235. 28. National Collaborating Centre for Mental Health: Nice clinical guidelines 90; Depression: the treatment and management of depression in adults Page 9 of 9 Page 9 of 9 Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 Lombardo et al. BMC Medicine 2011, 9:114 http://www.biomedcentral.com/1741-7015/9/114 (partial update of NICE clinical guideline 23).[http://www.nice.org.uk/ CG90]. 29. Kroenke K: Depression screening is not enough. Ann Intern Med 2001, 134:418-420. 30. Walters K, Buszewicz M, Weich S, King M: Help-seeking preferences for psychological distress in primary care: effect of current mental state. Br J Gen Pract 2008, 58:694-698. 31. Dowrick C, Leydon GM, McBride A, Howe A, Burgess H, Clarke P, Maisey S, Kendrick T: Patients’ and doctors’ views on depression severity questionnaires incentivised in UK quality and outcomes framework: qualitative study. BMJ 2009, 338:b663. 32. Kendrick T, Dowrick C, McBride A, Howe A, Clarke P, Maisey S, Moore M, Smith PW: Management of depression in UK general practice in relation to scores on depression severity questionnaires: analysis of medical record data. BMJ 2009, 338:b750. 32. Kendrick T, Dowrick C, McBride A, Howe A, Clarke P, Maisey S, Moore M, Smith PW: Management of depression in UK general practice in relation to scores on depression severity questionnaires: analysis of medical record data. BMJ 2009, 338:b750. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1741-7015/9/114/prepub doi:10.1186/1741-7015-9-114 Cite this article as: Lombardo et al.: The ‘help’ question doesn’t help when screening for major depression: external validation of the three- question screening test for primary care patients managed for physical complaints. BMC Medicine 2011 9:114. doi:10.1186/1741-7015-9-114 Cite this article as: Lombardo et al.: The ‘help’ question doesn’t help when screening for major depression: external validation of the three- question screening test for primary care patients managed for physical complaints. 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Редакционный совет: Б.А. Байтанаев – академик НАН РК, доктор исторических наук (Алматы, Казахстан) (председатель), Х.А. Амирханов – академик РАН, доктор исторических наук, профессор (Москва, Россия), С.Г. Бочаров – кандидат исторических наук (Севастополь, Россия), П. Георгиев – доктор наук, доцент (Шумен, Болгария), Е.П. Казаков – доктор истори- ческих наук (Казань, Россия), Н.Н. Крадин – член-корреспондент РАН, доктор исто- рических наук, профессор (Владивосток, Россия), А. Тюрк – PhD (Будапешт, Венгрия), А.А. Тишкин – доктор исторических наук профессор (Барнаул, Россия), В.С. Синика – кандидат исторических наук (Тирасполь, Молдова), Б.В. Базаров – академик РАН, доктор исторических наук, профессор (Улан-Удэ, Россия), Д.С. Коробов – доктор исторических наук, профессор РАН (Москва, Россия), О.В. Кузьмина – кандидат исторических наук (Самара, Россия), П. Дегри – профессор (Лёвен, Бельгия), Вэй Джан – Ph.D, профессор (Пекин, Китай). ПОВОЛЖСКАЯ АРХЕОЛОГИЯ e-ISSN 2500-2856 № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ Главный редактор член-корреспондент АН РТ, доктор исторических наук А.Г. Ситдиков Executive Editors: B. A. Baitanayev – Academician of the Nacional Academy of the RK, Doctor of Historical Sci- ences (Almaty, Republic of Kazakhstan) (chairman), Kh. A. Amirkhanov – Academician of RAS, Doctor of Historical Sciences, Professor (Moscow, Russian Federation), S. G. Bocharov – Candidate of Historical Sciences (Sevastopol, Russian Federation), P. Georgiev – Doctor of Historical Scienc- es (Shumen, Bulgaria), E. P. Kazakov – Doctor of Historical Sciences (Kazan, Russian Federation), N. N. Kradin – Doctor of Historical Sciences, Corresponding Member of the Russian Academy of Sci- ences (Vladivostok, Russian Federation), А. Türk – PhD (Budapest, Hungary), A.A. Tishkin – Doctor of Historical Sciences, Professor (Barnaul, Russian Federation), V. S. Sinika – Candidate of Historical Sciences (Tiraspol, Moldova), B. V. Bazarov – Academician of RAS, Doctor of Historical Sciences, Professor (Ulan-Ude, Russian Federation), D. S. Korobov – Doctor of Historical Sciences, Professor (Moscow, Russian Federation), O. V. Kuzmina – Candidate of Historical Sciences (Samara, Russian Federation), P. Degryse – Professor (Leuven, Belgium), Wei Jian – Ph.D, Professor (Beijing, China). Deputy Chief Editors: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences F. Sh. Khuzin Doctor of Historical Sciences Yu. A. Zeleneev Executive Secretary – Candidate of Veterinary Sciences G. Sh. Asylgaraeva Deputy Chief Editors: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences F. Sh. Khuzin Doctor of Historical Sciences Yu. A. Zeleneev Executive Secretary – Candidate of Veterinary Sciences G. Sh. Asylgaraeva Deputy Chief Editors: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences F. Sh. Khuzin Doctor of Historical Sciences Yu. A. Zeleneev Executive Secretary – Candidate of Veterinary Sciences G. Sh. Asylgaraeva POVOLZHSKAYA ARKHEOLOGIYA THE VOLGA RIVER REGION ARCHAEOLOGY e-ISSN 2500-2856 № 2 (40) 2022 Editor-in-Chief: Corresponding Member of the Tatarstan Academy of Sciences, Doctor of Historical Sciences A. G. Sitdikov © Академия наук Республики Татарстан, 2022 © ФГБОУ ВО «Марийский государственный университет», 2022 © Журнал «Поволжская археология», 2022 А.А. Выборнов – доктор исторических наук, профессор (Самара, Россия) М.Ш. Галимова – кандидат исторических наук (Казань, Россия) Р.Д. Голдина – доктор исторических наук, профессор (Ижевск, Россия) С.В. Кузьминых – кандидат исторических наук (Москва, Россия) А.Е. Леонтьев – доктор исторических наук (Москва, Россия) Т.Б. Никитина – доктор исторических наук (Йошкар-Ола, Россия) А.А. Чижевский – кандидат исторических наук (Казань, Россия) Ответственный за выпуск: Ф.Ш. Хузин – доктор исторических наук Адрес редакции: 420012 г. Казань, ул. Бутлерова, 30 Телефон: (843) 236-55-42 E-mail: arch.pov@mail.ru http://archaeologie.pro Индекс ПП753, электронный Каталог печатных изданий "ПОЧТА РОССИИ" Выходит 4 раза в год Издательство «Фəн» Казань, Татарстан Издательство «Фəн» Казань, Татарстан ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 Editorial Board: A. A. Vybornov – Doctor of Historical Sciences, Professor (Samara State Academy of Social Sciences and Humanities, Samara, Russian Federation) A. A. Vybornov – Doctor of Historical Sciences, Professor (Samara State Academy of Social Sciences and Humanities, Samara, Russian Federation) M. Sh. Galimova – Candidate of Historical Sciences (Institute of Archaeology named after A. Kh. Khalikov, Kazan, Russian Federation) . Sh. Galimova – Candidate of Historical Sciences (Institute of Archaeology named after A. Kh. Khalikov, azan, Russian Federation) Kazan, Tatarstan ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 CONTENT Seregin N.N. (Barnaul, Russian Federation), Vasyutin S.A. (Kemerovo, Russian Federation) Eastern Altai in the Early Turkic Time: “New” Materials of Excavation of the Kudyrge Complex (from the archaeological heritage of A.S. Vasyutin) .............................................8 Samashev Z.S. (Almaty, Republic of Kazakhstan), Aitkali A.K. (Nur-Sultan, Republic of Kazakhstan), Tolegenov Ye. (Ust-Kamenogorsk, Republic of Kazakhstan) To the Question of the Sacralization of the Image of the Kagan .............................21 Kubaev S.Sh. (Tashkent, Republic of Uzbekistan) Image of Medicinal Plants on Ceramic Products of Central Asia ...........................35 Ruslanova R.R., Ruslanov E.V. (Ufa, Russian Federation) Birsk and Kushnarenkovo Burial Grounds of the Early Middle Ages in the Light of New Field Researches .................................................................42 Kravchenko E.E. (Kazan, Russian Federation) About Cremation Burials in the Middle Reaches of the Seversky Donets River .............................................................................56 Podosenova Yu.A., Krylasova N.B., Danich A.V. (Perm, Russian Federation) Wooden Sheaths with Metallic Coating in the Middle Age Perm Cis-Urals ...........72 Akylbek S.Sh. (Shaulder, Republic of Kazakhstan), Gursoy M. (Turkestan, Republic of Kazakhstan) “Cultural Premıses” in Kultobe City ........................................................................89 Kazakov E.P. (Kazan, Russian Federation) The Post-Petrogrom in the System of the Medievel Ugrian Cultures of the Ural-Volga Region ..................................................................................102 Klenov M.V. (Syktyvkar, Russian Federation) The Initial Stage of the Establishment of the Orthodox Church in the European North-East...............................................................................114 Koval V.Yu., Badeev D.Yu. (Moscow, Russian Federation) External Lines of the Early Bolgar Fortifi cation ....................................................124 Valeev R.M., Bugarchev A.I. (Kazan, Russian Federation) Silver Coins of the 13th – Beginning of the 15th Centuries from the Excavation CLXXIX at the Bolgar Fortifi ed Settlement ...................135 Narozhny E.I. (Karachaevsk, Russian Federation), Tishchenko I.B. (Stavropol, Russian Federation) New Muslim Burials from the Golden Horde City of Madzhar (Stavropol Region) .........................................................................145 Sagidullaev D.Z. (Uralsk, Republic of Kazakhstan) Golden Horde Settlements of the North-Eastern Caspian Region: geography, topography, housing construction ..................................................159 СОДЕРЖАНИЕ Серегин Н.Н. (Барнаул, Россия), Васютин С.А. (Кемерово, Россия) Восточный Алтай в раннетюркское время: «новые» материалы раскопок комплекса Кудыргэ (из археологического наследия А.С. Васютина) .............................................8 Самашев З. (Алматы, Казахстан), Айткали А.К. (Нур-Султан, Казахстан), Толегенов Е. (Усть-Каменогорск, Казахстан) К вопросу о сакрализации образа кагана .............................................................21 Кубаев С.Ш. (Ташкент, Узбекистан) Изображение лекарственных растений на керамических изделиях Средней Азии ......................................................35 Русланова Р.Р., Русланов Е.В. (Уфа, Россия) Бирский и Кушнаренковский могильники эпохи раннего средневековья в свете новых полевых исследований ..........42 Кравченко Э.Е. (Казань, Россия) О захоронениях по обряду кремации в среднем течении р. Северский Донец ..........................................................56 Подосенова Ю.А., Крыласова Н.Б., Данич А.В. (Пермь, Россия) Деревянные ножны с металлическими обкладками в средневековом Пермском Предуралье .......................................................72 Акылбек С.Ш. (Шаульдер, Казахстан), Гурсой М. (Туркестан, Казахстан) «Культовые помещения» на городище Культобе .................................................89 Казаков Е.П. (Казань, Россия) Постпетрогром в системе средневековых угорских культур Урало-Поволжья ...............................................................102 Клёнов М.В. (Сыктывкар, Россия) Начальный этап становления православной церкви на Европейском Северо-Востоке ...................................................................114 Коваль В.Ю., Бадеев Д.Ю. (Москва, Россия) Внешняя линия фортификации раннего Болгара ..............................................124 Валеев Р.М., Бугарчев А.И. (Казань, Россия) Серебряные монеты XIII – начала XIV в. из раскопа CLXXIX Болгарского городища .................................................135 Нарожный Е.И. (Карачаевск, Россия), Тищенко И.Б. (Ставрополь, Россия) Новые мусульманские захоронения золотоордынского города Маджара (Ставрополье) .....................................145 Сагидуллаев Д.З. (Уральск, Казахстан) Золотоордынские поселения Северо-Восточного Прикаспия: география, топография, домостроительство ................................................159 POVOLZHSKAYA ARKHEOLOGIYA № 2 (40) 2022 Юдин Н.И., Масловский А.Н. (Азов, Россия), Каземпур М. (Тебриз, Иран) Глазурованная чаша с персидскими стихами из Азака ....................................175 Пигарёв Е.М. (Йошкар-Ола, Россия) Изменение уровня Каспийского моря и его влияние на исторические процессы на территории низовьев Волги в средневековье (анализ материалов гидрологии и археологии) ...............183 Зеленеев Ю.А. (Йошкар-Ола, Россия), Валеев Р.М. (Казань, Россия) Внутренние и внешние миграции в Примокшанье в XIII–XV веках ..............198 Мургабаев С.С., Малдыбекова Л.Д., Бахтыбаев М.М., Жетибаев К.М., Гурсой М., Сиздиков Б.С. (Туркестан, Казахстан) История орошения Сыганака ..............................................................................206 Мустафин Х.Х. (Долгопрудный, Россия), Энговатова А.В. (Москва, Россия), Альборова И.Э. (Долгопрудный, Россия), Тарасова А.А. (Москва, Россия) Палеогенетическая экспертиза останков из двух наиболее крупных массовых захоронений 1238 г. в ярославском детинце ...............................215 Бочаров С.Г. (Севастополь, Россия) Наследие Золотой Орды: возникновение городов Крымского ханства ..........231 Шайдуллин Р.В. (Казань, Россия) «Татарская энциклопедия» в контексте систематизации, обобщения и научной популяризации знаний об археологии Татарстана ...................242 Список сокращений .............................................................................................249 Правила для авторов ............................................................................................250 1 Работа выполнена при финансовой поддержке гранта РФФИ, проект № 20-49-590001 «Средневековое ювелирное наследие Пермского края: стилистические и химико-техно- логические особенности», при поддержке Министерства образования и науки Пермского края, соглашение № С-26/1192 от 19.12.2019 г. а также в рамках темы Государственного задания, номер регистрации темы АААА-А19-119032590066-2. ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 https://doi.org/10.24852/pa2022.2.40.72.88 https://doi.org/10.24852/pa2022.2.40.72.88 УДК 902.01, 903.01/.09 ДЕРЕВЯННЫЕ НОЖНЫ С МЕТАЛЛИЧЕСКИМИ ОБКЛАДКАМИ В СРЕДНЕВЕКОВОМ ПЕРМСКОМ ПРЕДУРАЛЬЕ1 © 2022 г. Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич В эпоху средневековья на территории Пермского края бытовали деревянные ножны, обложенные металлическими пластинами, в основном, из сплава с высоким содержа- нием серебра. Отдельные изделия были декорированы зернью, филигранью, вставками из стекла или камней, тиснеными антропоморфными и орнитоморфными изображе- ниями, позолотой. Целью статьи является введение в научный оборот источников и результатов их исследования для определения основных технологических характери- стик и особенностей функционирования ножен. На основе сбора и систематизации ис- точников сложилось представление о массовости и разнообразии ножен. Анализ мо- нетного материала позволил конкретизировать основной период их распространения: IX – первая половина X вв., хотя отдельные экземпляры встречаются и в XI в. Анализ особенностей залегания ножен в погребениях с учетом палеоантропологических дан- ных позволил сделать вывод, что ножны с металлическими обкладками были частью поясного набора и использовались в женском костюме как символический и декора- тивный элемент. Из Пермского Предуралья отдельные изделия проникли в Удмуртское Предуралье (поломская археологическая культура), в Ветлужско-Вятское междуречье и Волжскую Булгарию. Возможно, в Удмуртском Предуралье был свой центр изготов- ления подобных изделий со своими отличительными особенностями. Ключевые слова: археология, эпоха средневековья, Пермское Предуралье, ломо- ватовская культура, женский костюм, ножны, серебро, медь, зернь, филигрань, тисне- ние, позолота. CONTENT POVOLZHSKAYA ARKHEOLOGIYA № 2 (40) 2022 Iudin N.I., Maslovsky A.N. (Azov, Russian Federation), Kazempur M. (Tabriz, Iran) Glazed Bowl with Persian Pottery from Azak .......................................................175 Pigarev E.M. (Yoshkar-Ola, Russian Federation) Changes in the Level of the Caspian Sea and its Infl uence on Historical Processes in the Lower Reaches of the Volga in the Middle Ages (analysis of materials of hydrology and archaeology) ......................................183 Zeleneev Yu.A. (Yoshkar-Ola, Russian Federation), Valeev R.M. (Kazan, Russian Federation) The Internal and External Migrations in the Moksha Littoral During the 13th–15th Centuries ...........................................................................198 Murgabaev S.S., Maldybekova L.D., Bakhtybaev M.M., Zhetibaev K.M., Gursoy M., Sizdikov B.S. (Turkestan, Republic of Kazakhstan) History of the Syganak Irrigation ..........................................................................206 Mustafi n Kh.Kh. (Dolgoprudny, Russian Federation), Engovatova A.V. (Moscow, Russian Federation), Alborova I.E. (Dolgoprudny, Russian Federation), Tarasova A.A. (Moscow, Russian Federation) Genetic Examination of Remains from the Two Largest Mass Burials of 1238 in Yaroslavl Detynets .................215 Bocharov S.G. (Sevastopol, Russian Federation) Heritage of the Golden Horde: the origins of Crimean Khanat cities ....................231 Shaidullin R.V. (Kazan, Russian Federation) “Tatar Encyclopedia” in the Context of Systematization, Generalization and Scientifi c Popularization of Knowledge about the Archaeology of Tatarstan ...........................................242 List of Abbreviations ............................................................................................. 249 Submissions .......................................................................................................... 250 Контекст нахождения ножен В материалах ломоватовской куль- туры Пермского Предуралья (VII– XI вв.) известны деревянные ножны с металлическими обкладками. Пред- ставлены они широко, но к массовым находкам не принадлежат, т. к. носили статусный характер. Опыт их описа- ния и систематизации был (Голдина, 1985, с. 59; Крыласова, 2007, с. 229– 233), но детально ножны не анализи- ровались из-за недостаточной выбор- ки и плохой сохранности предметов. Раскопки Баяновского могильника существенно пополнили коллекцию ножен, что позволило исследовать их на новом уровне. Проанализировано 88 ножен с 11 памятников, из них 60 – с Баяновского могильника. Ножны с металлическими об- кладками характерны для «богатых» женских захоронений, где были при- надлежностью пояса (Крыласова, 2007, с. 229). Данные Баяновского могильника полностью подтвердили это: ножны сопровождают погребе- ния женщин2, умерших в возрасте 16–50 лет, редко захоронения дево- чек до 15 лет3. На других территори- ях такие ножны тоже характерны для женских комплексов (Мыдлань-Шай (п. 21, 64, 74, 79; Генинг, 1962, табл. VII: 1–3), Поломский II Красная горка (п. 70; Останина, 2011, с. 145, рис. 30: 6), Танкевский (п. 953; Казаков, 1992, рис. 31: 10), Больше-Тиганский (п. 30; Халиков, Халикова, 2018, табл. XX 72 72 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич с. 1. Детали: 1 – основа рукояти из двух деревянных элементов, 2 – торец руко рнутый берестой, 3 – основа ножен из двух деревянных элементов, 4 – метал ие пластины, соединенные пайкой, 5, 8 – проволочные кольца для дополнител иксации пластин 6 бронзовый подвес 7 торец рукояти покрытый серебря Рис. 1. Детали: 1 – основа рукояти из двух деревянных элементов, 2 – торец рукояти, обернутый берестой, 3 – основа ножен из двух деревянных элементов, 4 – металличе- ские пластины, соединенные пайкой, 5, 8 – проволочные кольца для дополнительной фиксации пластин, 6 – бронзовый подвес, 7 – торец рукояти, покрытый серебряной пластиной, 9 – остатки кожи между металлическими пластинами, 10 – шаровидный затыльник рукояти, 11 – декоративный затыльник рукояти, 12 – грушевидный нако- нечник ножен, 13 – фрагмент деревянной основы ножен со следами от проволоки или нити, 14 – шпенек с ромбической головкой, 15–16 – низки бус и пронизок для подве- шивания ножен. 1–6 – п.392, 7–9 – п.374, 10 – п.358 Баяновского м-ка, 11 – Огурдин- ский м-к, 12 – п.270, 13 – п.367 Баяновского м-ка, 14 – п.51 Редикарского м-ка, 15 – п.65, 16 – п.513 Баяновского м-ка Рис. 1. Fig. 1. Details: 1 – the base of the handle is made of two wooden elements; 2 – the butt of the handle, wrapped with birch bark, 3 – the base of the scabbard consists of two wooden elements, 4 – metal plates connected by soldering, 5, 8 – wire rings for additional fi xation of plates, 6 – bronze suspension, 7 – the end of the handle, covered with a silver plate, 9 – leftover leather between metal plates, 10 – spherical butt of the handle, 11 – decorative buttstock of the handle, 12 – pear-shaped scabbard tip, 13 – a fragment of the wooden base of the scabbard with traces of wire or thread, 14 - a peg with a rhombic head, 15–16 – beads for hanging scabbards. Конструкция ножен и их химико- технологические характеристики На Баяновском могильнике нож- ны часто уложены вдоль левого пред- плечья погребенной, реже – на месте ношения у левого бедра, тогда обыч- но сохраняется низка стеклянных и бронзовых бус, вытянутая от ножен к поясу (рис. 1: 15–16; 2: 5А; 3: 9, 19; 4: 1, 4; 5: 6, 8; 7: 7–8, 10, 15). Укладка но- жен у левого предплечья, возможно, была локальной погребальной тради- цией. Но Е.П. Казаковым отмечается, что и в Танкеевском могильнике ножи у женщин нередко положены у правой или левой руки, а в п. 953 ножны тоже располагались у левого плеча (Каза- ков, 1992, с. 102). Комплекс включал: нож, рукоять, футляр для ножа и подвес. Комплекс включал: нож, рукоять, футляр для ножа и подвес. Ножи, судя по доступным для анализа, универсальные, длиной 10– 15 см, с лезвием шириной 1–1,5 см. Ножи, судя по доступным для анализа, универсальные, длиной 10– 15 см, с лезвием шириной 1–1,5 см. Ножны имеют длину 23–43 см (с рукоятью), ширину 2–3, толщину 1–1,5 см, включают две части: руко- ять ножа и собственно ножны, обра- зующие единую композицию. Ножны имеют длину 23–43 см (с рукоятью), ширину 2–3, толщину 1–1,5 см, включают две части: руко- ять ножа и собственно ножны, обра- зующие единую композицию. В Лаборатории термических ана- лизов кафедры неорганической хи- мии, химической технологии и тех- носферной безопасности ПГНИУ под руководством к.х-т.н. И.Г. Мокрушина выполнено исследование основы но- жен (23 образца) по разработанной для археологических образцов мето- дике. Материал основы определен как береза, наряду с которой в трех образ- цах отмечена кость. Для сравнения, в Ветлужско-Вятском междуречье ос- нова ножен была из липы (Никитина, 2012, с. 11, 13). В Лаборатории термических ана- лизов кафедры неорганической хи- мии, химической технологии и тех- носферной безопасности ПГНИУ под руководством к.х-т.н. И.Г. Мокрушина выполнено исследование основы но- жен (23 образца) по разработанной для археологических образцов мето- дике. Материал основы определен как береза, наряду с которой в трех образ- цах отмечена кость. Для сравнения, в Ветлужско-Вятском междуречье ос- нова ножен была из липы (Никитина, 2012, с. 11, 13). ) Интересен вопрос о назначении таких ножен. В.Ф. Генинг предпола- гал их символическое, а не практи- ческое значение (Генинг 1962, с. 68). В.А. Оборин и Р.Д. Голдина считали их футлярами декоративных кинжа- лов. В.А. Оборин так описал нож- ны из п. 5 Плёсинского могильника: «Декоративный кинжал с серебряной обкладкой, наглухо скрепленный мед- ным стержнем, с кольцом для привя- зывания к поясу» (Оборин, 1962, с. 99). Контекст нахождения ножен Детали: 1 – основа рукояти из двух деревянных элементов, 2 – торец рукояти, обернутый берестой, 3 – основа ножен из двух деревянных элементов, 4 – металличе- ские пластины, соединенные пайкой, 5, 8 – проволочные кольца для дополнительной фиксации пластин, 6 – бронзовый подвес, 7 – торец рукояти, покрытый серебряной пластиной, 9 – остатки кожи между металлическими пластинами, 10 – шаровидный затыльник рукояти, 11 – декоративный затыльник рукояти, 12 – грушевидный нако- нечник ножен, 13 – фрагмент деревянной основы ножен со следами от проволоки или нити, 14 – шпенек с ромбической головкой, 15–16 – низки бус и пронизок для подве- шивания ножен. 1–6 – п.392, 7–9 – п.374, 10 – п.358 Баяновского м-ка, 11 – Огурдин- ский м-к, 12 – п.270, 13 – п.367 Баяновского м-ка, 14 – п.51 Редикарского м-ка, 15 – п.65, 16 – п.513 Баяновского м-ка 73 73 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 IVB: 17), Веселовский (п. 3), Нижняя стрелка м-ки (п. 17; Никитина, 2012, рис. 12: 7; 162: 7). IVB: 17), Веселовский (п. 3), Нижняя стрелка м-ки (п. 17; Никитина, 2012, рис. 12: 7; 162: 7). IVB: 17), Веселовский (п. 3), Нижняя стрелка м-ки (п. 17; Никитина, 2012, рис. 12: 7; 162: 7). железные модели ножей с очень корот- ким лезвием (Никитина, 2012, с. 11). железные модели ножей с очень корот- ким лезвием (Никитина, 2012, с. 11). Конструкция ножен и их химико- технологические характеристики Конструкция ножен и их химико- технологические характеристики Соединенные подвесом рукоять и футляр выявлены и в п. 328, 367, 406 Баяновского могильника. В этих случаях практическое использова- ние ножей невозможно, ножны были предметом символического и декора- тивного характера. Рукояти ножей обычно короткие (2,5–5 см), их было неудобно помещать в ладонь, драго- ценный декор на многих рукоятях не предполагал повседневного исполь- зования таких ножей. Иногда досто- верно зафиксировано отсутствие соб- ственно ножа. Рукояти имеют длину 2,5–5 см (чаще 3,5–4 см), лишь в двух случаях они были длиной 7–8 см. Основа вы- полнялась двумя способами: 1 – скле- ивалась из двух деревянных планок полусферического сечения с пазами для черешка ножа (рис. 1: 1); 2 – в цельной деревянной основе выреза- лось отверстие для черешка ножа. Основа футляра ножен складыва- лась из пары деревянных планок по- луовального сечения (рис. 1: 3) с вы- емкой для ножа. Они были длинными, и при склеивании их стягивали нитя- ми – на фрагментах двух ножен есть характерные следы в виде насечек (рис. 1: 13). Аналогично оцениваются ножны с других территорий. Так, описывая ножны из п. 3 Веселовского м-ка, Т.Б. Никитина отметила их декоративное или вотивное назначение: внутри были Металлические элементы обклад- ки ножен изготовлены из медных или серебряных пластин толщиной 0,13– 0,6 мм. Медные обкладки имеют глу- бокий слой окиси, их исследование с 74 74 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич гладкой или штампованной проволо- ки, дополнительно скреплявшие пла- стину. позиций химического состава метал- ла не проводилось. р У большинства ножен (64 экз.) об- кладки были серебряными. Они ис- следованы путем рентгено-флюорес- центного анализа (РФА, портативный РФА-анализатор Bruker S1) и методом сканирующей электронной микро- скопии с приставкой для локального микрорентгеноспектрального анализа (СЭМ РФлА; сканирующий электрон- ный микроскоп Hitachi S3400 с при- ставкой для локального микрорент- геноспектрального анализа Bruker). Первым методом получены предвари- тельные данные и определен химиче- ский состав обкладок ножен из фон- дов музеев. Второй способ дал более точные данные о составе металла. Пока в четырех случаях выявле- ны затыльники рукоятей, разные по форме и креплению. Простейший – тонкий берестяной лист, обернутый вокруг деревянной основы (рис. 1: 2, п. 392 Баяновского м-ка). Второй за- тыльник вырезан из тонкого листа ме- талла по форме сечения рукояти ножа с небольшим припуском, наложен на торец, припуск отогнут и зажат об- кладкой рукояти (рис. 1: 7; 4: 7 На од- них ножнах эта орнаментальная зона образована двумя пластинами,, п. 374 Баяновского м-ка). У затыльника рукояти из Огурдинского могильни- ка (рис. Конструкция ножен и их химико- технологические характеристики 1: 11) из пластины вырезана заготовка по форме сечения рукояти ножа с небольшим припуском; её кон- цы отогнуты, вставлены в готовую металлическую часть рукояти, припа- яны, потом нанесен декор. Затыльник в виде полого шарика (рис. 1: 10, п. 358 Баяновского м-ка) собран из двух спаянных полусфер и надет на руко- ять ножа. Подобный затыльник руко- яти есть в п. 30 Больше-Тиганского м-ка IX – н. X вв. (Халикова, Халиков, 2018, с. 40, табл. XXIVB: 17). Путем РФА в обкладках зафикси- ровано высокое содержание серебра (90–94%), небольшое количество меди (3–6%), золота (0,6–1,3 %), свин- ца (0,6–1,2%), цинка (0,6–1%), следы мышьяка и железа (до 1%). При СЭМ РФлА в обкладках вы- явлено высокое содержание серебра (92–95%), небольшое количество меди (5–7%). Расхождение в данных, полученных разными методами, на- блюдается по доле свинца, золота и цинка, что связано с особенностями проведения исследований. Общий результат показал, что материалом обкладок был сплав с высоким содер- жанием серебра. Сырьем, очевидно, служил монетный металл. Вероятно, затыльники были у большинства рукоятей, но так как их обычно не припаивали, они могли те- ряться ещё при использовании ножен. Обкладки футляра делались так же – пластина сгибалась, края заво- дились внахлест, спаивались, готовая пластина надевалась на деревянную основу, края дополнительно зажима- лись кольцами (рис. 1: 4–5). Иногда обкладка из цельной металлической пластины покрывала большую часть футляра, но чаще на деревянной осно- ве на некотором расстоянии помеща- ли 2–3 пластины. Иногда фон зерно-филигранного декора или тисненые изображения по- золочены. Методом СЭМ РФлА про- анализирован фрагмент с глубоким слоем позолоты, выявлено высокое содержание золота (88–95%) и ртути (5–12%), что свидетельствует о нане- сении позолоты путем амальгамиро- вания. Рукояти покрывали цельной пла- стиной, ее края сводили внахлест на тыльной стороне и спаивали. Снизу и сверху надевали колечки-обоймы из Пока есть единственный наконеч- ник ножен – полый грушевидный, слегка изогнутый, спаянный из двух половинок, тисненых из тонкого ли- 75 75 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 ста металла (рис 1: 12 п 270 Баянов глым изгибом вверху образующим Рис. 2. Ножны группы 1: 1 – п.4 Каневского, 2 – п.2 Важгортского, 3 – п.10 Урьинско- го, 4 – п.6 Важгорского, 5 – п.51 Редикарского м-ка Fig. 2. Sheath of group 1. Рис. 2. Ножны группы 1: 1 – п.4 Каневского, 2 – п.2 Важгортского, 3 – п.10 Урьинско- го, 4 – п.6 Важгорского, 5 – п.51 Редикарского м-ка Fig. 2. Sheath of group 1. Конструкция ножен и их химико- технологические характеристики глым изгибом вверху, образующим петлю, следом за которым конец был раскован (рис. 1: 6). Вдоль стержня имелись отверстия (5–6) для шпень- ков. Аналогичные подвесы имели ножны из поломской культуры (Семе- нов, 1980, табл. XXV: 10, 12; XXVI: 3, глым изгибом вверху, образующим петлю, следом за которым конец был раскован (рис. 1: 6). Вдоль стержня имелись отверстия (5–6) для шпень- ков. Аналогичные подвесы имели ножны из поломской культуры (Семе- нов, 1980, табл. XXV: 10, 12; XXVI: 3, ста металла (рис. 1: 12, п. 270 Баянов- ского м-ка). Такие наконечники из- вестны на ножнах сабель аланского и венгерского типа (Кирпичников, 1966, с. 68). Подвес ножен представлял собой литой бронзовый стержень с полукру- 76 76 76 76 76 76 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич Рис. 3. Ножны группы 2, тип 1: 1 – п.19 Урьинского, 2 – п.5 Плесинского, 3 – п.287 Агафоновского, 4 – п.45 Аверинского, 5 – п.26 Рождественского, 6 – п.20, 7 – п,51, 8 – п.55, 9 – п.65, 10 – п.97, 11 – п.252, 12 – п.266, 13 – п.283, 14 – п.345, 15 – п.358, 16 – п.359, 17 – п.435, 18 – п.476, 19 – п.281 367 Баяновского м-ка Fig. 3. Sheath of group 2, type 1. Рис. 3. Ножны группы 2, тип 1: 1 – п.19 Урьинского, 2 – п.5 Плесинского, 3 – п.287 Агафоновского, 4 – п.45 Аверинского, 5 – п.26 Рождественского, 6 – п.20, 7 – п,51, 8 – п.55, 9 – п.65, 10 – п.97, 11 – п.252, 12 – п.266, 13 – п.283, 14 – п.345, 15 – п.358, 16 – п.359, 17 – п.435, 18 – п.476, 19 – п.281 367 Баяновского м-ка Fig. 3. Sheath of group 2, type 1. Fig. 3. Sheath of group 2, type 1. 4, 5б, 6б, 7а; Генинг, 1962, табл. VII: 1), Ветлужско-Вятского междуречья (Никитина, 2012, рис. 12: 7), Танке- евского могильника (Казаков, 1992, рис. 30: 10). 5, 9; 7: 4, 9, 13, 15), шпеньки с таки- ми головками применялись нередко. В.Ф. Генинг по материалам п. 21 мо- гильника Мыдлань-Шай, где детально прослежены особенности крепления ножен, сделал реконструкцию, по ко- торой к этому шпеньку крепился ре- мешок, соединявший нижнюю часть ножен с поясом, а к верхнему концу ножен присоединялся ремешок с на- бором медных бус (Генинг, 1962, с. 68, рис. 29). За изготовлением основных дета- лей ножен следовал процесс сборки изделия. Конструкция ножен и их химико- технологические характеристики Основу обтягивали кожей – ее остатки видны под металлически- ми пластинами на многих ножнах (рис. 1: 9). Кожа служила для лучшей подгонки деталей и выполняла эсте- тическую функцию, когда обкладки покрывали футляр с промежутками. На полученную конструкцию наде- вали металлические части, затем на тыльную сторону шпеньками крепили подвес, скреплявший конструкцию и закрывавший места пайки на обклад- ках. Ножны, конструктивно и техно- логически схожие с ломоватовскими, известны в поломской культуре (Се- менов, 1980, с. 50) и у жителей Вет- лужско-Вятского междуречья (Ники- тина, 2012, с. 11, рис. 12: 5–7). р Классификация ножен что стало основанием для выделения основных таксономических единиц. без декора. Материал – медь, серебро. Декоративность придавали обжимные кольца из гладкой или штампованной проволоки, помещенные группами по низу рукояти и устью футляра, а ино- гда и по краям пластин в центральной части ножен, а также контраст ме- таллических пластин и промежутков между ними, затянутых кожей. Группа 1 (рис. 2, 5 экз.) – ниж- няя часть футляра обложена мед- ной или серебряной пластиной, верхняя – бронзовыми кольцами, со- гнутыми из литых заготовок полукру- глого сечения. Тип 1 (рис. 2: 1–3, 3 экз. – п. 40 Тип 1 (рис. 2: 1–3, 3 экз. – п. 40 Каневского, п. 2 Важгортского, п. 10 Урьинского м-ка) – без декора. Аналогичные ножны есть на Тан- кеевском (п. 30, 953) (Казаков, 1992, с. 102, рис. 31: 10, с. 137, рис. 50: 10– 11), Кочергинском (п. 5 (Талицкий, 1941, с. 167, рис. 8: 72)), Кузинских хуторах (п. 25 (Никитина, Акилбаев, 2019, с. 92, рис. 5: 13), на могильни- ке Уелги на Южном Урале (раскопки 2019 г.), где есть серия материалов прикамского происхождения (Бота- лов, 2019, с. 85–86). Каневского, п. 2 Важгортского, п. 10 Урьинского м-ка) – без декора. Тип 2 (рис. 2: 4, 1 экз. – п. 6 Важ- гортского м-ка) – верхний край обклад- ки ножен декорирован пластинчатой лентой с чеканным орнаментом в виде полугорошин. у р Тип 3 (рис. 2: 5, 1 экз. – п. 51 Реди- карского м-ка (Белавин, 2007, рис. 12: 13, 19)) – рукоять с зерно-филигран- ным декором: треугольники крупной серебряной зерни обрамляют колеч- ки-обоймы (снизу – из штампованной проволоки); в центре – треугольник мелкой золотой зерни, осмотр кото- рой при увеличении показал, что она напаяна на треугольную пластинку, которая затем припаяна к обкладке рукояти (рис. 2: 5В). Возможно, тре- угольник с зернью вырезан из какого- то ювелирного изделия. Ножны, обтянутые металлически- ми пластинами в сочетании с литыми бронзовыми кольцами-обоймами (тип 6 по В.А. Семенову), известны в Вар- нинском могильнике (Семенов, 1980, с. 50). Тип 3 (рис. 2: 5, 1 экз. – п. 51 Реди- карского м-ка (Белавин, 2007, рис. 12: 13, 19)) – рукоять с зерно-филигран- ным декором: треугольники крупной серебряной зерни обрамляют колеч- ки-обоймы (снизу – из штампованной проволоки); в центре – треугольник мелкой золотой зерни, осмотр кото- рой при увеличении показал, что она напаяна на треугольную пластинку, которая затем припаяна к обкладке рукояти (рис. 2: 5В). Возможно, тре- угольник с зернью вырезан из какого- то ювелирного изделия. Тип 2 (14 экз.) – с зерно-филигран- ным декором. Классификация ножен Почти каждые ножны индивиду- альны, тем не менее выборка позво- ляет провести классификацию этой категории ювелирных изделий. От- личие ножен заключается в способах наложения металлических обкладок и в особенностях их декорирования, Нижняя часть ножен укрепля- лась сквозным шпеньком. На нож- нах из п. 51 Редикарского могильни- ка его головка имела ромбическую форму (рис. 1: 14), судя по следам на отдельных предметах (рис. 5: 1, 77 77 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 без декора. Материал – медь, серебро. Декоративность придавали обжимные кольца из гладкой или штампованной проволоки, помещенные группами по низу рукояти и устью футляра, а ино- гда и по краям пластин в центральной части ножен, а также контраст ме- таллических пластин и промежутков между ними, затянутых кожей. что стало основанием для выделения основных таксономических единиц. Материал – серебро. Подтип 2а (рис. 4: 1–2; 5: 1–13, 13 экз.) – с зерно-филигранным декором и вставками. Вариант 1 (рис. 4: 1–2; 5: 1–11; 11 экз.: Пермский край, д. Зобачева (Спи- цын, 1902, т. III: 7), п. 86, 136, 328, 365, 392, 400, 433, 502 Баяновского м-ка), в основе декоративной композиции – элемент в виде «якоря» высотой 3– 5,5 см: каст с каплевидной вставкой обрамлен штампованной проволоч- кой с концами, спускающимися вниз и расходящимися в стороны завитка- ми (рис. 6: 1–9). «Якорьки» дополне- ны треугольниками зерни на концах завитков, по бокам и вокруг каста. Иногда вставку заменяла розетка из зерни (п. 86, 433 – рис. 5: 4, 10; 6: 6, 9). Ножны, обтянутые металлически- ми пластинами в сочетании с литыми бронзовыми кольцами-обоймами (тип 6 по В.А. Семенову), известны в Вар- нинском могильнике (Семенов, 1980, с. 50). Ножны, обтянутые металлически- ми пластинами в сочетании с литыми бронзовыми кольцами-обоймами (тип 6 по В.А. Семенову), известны в Вар- нинском могильнике (Семенов, 1980, с. 50). Группа 2 (83 экз.) – ножны покры- ты металлическими пластинами. Они либо полностью закрывали рукоять и футляр, либо покрывали рукоять и окончание ножен, а между ними яру- сами располагались горизонтальные пластины, в промежутках между ко- торыми было видно основание, обтя- нутое кожей. Группа 2 (83 экз.) – ножны покры- ты металлическими пластинами. Они либо полностью закрывали рукоять и футляр, либо покрывали рукоять и окончание ножен, а между ними яру- сами располагались горизонтальные пластины, в промежутках между ко- торыми было видно основание, обтя- нутое кожей. «Якорьковый» элемент повторялся на рукояти (за одним исключением, где есть только каст со вставкой – п. 392, рис. 4: 1; 5: 8; 6: 4) и в верхней части футляра ножен, где выделялась декоративная зона в виде широкой (10–12 см) пластины, обрамленной вверху и внизу рядами гладкой или штампованной проволоки, вдоль кото- рой обычно помещены треугольники Тип 1 (рис. 3; 46 экз. – п. 19 Урьин- ского, п. 81, 118 Деменковского, п. 287 Агафоновского, п. 45 Аверинского, п. 5 Плесинского, п. 26 Рождествен- ского, остальные – Баяновский м-к) – 78 78 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич Рис. 4. Примеры ножен группы 2 с декоративной отделкой: 1 – п.392, 2 – п.328, 3 – п.517, 4 – п.492, 5 – п.124, 6 – п.480, 7 – п.374 Баяновского м-ка. Fig. 4. Examples of Group 2 sheath with decorative trim. Рис. 4. что стало основанием для выделения основных таксономических единиц. Примеры ножен группы 2 с декоративной отделкой: 1 – п.392, 2 – п.328, 3 – п.517, 4 – п.492, 5 – п.124, 6 – п.480, 7 – п.374 Баяновского м-ка. Fig. 4. Examples of Group 2 sheath with decorative trim. зерни. На одних ножнах эта орнамен- тальная зона образована двумя пла- стинами, каждая обрамлена гладкой проволокой с треугольниками зерни вдоль нее (п. 86, рис. 5: 4). В отдель- ных изделиях орнаментальная зона ограничивалась по бокам «рамкой» из припаянных вертикально проволочек (п. 400, 433, 502, рис. 5: 9–10; 6: 5, 9). фектов (отсутствие пропуска насечек, равномерное расстояние между на- сечками) (рис. 6). Касты для вставок открытые, из тонкой металлической полоски, на- паянной под прямым углом к поверх- ности ножен. После крепления встав- ки края каста обжимались. Вставка дополнительно подклеивалась – на одном изделии между стеклянной вставкой и металлом заметно веще- ство темного цвета (смола?). Матери- Зернь на ножнах калиброванная, филигрань в виде штампованной зер- неной проволочки без видимых де- 79 79 № 1 (39) 2022 № 2 (40) 2022 № 1 (39) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ Рис. 5. Ножны группы 2, тип 2: 1–2 – Пермский край, 3 – д. Зобачева, 4 – п.86, 5 – п.328, 6 – п.365, 7 – п.136, 8 – п.392, 9 – п.400, 10 – п.433, 11 – п.502, 13 – п.517 Бая- новского, 12 – п.6 Каневского м-ка. Fig. 5. Sheath of group 2, type 2. Рис. 5. Ножны группы 2, тип 2: 1–2 – Пермский край, 3 – д. Зобачева, 4 – п.86, 5 – п.328, 6 – п.365, 7 – п.136, 8 – п.392, 9 – п.400, 10 – п.433, 11 – п.502, 13 – п.517 Бая- новского, 12 – п.6 Каневского м-ка. Fig. 5. Sheath of group 2, type 2. Fig. 5. Sheath of group 2, type 2. ал вставок – янтарь (п. 136, 328, 365, 502), стекло или камень зеленого цве- та (п. 392, 400), черная смола с мато- вой поверхностью (п. 392) (рис. 6: 11). Ближайшие аналоги ножнам с зер- но-филигранным декором, в том чис- ле с «якорьками», есть в поломской культуре: в погребениях VIII–IX вв. Варнинского могильника (Семенов, 1980, с. 50, 132, табл. XXVI: 3–6; Ива- нова, Куликов, 2000, с. 31, 190, № 56), погребениях вт. пол. VIII – перв. пол. IX вв. могильника Мыдлань-Шай (п. 21, 74, 64, 79; Генинг, 1962, табл. VII: 1–3), погребениях VIII–IX вв. что стало основанием для выделения основных таксономических единиц. По- ломского могильника (Руденко, 2015, рис. 44). Аналогичные изделия, место находок которых локализуется в При- камье, принадлежат Фонду Марджа- ни (ИМ/М-80-81) (Путешествие…, с. 375–378). рис. 44). Аналогичные изделия, место находок которых локализуется в При- камье, принадлежат Фонду Марджа- ни (ИМ/М-80-81) (Путешествие…, с. 375–378). рис. 44). Аналогичные изделия, место находок которых локализуется в При- камье, принадлежат Фонду Марджа- ни (ИМ/М-80-81) (Путешествие…, с. 375–378). Вариант 2 (рис. 5: 12, 1 экз., п. 6 Каневского м-ка) – на рукояти каст с каплевидной вставкой, в верхней ча- сти футляра – выложенная из прово- локи «стрелочка»; орнаментальные зоны на рукояти и в верхней части футляра ограничены проволочны- ми поясками, вдоль которых, судя по рисунку, зигзагами выложена зернь. Прорисовка ножен дана в моногра- фии Р.Д. Голдиной (Голдина, 1985, 80 80 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич Рис. 6. 1–10 – декоративные элементы на ножнах с зерно-филигранным декором и вставками: 1 – п.136, 2 – п.365, 3 – п.502, 4 – п.392, 5 – п.400, 6 – п.86, 7 – п.328, 9 – п.433, 10 – п.517 Баяновского м-ка, 8 – Пермский край. 11 – вставки: А – янтарь, Б – стекло; В – смола: А – п.328, Б–В – п.392 Баяновского м-ка. Fig. 6. 1–10 – Decorative elements on the scabbard with grain-fi ligree decor and inserts. 11 – Inserts: A – amber, Б – glass; B – resin.. Рис. 6. 1–10 – декоративные элементы на ножнах с зерно-филигранным декором и вставками: 1 – п.136, 2 – п.365, 3 – п.502, 4 – п.392, 5 – п.400, 6 – п.86, 7 – п.328, 9 – п.433, 10 – п.517 Баяновского м-ка, 8 – Пермский край. 11 – вставки: А – янтарь, Б – стекло; В – смола: А – п.328, Б–В – п.392 Баяновского м-ка. Fig. 6. 1–10 – Decorative elements on the scabbard with grain-fi ligree decor and inserts. 11 – Inserts: A – amber, Б – glass; B – resin.. т. XXIX: 22), в публикации по резуль- татам раскопок Урьинского, Канев- ского и Важгортского могильников (Генинг, Голдина, 1970) ее нет. Нож- ны не сохранились, и сейчас сложно судить, насколько адекватно худож- ник воспроизвел декор (отметим, что прорисовки ножен из одних и тех же погребений в публикациях 1970 и т. XXIX: 22), в публикации по резуль- татам раскопок Урьинского, Канев- ского и Важгортского могильников (Генинг, Голдина, 1970) ее нет. Нож- ны не сохранились, и сейчас сложно судить, насколько адекватно худож- ник воспроизвел декор (отметим, что прорисовки ножен из одних и тех же погребений в публикациях 1970 и 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. Вариант 3 (рис. 4: 3; 5: 13; 6: 10, 1 экз., п. 517 Баяновского м-ка) – на пластине в верхней части ножен, об- рамленной узкими серебряными по- лосами с чеканным орнаментом в 81 81 № 1 (39) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 Рис. 7. Ножны группы 2, типы 3–4: 1 – п.80, 2 – п.106, 3 – п.92, 4 – п.124, 5 – п.127, 6 – п.240, 7 – п.276, 8 – п.401, 9 – п.404, 10 – п.425, 11 – п.440, 12 – п.473, 13 – п.480, 14 – п.481, 15 – п.492, 17 – п.474, 18 – п.99 Баяновского м-ка, 16 – Пермский край. Fig. 7. Sheath of group 2, type 3–4. Рис. 7. Ножны группы 2, типы 3–4: 1 – п.80, 2 – п.106, 3 – п.92, 4 – п.124, 5 – п.127, 6 – п.240, 7 – п.276, 8 – п.401, 9 – п.404, 10 – п.425, 11 – п.440, 12 – п.473, 13 – п.480, 14 – п.481, 15 – п.492, 17 – п.474, 18 – п.99 Баяновского м-ка, 16 – Пермский край. Fig. 7. Sheath of group 2, type 3–4. с. 155–156; Krylasova, Podosenova, Sarapylov, 2015, р. 203–219). Это под- тверждают даты монет и брактеатов, найденных в погребениях с ножнами и соседних захоронениях4. виде точек, вверху и внизу – два треу- гольника вершинами друг к другу, вы- ложенные из треугольников зерни, по- мещенных в шахматном порядке, фон позолочен. На пластине в нижней ча- сти ножен сверху – овальная площад- ка с бронзовой «рамкой», где остались следы припоя, очевидно, здесь был какой-то декоративный элемент, пред- положительно, вставка с филигран- ным обрамлением (рис. 6: 10). Подтип 2б (рис. 1: 11, 1 экз., Огур- динский м-к) – обкладка рукояти ножа с обильным зерно-филигранным де- кором: вертикальные ряды треуголь- ников зерни перемежаются рядами гладкой и торсированной проволо- ки. Ряды торсированной проволочки расположены разнонаправленными витками, создавая эффект «косич- ки». Затыльник рукояти декорирован крупной зернью на колечках торси- рованной проволоки. Применение в декорировании ювелирных изделий Пермского Предуралья крупной зерни на филигранных подложках, а также торсированной проволоки и глади в комплексе началось с XI в. (Подосё- По сопутствующему инвентарю погребений Баяновского и Канев- ского могильников ножны с зерно- филигранным декором и вставками датированы IX–X вв. 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. Приемы, ис- пользованные в их декоре, позволяют сузить дату до IX – первой половины X вв., когда в ювелирных изделиях Пермского Предуралья сочетались укладка зерни, штампованной прово- лочки и позолота (Подосёнова 2021, 82 82 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич нова, 2021, с. 156–157). Огурдинский могильник датирован к. X – перв. по- ловиной XI вв. (Белавин, Крыласова, 2012, с. 233). Данное изделие – самое позднее из категории ножен с метал- лическими обкладками, свидетель- ствующее о верхней границе периода их распространения. мог проникнуть в Прикамье на пред- метах торевтики. мог проникнуть в Прикамье на пред- метах торевтики. Подтип 3б (рис. 4: 7; 7: 16–17, 2 экз., Пермский край, п. 374 Баяновско- го м-ка) – с изображением птичек. На ножнах из п. 374 (рис. 4: 7; 7: 17) ниж- няя часть обтянута цельной пластиной шириной 16,5 см, верхняя – пласти- ной шириной 8 см, между ними – пластина шириной 3,3 см. По всем этим элементам размещались тисне- ные птички: одна – на рукояти, две – на верхней пластине футляра, одна – на средней пластине, 4 – на конце но- жен. Фигурки на рукояти и двух верх- них пластинах ножен позолочены. Птички высотой 2,3 см повернуты в профиль влево; имеют овальную го- лову с овальным глазом и хохолком, загнутым в крутую петлю, S-образно выгнутую шею, которая внизу пере- ходит в пару лапок, овальное тулови- ще, в центре которого помещен ромб с косой решеткой, обрамленный вали- ками, имитирующими крыло, оваль- ный поднятый вверх хвост обрамлен гладким валиком, внутри покрыт горизонтальными линиями (рис. 8: 12–13). Тип 3 (17 экз.) с тисненым орна- ментом. Материал – серебро. Подтип 3а (рис. 4: 4–6; 7: 1–15, 16 экз., п. 62, 80, 92, 106, 124, 127, 240, 276, 401, 404, 425, 440, 473, 480, 481, 492 Баяновского м-ка) – с изображе- нием антропоморфов в позе коитуса. Тисненые фигурки высотой 3–3,8 см помещены на рукояти, на пластине, обтягивающей верхнюю часть фут- ляра, и в верхней части пластины на конце ножен. На ножнах из п. 124 и 425 тисненые фигурки позолочены (рис. 4: 5; 7: 4, 10; 8: 3). При первых находках ножен с этим декором тисненые фигурки интерпре- тировались как «рыбки» или «фили- ны». Последующие находки с более четкими оттисками показали, что они имеют общий сюжет, по изображе- ниям выделено три штампа. 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. 1 – Ан- тропоморфные фигурки повернуты в профиль друг к другу; головы смы- каются – показан один нос, два глаза; ноги перекрещены; руки протянуты друг к другу; у левой фигуры выделен признак мужского пола (рис. 8: 1–3). 2 – Головы как в предыдущем вари- анте, фигуры слиты в одно туловище, ноги не перекрещены, в центре туло- вища – овал, где сверху обозначены две руки по диагонали вниз, в цен- тре – сердцевидная фигура (рис. 8: 4). 3 – Фигуры вполоборота, головы смыкаются, обозначено два носа и два глаза; руки вытянуты горизонтально в позе объятия, на более отчетливых оттисках видны пальцы; ноги пере- крещены (рис. 8: 8–11) – этот штамп наиболее распространен (9 ножен из 16). Сюжет изображения известен в восточном декоративном искусстве и На ножнах из грабительских рас- копок в Пермском крае птичка слег- ка отличается: шея выгнута дугой, туловище и внутренняя часть хвоста покрыты горизонтально вытянутыми овалами (рис. 8: 14). Птички напоминают павлина или фазана, скорее всего, они также заим- ствованы с предметов восточной то- ревтики или с привозных тканей. Пока ножны с тисненым орнамен- том найдены лишь на Баяновском мо- гильнике. Очевидно, они были про- дукцией одного ремесленного центра, о чем свидетельствует конструктив- ное сходство изделий и использова- ние стандартных штампов. Судя по датам монет, найденных в комплексах с ножнами и соседних погребениях, они бытовали в период второй поло- вины IX – первой половины X вв.5 83 83 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 Рис. 8. 1–11 – тисненые изображения антропоморфов в позе коитуса: 1–2 – п.492, 3 – п.124, 4 – п.127, 5 – п.106, 6 – п.404, 7 – п.62, 8 – п.92, 9 – п.480, 10 – п.440, 11 – п.401. 12–14 – тисненые изображения птичек: 12–13 – п.374 Баяновского м-ка, 14 – Пермский край; 15 – оформление чеканным орнаментом, п.99 Баяновского м-ка. Fig. 8. 1–11 – Embossed images of anthropomorphs in the coitus pose. 12–14 – Embossed images of birds, 15 – decoration with hammered ornament. Рис. 8. 1–11 – тисненые изображения антропоморфов в позе коитуса: 1–2 – п.492, 3 – п.124, 4 – п.127, 5 – п.106, 6 – п.404, 7 – п.62, 8 – п.92, 9 – п.480, 10 – п.440, 11 – п.401. 12–14 – тисненые изображения птичек: 12–13 – п.374 Баяновского м-ка, 14 – Пермский край; 15 – оформление чеканным орнаментом, п.99 Баяновского м-ка. Fig. 8. 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. 20: 8; 117: 11; 124: 7) могильни- ков. витии традиции изготовления ножен, истоки которой установить трудно. К.А. Руденко, также предполагая при- камское происхождение этих изделий, видит их прототипы в ножнах усече- но-конической формы VI в. (Руденко 2015, с. 158, илл. 244). Но конструк- тивное отличие этих ножен, хроно- логический разрыв почти в 2 века не позволяет выстраивать эти изделия в один типологический ряд. Не исклю- чено, что прототипом прикамских но- жен стали хазарские образцы, так же как для многих ломоватовских укра- шений (Белавин, Крыласова, 2020). В Верхне-Салтовском могильнике собрана серия ножен с деревянной основой и металлическими оковками, но по структуре и способу крепления они отличаются от ломоватовских (Покровский, 1905, с. 465–492; Михе- ев, 1985, рис. 34). витии традиции изготовления ножен, истоки которой установить трудно. К.А. Руденко, также предполагая при- камское происхождение этих изделий, видит их прототипы в ножнах усече- но-конической формы VI в. (Руденко 2015, с. 158, илл. 244). Но конструк- тивное отличие этих ножен, хроно- логический разрыв почти в 2 века не позволяет выстраивать эти изделия в один типологический ряд. Не исклю- чено, что прототипом прикамских но- жен стали хазарские образцы, так же как для многих ломоватовских укра- шений (Белавин, Крыласова, 2020). В Верхне-Салтовском могильнике собрана серия ножен с деревянной основой и металлическими оковками, но по структуре и способу крепления они отличаются от ломоватовских (Покровский, 1905, с. 465–492; Михе- ев, 1985, рис. 34). Ножны с пластинами, имеющими подобный треугольный выступ, най- дены в п. 953 Танкеевского (Казаков, 1992, рис. 31: 10), п. 5 Веселовского, п. 2 и 6 Юмского (Никитина, 2012, рис. 20: 8; 117: 11; 124: 7) могильни- ков. Заключение Ножны с металлическими обклад- ками бытовали в Пермском Пред- уралье в IX – первой половине X вв., хотя, судя по находке из Огурдинского могильника, они сохранялись до кон- ца ломоватовской культуры (XI в.). Их химико-технологическое единство с иными категориями ювелирных из- делий местного производства, при- сутствие серий ножен на памятниках Пермского Предуралья свидетель- ствуют о местном зарождении и раз- Подобные ножны в металлических обкладках известны в культурах, тес- но взаимосвязанных с ломоватовской. Часть ножен с памятников ранней Волжской Булгарии, бассейна р. Чепу- цы и Ветлужско-Вятского междуречья могла быть продукцией ювелирных мастерских Пермского Предуралья. Но отличающиеся декором ножны производились за его пределами, ве- роятнее всего, на территории полом- ской культуры. 1985 гг. заметно различаются), в част- ности, выкладывание зерни зигзагами неизвестно ни на одном ювелирном изделии. 1–11 – Embossed images of anthropomorphs in the coitus pose. 12–14 – Embossed images of birds, 15 – decoration with hammered ornament. Тисненый орнамент известен в оформлении ножен из п. 163, 191, 218 Варнинского (Семенов, 1980, табл. XXVI: 5–7), п. 70 Поломского II (Останина, 2011, с. 72, 94, 145, рис. 30: 6), п. 3 и 5 Веселовского (Никитина, 2012, с. 11, рис. 12: 5–6; с. 13, рис. 20: 8) могильников. Но его элементами были петельки с завитками на кон- цах, обрамляющие точку или розетку, отдаленно схожие с «якорьками» на прикамских ножнах, возможно, объ- Тисненый орнамент известен в оформлении ножен из п. 163, 191, 218 Варнинского (Семенов, 1980, табл. XXVI: 5–7), п. 70 Поломского II (Останина, 2011, с. 72, 94, 145, рис. 30: 6), п. 3 и 5 Веселовского (Никитина, 2012, с. 11, рис. 12: 5–6; с. 13, рис. 20: 8) могильников. Но его элементами были петельки с завитками на кон- цах, обрамляющие точку или розетку, отдаленно схожие с «якорьками» на прикамских ножнах, возможно, объ- единенные с ними символическим значением. На ножнах Варнинского могильника «поверхность наружной стороны украшена тисненым орна- ментом, устьевая и концевая наклад- ки – пирамидками зерни» (тип 8 по В.А. Семенову) (Семенов, 1980, с. 50, табл. XXVI: 4–7). В ножнах Пермско- го Предуралья никогда не сочетались тисненый и зерно-филигранный орна- мент, ни разу не встречен декор в кон- цевой части ножен. единенные с ними символическим значением. На ножнах Варнинского могильника «поверхность наружной стороны украшена тисненым орна- ментом, устьевая и концевая наклад- ки – пирамидками зерни» (тип 8 по В.А. Семенову) (Семенов, 1980, с. 50, табл. XXVI: 4–7). В ножнах Пермско- го Предуралья никогда не сочетались тисненый и зерно-филигранный орна- мент, ни разу не встречен декор в кон- цевой части ножен. 84 84 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич Тип 4 (рис. 7: 18, 1 экз., п. 99 Ба- яновского м-ка) с чеканным орнамен- том в виде полугорошин по краям пластины в верхней части футляра. На лицевой стороне нижний край пластины имел треугольный выступ (рис. 8: 15). Чеканный орнамент в виде полугорошин распространился в Пермском Предуралье в IX – первой половине XI вв. Его применяли при декорировании погребальных масок, пластинчатых медальонов (Подосё- нова, 2017, с. 100–105), лицевых пла- стин сумочек, сабельных ножен и пр. Ножны с пластинами, имеющими подобный треугольный выступ, най- дены в п. 953 Танкеевского (Казаков, 1992, рис. 31: 10), п. 5 Веселовского, п. 2 и 6 Юмского (Никитина, 2012, рис. 3 Из 57 погребений по полу определено 55, по полу и возрасту – 37, из которых 33 взрос лых, 4 – до 15 лет. р 2 Пол установлен на основе палеоантропологических определений, сделанных н.с. ПФИЦ УрО РАН Н.Г. Брюховой, и по сопровождающему инвентарю. 3 Из 57 погребений по полу определено 55, по полу и возрасту – 37, из которых 33 взрос- лых, 4 – до 15 лет. 4 Определение монет к.и.н. В.С. Кулешова. П. 434 – дирхам 'Аббасиды, ал-Му'тасим би- ллах, аш-Шаш, 219 г. х. (834 г. ч.) и дирхам среднеаббасидского периода (ок. 850–865 гг.). П. 356 – 2 бронзовые монетовидные подвески с приклеенными серебряными брактеатами под Сасанидов, распространенными во вт. половине VIII – перв. половине IX вв. 5 Монеты определены к.и.н. В.С. Кулешовым. Погр. 64 – дирхам ‘Аббасиды, ал-Ма’мун, Мадинат ас-Салам, 217 г. х. (823/833 гг. ч.) – тип, обращавшийся только во второй трети IX века. Погр. 69 – Саманиды, Наср б. Ахмад, Самарканд (?), год потёрт, с именем халифа ал-Муктадира би-ллаха (908–932 гг.), год чеканки в интервале 914–932 гг.; Саманиды, Наср б. Ахмад (914–943 гг.), место чеканки и год стёрты; Саманиды, Наср б. Ахмад, Самарканд, 308 г. х. (920/921 гг. ч.). Погр. 70 – Саманиды, Наср б. Ахмад, Самарканд (?), 32х г. х. (932– 941 гг.), Саманиды, Наср б. Ахмад, Самарканд, 301 г. х. (914 гг.). Погр. 92 – 9 медных мо- нетовидных подвесок с серебряными брактеатами, подражающими сасанидским драхмам Хосрова II, период распространения – вторая половина VIII – первая половина IX вв. Погр. , 4 Определение монет к.и.н. В.С. Кулешова. П. 434 – дирхам 'Аббасиды, ал-Му'тасим би- ллах, аш-Шаш, 219 г. х. (834 г. ч.) и дирхам среднеаббасидского периода (ок. 850–865 гг.). П. 356 – 2 бронзовые монетовидные подвески с приклеенными серебряными брактеатами под Сасанидов, распространенными во вт. половине VIII – перв. половине IX вв. 5 Монеты определены к.и.н. В.С. Кулешовым. Погр. 64 – дирхам ‘Аббасиды, ал-Ма’мун, Мадинат ас-Салам, 217 г. х. (823/833 гг. ч.) – тип, обращавшийся только во второй трети IX века. Погр. 69 – Саманиды, Наср б. Ахмад, Самарканд (?), год потёрт, с именем халифа ал-Муктадира би-ллаха (908–932 гг.), год чеканки в интервале 914–932 гг.; Саманиды, Наср б. Ахмад (914–943 гг.), место чеканки и год стёрты; Саманиды, Наср б. Ахмад, Самарканд, , р р р р 5 Монеты определены к.и.н. В.С. Кулешовым. Погр. 64 – дирхам ‘Аббасиды, ал-Ма’мун, Мадинат ас-Салам, 217 г. х. (823/833 гг. ч.) – тип, обращавшийся только во второй трети IX века. Погр. 69 – Саманиды, Наср б. Ахмад, Самарканд (?), год потёрт, с именем халифа ал-Муктадира би-ллаха (908–932 гг.), год чеканки в интервале 914–932 гг.; Саманиды, Наср б. Ахмад (914–943 гг.), место чеканки и год стёрты; Саманиды, Наср б. Ахмад, Самарканд, 308 г. х. (920/921 гг. ч.). Погр. 70 – Саманиды, Наср б. Ахмад, Самарканд (?), 32х г. х. (932– 941 гг.), Саманиды, Наср б. Ахмад, Самарканд, 301 г. х. (914 гг.). Погр. 92 – 9 медных мо- нетовидных подвесок с серебряными брактеатами, подражающими сасанидским драхмам Хосрова II, период распространения – вторая половина VIII – первая половина IX вв. Погр. 2 Пол установлен на основе палеоантропологических определений, сделанных н.с. ФИЦ УрО РАН Н.Г. Брюховой, и по сопровождающему инвентарю. 4 Определение монет к.и.н. В.С. Кулешова. П. 434 – дирхам 'Аббасиды, ал-Му'тасим би- ллах, аш-Шаш, 219 г. х. (834 г. ч.) и дирхам среднеаббасидского периода (ок. 850–865 гг.). П. 356 – 2 бронзовые монетовидные подвески с приклеенными серебряными брактеатами под Сасанидов, распространенными во вт. половине VIII – перв. половине IX вв. Примечания: 2 2 Пол установлен на основе палеоантропологических определений, сделанных н.с. ПФИЦ УрО РАН Н.Г. Брюховой, и по сопровождающему инвентарю. 3 Из 57 погребений по полу определено 55, по полу и возрасту – 37, из которых 33 взрос- лых, 4 – до 15 лет. 5 Монеты определены к.и.н. В.С. Кулешовым. Погр. 64 – дирхам ‘Аббасиды, ал-Ма’мун, Мадинат ас-Салам, 217 г. х. (823/833 гг. ч.) – тип, обращавшийся только во второй трети IX века. Погр. 69 – Саманиды, Наср б. Ахмад, Самарканд (?), год потёрт, с именем халифа ал-Муктадира би-ллаха (908–932 гг.), год чеканки в интервале 914–932 гг.; Саманиды, Наср б. Ахмад (914–943 гг.), место чеканки и год стёрты; Саманиды, Наср б. Ахмад, Самарканд, 308 г. х. (920/921 гг. ч.). Погр. 70 – Саманиды, Наср б. Ахмад, Самарканд (?), 32х г. х. (932– 941 гг.), Саманиды, Наср б. Ахмад, Самарканд, 301 г. х. (914 гг.). Погр. 92 – 9 медных мо- нетовидных подвесок с серебряными брактеатами, подражающими сасанидским драхмам Хосрова II, период распространения – вторая половина VIII – первая половина IX вв. Погр. 85 85 № 1 (39) 2022 № 2 (40) 2022 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ 266 – дирхам Саманидов, Наср ибн Ахмад, Андабара, 300 г. х. (единицы года пропущены резчиком штемпеля) (914–922 гг.); дирхам Волжской Булгарии, подражание Саманидам, Насру ибн Ахмаду, датируемый второй четвертью X в.; дирхам Саманидов, Наср ибн Ах- мад, Балх, 302 г. х. (914/915). 266 – дирхам Саманидов, Наср ибн Ахмад, Андабара, 300 г. х. (единицы года пропущены резчиком штемпеля) (914–922 гг.); дирхам Волжской Булгарии, подражание Саманидам, Насру ибн Ахмаду, датируемый второй четвертью X в.; дирхам Саманидов, Наср ибн Ах- мад, Балх, 302 г. х. (914/915). ЛИТЕРАТУРА Погребальный инвентарь могильника «Кузинские ху- тора» // Поволжская археология. 2019. № 4 (30). С. 82–98. р р 14. 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Археология повседневности: материальная культура средневеково- го Предуралья. Пермь: ПГГПУ, 2007. 352 с. р ур р 12. Михеев В.К. Подонье в составе Хазарского каганата. Харьков: Вища школа, 198 148 с. . ихеев . . одо е сос а е азарс о о а а а а. ар о : ща о а, 985. 148 с. 13. Никитина Т.Б. Погребальные памятники IX–XI вв. Ветлужско-Вятского междуречья 148 с. 13. Никитина Т.Б. Погребальные памятники IX–XI вв. Ветлужско-Вятского междуречья // А й й В 14 К О 2012 408 13. Никитина Т.Б. Погребальные памятники IX–XI вв. Ветлужско-Вятского междуречья // Археология eвразийских степей. Вып. 14. Казань: Отечество, 2012. 408 с. // Археология eвразийских степей. Вып. 14. Казань: Отечество, 2012. 408 с. 14. Никитина Т.Б., Акилбаев А.В. Погребальный инвентарь могильника «Кузинские ху- // П 2019 №4 (30) С 82 98 р р , 14. Никитина Т.Б., Акилбаев А.В. Информация об авторах: Подосенова Юлия Александровна, кандидат исторических наук, Пермский фе- деральный исследовательский центр УрО РАН (г. Пермь, Россия), старший научный сотрудник, доцент. Пермский государственный гуманитарно-педагогический универ- ситет (г. Пермь, Россия); podosenka@yandex.ru Крыласова Наталья Борисовна, доктор исторических наук, профессор, Перм- ский государственный гуманитарно-педагогический университет (г. Пермь, Россия); главный научный сотрудник, ПФИЦ УрО РАН (г. Пермь, Россия); n.krylasova@mail.ru Данич Андрей Васильевич, научный сотрудник Камской археолого-этнографиче- ской экспедиции, Пермский государственный гуманитарно-педагогический универси- тет (г. Пермь, Россия); adanich@yandex.ru Y.A. Podosenova, N.B. Krylasova, A.V. Danich On the territory of the Perm cis-Urals during the Middle Ages wooden knife sheaths coated with metallic plates became popular. Most sheaths’ decorations were made of metal alloys with a high percentage of silver. Some specimens were decorated with grains, fi ligree, glass or stone encrustations, gild, and printed anthropomorphic and ornitomorphic images. The object of the article is to introduce into scientifi c discourse the sources and research results that will facilitate determining basic technological characteristics and individual features in the sheaths’ functions. Based on source collection and categorization, the authors reveal how diverse the items were, as well as the scale of their production. Coin material facilitated determining the period of the sheaths’ highest popularity, which is the 9th and the fi rst half of the 10th centuries. Nevertheless, there are specimens dating back to the 11th century as well. The analysis of the features of the occurrence in burials, taking into account paleoanthropological data, allowed us to conclude that the sheaths with metal plates was part of a belt set and was used in women's costume as a symbolic and decorative element. Some items made their way from the Perm cis-Urals to the Udmurt cis-Urals (the Polom archaeological culture) and to the territory of Volga Bulgaria. It is possible that there were local production centres for these items in the Udmurt cis-Urals and on the territory of the ancient Mari people. Keywords: archaeology, the Middle Ages, the Perm cis-Urals, Lomovatovo culture, women’s costume, sheath, silver, copper, grain, fi ligree, printing, gilding. ЛИТЕРАТУРА Казань: Фэн, 2018. 144 с. ) р р 25. Krylasova N.B., Podosenova Y.A., Sarapulov A.N. Jewellery techniques in the territory o Рerm Сis-Ural region in the middle ages // Terra Sebvs. 2015. Т. 7. С. 203–219. 86 86 Ю.А. Подосёнова, Н.Б. Крыласова, А.В. Данич Информация об авторах: ) y y ( ) 5. Gening, V. F. 1962. In Surov, E. G. (ed.). Voprosy arkheologii Urala (Problems of the Urals Archaeology) 3. Sverdlovsk: Ural State University, 7–111 (in Russian). REFERENCES Puteshestvie ibn Fadlana: Volzhskii put' ot Bagdada do Bulgara (The Journey of Ibn Fadlan: the Volga Route from Baghdad to Bolgar). Moscow: “Izdatel'skii dom Mardzhani” Publ. (in Russian). ) 21. Rudenko, K. A. 2015. Bulgarskoe serebro (Bulgarian Silver). Series: Drevnosti Biliara (Antiquities of Biliar) II. Kazan: “Zaman” Publ. (in Russian). ) 21. Rudenko, K. A. 2015. Bulgarskoe serebro (Bulgarian Silver). Series: Drevnosti Biliara (Antiquities of Biliar) II. Kazan: “Zaman” Publ. (in Russian). i ( d ) k l k k l ( f 22. Semenov, V. A. 1980. In Gening, V. F. (ed.). Novyi pamiatnik polomskoi kul’tury (New Site of the Polom Culture). Izhevsk: Research Institute at the Council of Ministers of Udmurt ASSR, 5–135 (in Russian). ( ) 23. Talitskiy, M. V. 1940. In Tretyakov P. N. (ed.). Materialy i issledovaniia po arkheologii (Materials and Studies in the Archaeology) 1. Moscow-Leningrad: Academy of Sciences of the USSR, 159–168 (in Russian). ( ) 24. Khalikova, E. A. Khalikov, A. Kh. 2018. In Early Hungarians on the Kama and in the Urals (Bolshie-Tigany burial ground) Series: Arkheologiia evraziiskikh stepei (Archaeology of Eurasian Steppes) 25. Kazan: “Fen” Publ. (in Russian). pp ) ( ) 25. Krylasova, N. B., Podosenova, Y. A., Sarapulov, A. N. 2015. In Terra Sebvs. Vol. 7, 203–219 pp ) ( ) 25. Krylasova, N. B., Podosenova, Y. A., Sarapulov, A. N. 2015. In Terra Sebvs. Vol. 7, 203–219. About the Authors: REFERENCES 1. Belavin, A. M. 2007. In Belavin, A. M. (ed.). Trudy Kamskoi arkheologo-etnografi cheskoi ekspeditsii Permskogo gosudarstvennogo gumanitarno-pedagogicheskogo universiteta (Proceedings of the Kama Archaeological and Ethnographical Expedition of the Perm State Humanitarian Pedagogical University) 4. Perm: Perm State Humanitarian Pedagogical University, 23–50 (in Russian). 1. Belavin, A. M. 2007. In Belavin, A. M. (ed.). Trudy Kamskoi arkheologo-etnografi cheskoi ekspeditsii Permskogo gosudarstvennogo gumanitarno-pedagogicheskogo universiteta (Proceedings of the Kama Archaeological and Ethnographical Expedition of the Perm State Humanitarian Pedagogical University) 4. Perm: Perm State Humanitarian Pedagogical University, 23–50 (in Russian). y) g g y ( ) 2. Belavin, A. M., Krylasova, N. B. 2012. Ogurdinskii mogil'nik (Ogurdino Burial Ground). Perm: Perm State Humanitarian Pedagogical University (in Russian). y) g g y ( ) 2. Belavin, A. M., Krylasova, N. B. 2012. Ogurdinskii mogil'nik (Ogurdino Burial Ground). Perm: Perm State Humanitarian Pedagogical University (in Russian). 3. Belavin, A. M., Krylasova, N. B. 2020. In “Hadak útján” A népvándorláskor fi atal kutatóinak XXIX. konferenciája 29th Conference of Young Scholars on the Migration Period. Főszerk.: Türk A. Ed/Szerk.: Jancsik B. – Sudár B. Studia ad Archaeologiam Pazmaniensia 21. Magyar Őstörténeti Témacsoport Kiadványok 10. Budapest, 73–84 University (in Russian). 4. Botalov, S. G. 2019. U istokov yuzhnoural'skikh narodov. Yuzhnyy Ural v epokhu Zolotoy Ordy (IX – nachalo XV veka)(At the Origins of the South Ural Peoples. Southern Urals in the Era of the Golden Horde (9th – Early 15th Century)) Series: Istoriya Yuzhnogo Urala: v 8 t (History of the Southern Urals: in 8 volumes) 5. Chelyabinsk: South Ural State University (in Russian). ) y y ( ) 5. Gening, V. F. 1962. In Surov, E. G. (ed.). Voprosy arkheologii Urala (Problems of the Ura Archaeology) 3. Sverdlovsk: Ural State University, 7–111 (in Russian). ) y y ( ) 5. Gening, V. F. 1962. In Surov, E. G. (ed.). Voprosy arkheologii Urala (Problems of the Urals Archaeology) 3. Sverdlovsk: Ural State University, 7–111 (in Russian). This work was carried out with the fi nancial support of the RFBR grant, project No. 20-49- 590001 "Medieval jewelry heritage of Perm Krai: stylistic and chemical-technological features", with the support of the Ministry of Education and Science of Perm Krai, agreement No. C-26/1192 dated 19.12.2019, and also within the framework of the topic of the State assignment, the registra- tion number of the topic AAAA19-119032590066-2 87 87 ПОВОЛЖСКАЯ АРХЕОЛОГИЯ ПОВОЛЖСКАЯ АРХЕОЛОГИЯ № 1 (39) 2022 № 2 (40) 2022 6. REFERENCES Mikheev, V. K. 1985. Podon'e v sostave Khazarskogo kaganata (The Don Region in Khazar Khaganate). Kharkiv: “Vishcha shkola” Publ. (in Russian). g g y ( ) 12. Mikheev, V. K. 1985. Podon'e v sostave Khazarskogo kaganata (The Don Region in Khazar Khaganate). Kharkiv: “Vishcha shkola” Publ. (in Russian). g ) ( ) 13. Nikitina, T. B. 2012. Pogrebal’nye pamiatniki IX–XI vv. Vetluzhsko-Viatskogo mezhdurech’ia (Burial Sites of the 9th – 11th Centuries in the Vetluga-Vyatka Interfl uvial Area). Series: Arkheologiya evraziiskikh stepei (Archaeology of Eurasian Steppes) 14. Kazan: “Otechestvo” Publ. (in Russian). 14 Nikiti T B Akilb A V 2019 I P l h k kh l i (V l Ri R i A g ) ( ) 13. Nikitina, T. B. 2012. Pogrebal’nye pamiatniki IX–XI vv. Vetluzhsko-Viatskogo mezhdurech’ia (Burial Sites of the 9th – 11th Centuries in the Vetluga-Vyatka Interfl uvial Area). Series: Arkheologiya evraziiskikh stepei (Archaeology of Eurasian Steppes) 14. Kazan: “Otechestvo” Publ. (in Russian). p ( gy pp ) ( ) 14. Nikitina, T. 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Perm: Perm State Humanitarian Pedagogical University Publ. (in Russian). g g y ( ) 19. Pokrovskiy, A. M. 1905. In Trudy XII Arkheologicheskogo s"ezda v Khar'kove (Proceedings of the 12th Archaeological Congress in Kharkov) 1. Moscow: Tovarishchestvo tipografi i A.I. Mamontova, 465–492 (in Russian). ( ) 20. 2016. Puteshestvie ibn Fadlana: Volzhskii put' ot Bagdada do Bulgara (The Journey of Ibn Fadlan: the Volga Route from Baghdad to Bolgar). Moscow: “Izdatel'skii dom Mardzhani” Publ. (in Russian). ( ) 20. 2016. REFERENCES Gening, V. F., Goldina, R. D. 1970. In Gening, V. F. (ed.). Voprosy arkheologii Urala (Issues of the Ural Archaeology) 9. Pamiatniki lomovatovskoi kul’tury (Sites of the Lomovatovo Culture). Sverdlovsk, 30–56 (in Russian). , ( ) 7. Goldina, R. D. 1985. Lomovatovskaia kul’tura v Verkhnem Prikam’e (The Lomovatovo Culture in the Upper Kama Area). Irkutsk: Irkutsk State University (in Russian). pp ) y ( ) 8. Ivanova, M. G., Kulikov, K. I. 2000. Drevnee iskusstvo Udmurtii. Al'bom (The ancient art Udmurtia. Album). Izhevsk: Udmurt Institute of History, Language and Literature Publ. (in Russian) 9 K k E P 1992 K l’ i V l h k i B l ii ( k l’ i i ii) (C l 8. Ivanova, M. G., Kulikov, K. I. 2000. Drevnee iskusstvo Udmurtii. Al'bom (The ancient art of Udmurtia. Album). Izhevsk: Udmurt Institute of History, Language and Literature Publ. (in Russian). 9. Kazakov, E. P. 1992. Kul’tura rannei Volzhskoi Bolgarii (etapy etnokul’turnoi istorii) (Culture of 8. Ivanova, M. G., Kulikov, K. I. 2000. Drevnee iskusstvo Udmurtii. Al bom (The ancient art of Udmurtia. Album). Izhevsk: Udmurt Institute of History, Language and Literature Publ. (in Russian). 9. Kazakov, E. P. 1992. Kul’tura rannei Volzhskoi Bolgarii (etapy etnokul’turnoi istorii) (Culture of the Early Volga Bulgaria: Stages of the Ethnic-Cultural History). Moscow: “Nauka” Publ. (in Russian). 9. Kazakov, E. P. 1992. Kul’tura rannei Volzhskoi Bolgarii (etapy etnokul’turnoi istorii) (Culture the Early Volga Bulgaria: Stages of the Ethnic-Cultural History). Moscow: “Nauka” Publ. (in Russian the Early Volga Bulgaria: Stages of the Ethnic-Cultural History). Moscow: “Nauka” Publ. (in Russian). 10. Kirpichnikov, A. N. 1966. Drevnerusskoe oruzhie (Early Russian Weapons) 1. Mechi i sabli IX-XIII vv. (Swords and Sabers of 9th–13th Centuries). Series: Svod Arkheologicheskikh Istochnikov (Corpus of Archaeological Sources) E1-36 Moscow; Leningrad: “Nauka” Publ (in Russian) 10. Kirpichnikov, A. N. 1966. Drevnerusskoe oruzhie (Early Russian Weapons) 1. Mechi i sab IX-XIII vv. (Swords and Sabers of 9th–13th Centuries). Series: Svod Arkheologicheskikh Istochniko (Corpus of Archaeological Sources) E1-36. Moscow; Leningrad: “Nauka” Publ. (in Russian). 11. Krylasova, N. B. 2007. Arkheologiya povsednevnosti: material'naya kul'tura srednevekovogo Predural'ya (Archaeology of Everyday Life: The Material Culture of the Medieval Urals). Perm: Perm State Humanitarian Pedagogical University (in Russian). 11. Krylasova, N. B. 2007. Arkheologiya povsednevnosti: material'naya kul'tura srednevekovogo Predural'ya (Archaeology of Everyday Life: The Material Culture of the Medieval Urals). Perm: Perm State Humanitarian Pedagogical University (in Russian). g g y ( ) 12. About the Authors: Podosenova Yulia A. Perm Federal Research Centre of the Uralski branch of the Russian Academy of Sciences. Lenina st., 13A, Perm, 614990, Russian Federation; Perm State Humanitarian Pedagogical University. Sibirskaya Str., 24, Perm, 614990, Russian Federation; podosenka@yandex.ru Podosenova Yulia A. Perm Federal Research Centre of the Uralski branch of the Russian Academy of Sciences. Lenina st., 13A, Perm, 614990, Russian Federation; Perm State Humanitarian Pedagogical University. Sibirskaya Str., 24, Perm, 614990, Russian Federation; podosenka@yandex.ru Krylasova Natalia B. Doctor of Historical Sciences, Associate Professor. Perm State Humanitarian Pedagogical University (PSHPU). Sibirskaya Str., 24, Perm, 614990, Russian Federation. Perm scientifi c center Ural branch of the Russian Academy of Sciences. Lenina st., 13A, Perm, 614990, Russian Federation; n.krylasova@mail.ru Krylasova Natalia B. Doctor of Historical Sciences, Associate Professor. Perm State Humanitarian Pedagogical University (PSHPU). Sibirskaya Str., 24, Perm, 614990, Russian Federation. Perm scientifi c center Ural branch of the Russian Academy of Sciences. Lenina st., 13A, Perm, 614990, Russian Federation; n.krylasova@mail.ru Danich Andrey V. Perm State Humanitarian Pedagogical University. Sibirskaya St., 24, Perm, 614990, Russian Federation; adanich@yandex.ru 88 88
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A test of species’ mobility hypothesis in ecological niche modeling
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Author: Xiao Feng1* 1Department of Geography, Florida State University, Tallahassee, Florida, USA *Correspondence to fengxiao.sci@gmail.com 1 1 1 Title: A test of species’ mobility hypothesis in ecological niche modeling Title: A test of species’ mobility hypothesis in ecological niche modeling Running title: Mobility hypothesis in ENM Author: Xiao Feng1* Keywords: BAM, GIS, SDM, species’ mobility hypothesis, Maxent Abstract Aim: Ecological niche model (ENM) or species distribution model is a modeling technique broadly used in ecology and biogeography and is increasingly used in decision-making regarding land use and biodiversity conservation. The methodology behind ENM applications is critical for model accuracy. One critical question that every ENM study faces is how to define a model training area. Theories have suggested designing a training domain based on species’ dispersal ability for improved model performance (species’ mobility hypothesis). While this idea has been well perceived, there still lacks direct quantitative evidence that whether this approach leads to optimal model performance. Here I conducted a modeling experiment to investigate species’ mobility hypothesis. Location: North and South America Time period: 1950-present Time period: 1950-present Major taxa studied: hummingbird Major taxa studied: hummingbird 2 2 Methods: The modeling experiment was based on a group of hummingbird species. A series of spatial buffers (from 5 to 5000km) were created around occurrences, where background data were sampled and used as input for model calibration. The models calibrated with spatial buffers were compared with models calibrated with training domains that considered species’ dispersal abilities (bioM). Methods: The modeling experiment was based on a group of hummingbird species. A series of spatial buffers (from 5 to 5000km) were created around occurrences, where background data were sampled and used as input for model calibration. The models calibrated with spatial buffers were compared with models calibrated with training domains that considered species’ dispersal abilities (bioM). Results: The experiment showed that model performance increased when the size of the training domain was larger, though the model performance reached saturation when size of the training domain passed a certain threshold. The model performance based on bioM was comparable to the saturation performance of models when spatial buffers were used. Main conclusions: This study provided positive evidence that supports the species’ mobility hypothesis that designing a training domain based on species’ dispersal ability could lead to optimal or near-optimal model performance. When no information of dispersal is available, modelers may use a tuning strategy to identify the size of the training domain for optimized model performance. Keywords: BAM, GIS, SDM, species’ mobility hypothesis, Maxent Keywords: BAM, GIS, SDM, species’ mobility hypothesis, Maxent 3 Introduction Ecological niche model or species distribution model is a modeling technique broadly used in the field of ecology and biogeography. Ecological niche model uses the associations between species’ point observations and environmental conditions of those locations to estimate species’ ecological niche and potential distributions. Typical applications of ecological niche model include: species’ range shifts under climate change (Blowes et al., 2019), range reductions due to habitat loss and anthropogenic disturbance (Doughty et al. 2016), biological invasions (Park and Potter 2015), and the conservation of rare or endangered species (Hannah et al., 2020). While the ecological niche model has had rapid developments over the past decade and is increasingly used in decision-making regarding land use and biodiversity conservation (Araújo et al., 2019), the theory and critical assumptions behind this technique also warrant our attention. While the ecological niche model has had rapid developments over the past decade and is increasingly used in decision-making regarding land use and biodiversity conservation (Araújo et al., 2019), the theory and critical assumptions behind this technique also warrant our attention. The ecological niche theory forms the basis of the ecological niche model, and the more commonly adopted concept of ecological niche is the one by Hutchinson -- a set of environmental conditions permitting the species to live or to exist indefinitely (Hutchinson, 1957). More recently, Soberon and Peterson (2005) conceptualized a BAM framework that greatly influenced the development and use of ecological niche models. The BAM framework classifies the various factors that determine species’ distributions as biotic interactions (B), abiotic conditions (A), and dispersal ability (or mobility; M), and a species is expected to be present in accessible areas with suitable sets of abiotic conditions and biotic interactions. 4 It is critical to use theories to guide the practices of ecological niche modeling to achieve the fullest potential (Peterson & Soberón, 2012). One important assumption in ecological niche modeling is the equilibrium status between environmental conditions and species’ occurrences (Araújo & Pearson, 2005), in other words, a species is present in all suitable locations and absent from unsuitable locations (Soberon & Peterson, 2005). Considering species’ dispersal ability (or mobility/M in BAM) has been broadly acknowledged and used in designing the training domain (or modeling domain) to better fit the equilibrium assumption. Introduction Cooper and Soberon (2018) implemented a series of ecological niche models for hummingbirds based on training domains that reflect topography, ecoregions and known occurrences, refined by significant abiotic barriers of dispersal (e.g. rivers, deep valleys, crests of mountains). They concluded that restricting a training domain to a theoretically accessible area can improve model performance; however, the model assessment was conducted at a stacked level (i.e. community composition) instead of individual species level, whereas the latter was more commonly used in assessing the performance of ecological niche models. To summarize, there is no doubt of the importance of species’ dispersal ability in designing a training domain Barve et al. (2011) conducted simulations of virtual species and training domains of varied sizes, and demonstrated the positive association between size of training domain and (the potentially inflated) the area under the receiver operating characteristic curve (ROC AUC). They provided insightful discussion of the role of considering species’ dispersal ability in model training, validation, comparison, though there was no direct demonstration of those effects. Saupe et al. (2012) used virtual species simulations to compare models calibrated with training domains based on species’ dispersal ability and models calibrated with much broader regions, but the conclusion that the former provided more reliable model performance than the latter was not reached. Their interpretation was that the information of species’ dispersal ability did not directly inform the model fitting process, but only indirectly influenced the model fitting through the selection of pseudo-absences or background data. Owens et al. (2013) proposed a potential modification of how to design a training domain based on species’ dispersal ability; that is to further refine the training domain based on sampling effort, and one simple example is to exclude areas from the training domain that have not been sampled thus can not produce occurrence records. Cooper and Soberon (2018) implemented a series of ecological niche models for hummingbirds based on training domains that reflect topography, ecoregions and known occurrences, refined by significant abiotic barriers of dispersal (e.g. rivers, deep valleys, crests of mountains). They concluded that restricting a training domain to a theoretically accessible area can improve model performance; however, the model assessment was conducted at a stacked level (i.e. community composition) instead of individual species level, whereas the latter was more commonly used in assessing the performance of ecological niche models. Introduction When true absence data are unavailable (Mackenzie, 2005), selecting pseudo-absences or background data from the training domain has been broadly adopted in applications of ecological niche modeling using a variety of algorithms, such as Maxent (Phillips et al. 2009) and generalized linear model (Wintle et al. 2005). In particular, when species’ dispersal ability is considered in designing a training domain, the selected background data are expected to represent the environmental conditions that a species has explored or been exposed to but did not preferably select compared to the environmental conditions of occurrences. The idea of designing a training domain based on species’ dispersal ability has been well perceived and used in the current literature, but there still lacks direct quantitative evidence that whether this approach leads to optimal model performance. For example, Anderson and Raza (2010) compared models using a large training domain and a smaller training domain directly surrounding the localities of the focal species. They concluded that the smaller training domain led to more realistic predictions of species’ potential distributions, though this was mainly based on expert opinions of the species’ natural history and biogeographic knowledge of the region. 5 Barve et al. (2011) conducted simulations of virtual species and training domains of varied sizes, and demonstrated the positive association between size of training domain and (the potentially inflated) the area under the receiver operating characteristic curve (ROC AUC). They provided insightful discussion of the role of considering species’ dispersal ability in model training, validation, comparison, though there was no direct demonstration of those effects. Saupe et al. (2012) used virtual species simulations to compare models calibrated with training domains based on species’ dispersal ability and models calibrated with much broader regions, but the conclusion that the former provided more reliable model performance than the latter was not reached. Their interpretation was that the information of species’ dispersal ability did not directly inform the model fitting process, but only indirectly influenced the model fitting through the selection of pseudo-absences or background data. Owens et al. (2013) proposed a potential modification of how to design a training domain based on species’ dispersal ability; that is to further refine the training domain based on sampling effort, and one simple example is to exclude areas from the training domain that have not been sampled thus can not produce occurrence records. Introduction To summarize, there is no doubt of the importance of species’ dispersal ability in designing a training domain Barve et al. (2011) conducted simulations of virtual species and training domains of varied sizes, and demonstrated the positive association between size of training domain and (the potentially inflated) the area under the receiver operating characteristic curve (ROC AUC). They provided insightful discussion of the role of considering species’ dispersal ability in model training, 6 6 and its implications in model training and validation, but, to my understanding, there is no direct quantitative evidence supporting the optimal model performance by considering species’ dispersal ability. The design of the training domain can also be thought from the perspective of modeling algorithms, which may lead to different expectations of the optimal training domain. As discussed in Saupe et al. (2012), the information of species’ dispersal is used “indirectly” by modeling algorithms, and a training domain is used to generate pseudo-absences or background data, which are used differently as input by different algorithms. Take the popular algorithm of Maxent as an example, background data can be randomly sampled from the training domain. Maxent uses the environmental conditions of the background data to characterize the environmental profile of the training domain. The model prediction of the (relative) probability of presences depends on the contrast between the environmental profile of the background data and occurrences (Merow et al., 2013). In this manner, the hypothesized optimal training domain would be the one that helps the modeling algorithm best distinguish occurrences vs. background data. If so, the question of whether the training domain based on species’ dispersal ability is optimal becomes whether this training domain helps the algorithm distinguish occurrences vs. background data. Following previous explorations, here I continue to investigate the role of species’ dispersal ability in defining a training domain, size of a training domain, and model performance. I proposed three hypotheses regarding the relationship between training domain and model 7 7 performance (Fig. 1). The unimodal hypothesis predicts that model performance shows a unimodal relationship with size of the training domain, whereas a medium sized training domain would lead to an optimal model performance. The linear hypothesis predicts that the model performance increases with increased size of training domain, whereas a larger domain provides more useful information thus better model performance. Introduction The saturation hypothesis predicts increased model performance with increased size of training domain, though there is a bottleneck for the amount of useful information obtained from the training domain, thus model performance will reach saturation when the training domain reaches a certain size. The species’ mobility hypothesis predicts that the training domain designed from species’ mobility would lead to optimal model performance. The species’ mobility hypothesis could be compatible with the unimodal and saturation hypotheses whereas the model performance based on bioM could be either at the peak of the unimodal curve or the plateau of the saturation curve. Here I designed a modeling experiment to test these hypotheses. The experiment used a series of training domains with different size and training domains delineated based on species’ mobility (Cooper & Soberón, 2018). Specifically, I aim to investigate two questions: 1) How would different sized training domains affect model performance? 2) How are the models calibrated with training domains that considered species’ dispersal abilities (bioM) compared with models calibrated with different sized training domains? Methods 8 The occurrences of hummingbird species were obtained from Cooper & Soberón (2018). Briefly, this dataset included occurrences of all hummingbird species (Aves: Trochilidae) that were available from GBIF and eBird in December 2013. This dataset has been filtered and curated by (Cooper & Soberón, 2018) (2018) based on multiple criteria, including taxonomy, ebird observation effort, spatial clustering, and spatial outlier. Here I only kept species with adequate (>100) spatially unique occurrences as a way to guarantee reliable model performance (Proosdij et al., 2016). Eight species that are mainly distributed in islands were excluded from this study, as their restricted range conflict with the study design when large spatial buffers were used (see next section). The final dataset included 87 species. Training domain Two approaches were used to design the training domains, where background points were randomly sampled to reflect the environmental profile of the study area (Fig. 2). In the first approach, the training domain was designed to reflect each species’ accessible aera (or mobility in BAM framework; here termed bioM). The bioM for each species was manually delineated by (Cooper & Soberón, 2018) (2018) to reflect topography, ecoregions and known occurrences, refined by significant abiotic barriers of dispersal (e.g. rivers, deep valleys, crests of mountains). The second approach created a series of 26 spatial buffers of each species’ occurrences to approximate accessible areas with varied dispersal capacities (here termed spatial buffers). The sizes of the spatial buffers were 5km, 7km, 9km, from 10km to 100km with increment of 10km, from 100km to 1000km with increment of 100km, and from 1000 to 5000 with increment of 1000km. The bioM used here represents the hypothesized accessible area, and is expected to lead 9 9 to optimal model performance based on the species’ mobility hypothesis (Barve et al., 2011; Saupe et al., 2012; Owens et al., 2013). In contrast, the spatial buffers cover the locations “around” known occurrences and assume uniform dispersal capacity along all directions. Model training a continent), and random background data from bioM, reflecting a scenario when the model is projected to an area that species is known to occur. Model performance was measured by sensitivity, specificity, and true skill statistics (TSS) at the threshold of 5% training omission rate. Model training Four bioclimatic variables that represent the climatic extremes that often constrain species distributions were used to train models; the variables were the same as those used in Cooper & Soberón (2018). The four variables were: bio10 (mean temperature of the warmest quarter), Four bioclimatic variables that represent the climatic extremes that often constrain species distributions were used to train models; the variables were the same as those used in Cooper & Soberón (2018). The four variables were: bio10 (mean temperature of the warmest quarter), bio11 (mean temperature of the coldest quarter), bio16 (precipitation of the wettest quarter), and bio17 (precipitation of the driest quarter). The climatic data at the resolution of 2.5 arc-minutes were downloaded from Worldclim version 1.4 (Hijmans et al., 2005). From each training domain, ten thousand pixels were randomly selected as background points used for model training. All pixels would be used if the training domain has less than ten thousand pixels. bio11 (mean temperature of the coldest quarter), bio16 (precipitation of the wettest quarter), and bio17 (precipitation of the driest quarter). The climatic data at the resolution of 2.5 arc-minutes were downloaded from Worldclim version 1.4 (Hijmans et al., 2005). From each training domain, ten thousand pixels were randomly selected as background points used for model training. All pixels would be used if the training domain has less than ten thousand pixels. The models were trained using Maxent, a broadly used modeling algorithm in the field of ENM/SDM, using maxnet package version 0.1.4. Linear, quadratic, and hinge features were selected to allow adequate model complexity. Default regularization parameters were used. Fine tuning of model performance was not implemented, as the training domain was the objective of investigation. Clamping was turned off, as its effect could depend on the point where clamping begins (Qiao et al., 2018). 10 The occurrences of each species were separated into four sets to perform cross-validations with three sets for training and one set for testing. The separation was performed with random separation and environmental blocking, to mimic the scenario of with or without model extrapolation. The four sets of occurrences generated by random separation generally had similar spatial extent and environmental conditions, thus no or very little extrapolation would be involved in model prediction. Model training The environmental blocking grouped occurrences into clusters based on their Euclidean distance in the environmental space (Valavi et al., 2019), thus strict or combinational extrapolation could be involved during model prediction (Qiao et al., 2018). Environmental blocking was performed using the blockCV package (version 2.1.4) (Valavi et al., 2019). Two methods were used to generate background data for model evaluation: random background data from the largest buffer (5000km), reflecting a scenario when a calibrated model is projected to a large spatial extent (e.g. a continent), and random background data from bioM, reflecting a scenario when the model is projected to an area that species is known to occur. Model performance was measured by sensitivity, specificity, and true skill statistics (TSS) at the threshold of 5% training omission rate. The occurrences of each species were separated into four sets to perform cross-validations with three sets for training and one set for testing. The separation was performed with random separation and environmental blocking, to mimic the scenario of with or without model extrapolation. The four sets of occurrences generated by random separation generally had similar spatial extent and environmental conditions, thus no or very little extrapolation would be involved in model prediction. The environmental blocking grouped occurrences into clusters based on their Euclidean distance in the environmental space (Valavi et al., 2019), thus strict or combinational extrapolation could be involved during model prediction (Qiao et al., 2018). The occurrences of each species were separated into four sets to perform cross-validations with three sets for training and one set for testing. The separation was performed with random separation and environmental blocking, to mimic the scenario of with or without model extrapolation. The four sets of occurrences generated by random separation generally had similar spatial extent and environmental conditions, thus no or very little extrapolation would be involved in model prediction. The environmental blocking grouped occurrences into clusters based on their Euclidean distance in the environmental space (Valavi et al., 2019), thus strict or combinational extrapolation could be involved during model prediction (Qiao et al., 2018). Environmental blocking was performed using the blockCV package (version 2.1.4) (Valavi et al., 2019). Two methods were used to generate background data for model evaluation: random background data from the largest buffer (5000km), reflecting a scenario when a calibrated model is projected to a large spatial extent (e.g. Regression analyses Stepwise regression was performed to analyze the relationship between buffer size (independent variable) and model performance (dependent variable) using segmented package 1.6-0 (Muggeo, 2003). Preliminary analysis of the relationship showed an increase of model performance with buffer size, though the pattern was commonly accompanied with performance saturation when the buffer size passed a certain threshold. Logistic regression was not used here, because the 11 logistic curve is monotonic (continuously increasing or decreasing) that would not allow the fitting of a unimodal trend. Instead, two segments stepwise regression was used, as it allows the fitted curve to have two segments with different slopes. The buffer size was log10 transformed so that the independent variable could be more evenly sampled. The model performance based on bioM was compared with model performance based on a spatial buffer that was similar to bioM in size (area) using t test; model performance based on bioM was also compared with model performance based on a spatial buffer that had the highest evaluation score among all buffers using t test. These analyses were implemented respectively for each species and the mean of all species, different data separation methods, and different model performance indices. The analyses were performed in R (version 4.1.2). Results The patterns were the same for sensitivity when background points generated from the bioM were used for model evaluation, but specificity showed slightly different patterns (Fig. 3b). Results The model evaluations were based on background data from the largest buffer or bioM, and the two methods showed slightly different but similar patterns. When background points generated from the largest buffer were used for model evaluation, the buffer size used to define the modeling domain was positively associated with model performance (sensitivity, specificity, and TSS). The model performance increased more rapidly during the initial increase of buffer size (e.g from 5km to 50km), after which the model performance gradually reached saturation (Fig. 3a). The same trend was found in random separation and environmental block separation, though environmental block separation generally showed lower sensitivity and TSS but higher specificity compared with random separation. The turning point of the stepwise regression was estimated to be 42-154 km based on mean model performance of all species, or between 42 and 12 130km based on the median of model estimations of individual species (Table 1). The model sensitivity based on bioM was comparable to that of the saturation stage when using spatial buffers (Fig. 3a). There was no significant difference in sensitivity between models based on bioM and models based on spatial buffers with similar size as bioM (Table 2), or between models based on bioM and models based on spatial buffers that had highest sensitivity (Table 3). For a small number of cases, there were significant difference in specificity and TSS between between models based on bioM and models based on spatial buffers with similar size as bioM (Table 2); the number of cases were doubled when comparing models based on bioM to models based on spatial buffers that had highest sensitivity (Table 3). Similar pattern was found for different occurrence separation methods (Fig. 3a). The patterns were the same for sensitivity when background points generated from the bioM were used for model evaluation, but specificity showed slightly different patterns (Fig. 3b). Specificity showed a decreasing trend with buffer size when buffer size reached the turning point (Fig. 3b). The combined effect of sensitivity and specificity led to a more stable saturation status for TSS after buffer size reached the turning point (Fig. 3b). Also, when using background points generated from the bioM for model evaluation (instead of the largest buffer), most of the significant differences in specificity and TSS (between models based on bioM and spatial buffer) disappeared (Tables 2 & 3). Which hypothesis is supported? 13 This study conducted an experiment to investigate the relationship between size of training domain and model performance, as well as whether defining training domain based on species mobility leads to optimal model performance. Overall, I concluded that the saturation hypothesis was supported (Fig. 1); i.e. model performance (here, sensitivity and TSS) showed a rapid increase when buffer size, and stabilized at a certain level after buffer size reached the turning point (Fig. 3). The results of specificity showed a trend of increase and decrease when bioM was used in model evaluation, seemingly support the unimodal hypothesis, but together with the results of specificity when largest buffer was in model evaluation, it can be interpreted as that the background data used in model training would have the highest specificity in recognizing itself; in other words, highest specificity occurs when the same background data are used in training and testing. Interestingly, the model performance based on species’ mobility (bioM) was almost always similar to the saturation performance obtained from spatial buffers, suggesting bioM provided reasonably good training domains, thus providing positive evidence to support the species’ mobility hypothesis. Does species’ mobility provide the optimal training domain? The experiment provided positive evidence to support the hypothesized role of species’ mobility in defining the training domain of ENM. The model sensitivity based on species’ mobility (bioM) generally fell in the saturation sensitivity obtained from spatial buffers. In most cases, the model based on bioM was not significantly different from that based on similar sized buffers or buffers with the highest evaluation index. This suggested that bioM can help define a training domain that leads to optimal or near-optimal performance (especially sensitivity), though not necessarily superior to spatial buffers. Is it reasonable to use spatial buffers as training domain? larger area can yield higher specificity, and subsequently inflated TSS or AUC values, compared with that of a smaller area for the same model. Is it reasonable to use spatial buffers as training domain? In view of considering species’ mobility in the application of ENM/SDM, multiple approaches of designing the training domain have been used in literature, such as using ecoregions, convex hull of species’ occurrences, or expert range maps. Building spatial buffers around known occurrences is another approach that is commonly used in literature. It provides a relatively simple way to delineate an area around known occurrences as the training domain. The size of the spatial buffer (or radius) can be set to reflect the dispersal ability of a species, thus better 14 meet the theoretical consideration of species’ mobility. The spatial buffer could lead to relatively smoothed boundaries (e.g. a circle) that assume uniform dispersal ability along all directions and do not capture fine scale dispersal barriers, compared with fine tuned area based on dispersal simulations (Machado-Stredel et al., 2021). The spatial buffer appcoach is a relatively simple method, but is it a reasonable approach for defining the training domain? The experiment showed models built with small spatial buffers had lowest sensitivity, suggesting using background points immediately next to known occurrences does not provide enough useful information for the model to recognize the difference between the environmental conditions a species preferred vs. background conditions. On the other hand, models with intermediate and extremely large buffers had similar sensitivity, suggesting a larger buffer can provide more useful information for model calibration, but this improvement saturates at a certain point. Therefore, the buffer method can be considered as a reasonable way to construct a training domain when an appropriate size is used. A caveat to note is that, when using a spatial buffer to generate background data, an extremely large buffer could potentially lead to inflated model specificity, and may mislead model evaluation or model comparison. There are several reasons for this caveat. First, when specificity is calculated from background (pseudo-absence) data, its interpretation becomes the proportion of areas predicted as absence, thus it should be given less weight compared with sensitivity, which is calculated from presence data (Peterson et al., 2008). Second, the value of specificity can depend on the selection of background data used in evaluation. Background data from a 15 larger area can yield higher specificity, and subsequently inflated TSS or AUC values, compared with that of a smaller area for the same model. Implications for future studies Ecological niche modelers always face the question of how to define the training domain. The ecological theory suggests the training domain shall consider species’ dispersal ability, though the information and knowledge of dispersal are largely inadequate (Driscoll et al., 2014), thus limiting the implementation of this idea in ecological niche model applications. Two findings of this study (i.e. the stratution relationship of spatial buffers and model performance, and the similarity between bioM and the stratution performance of spatial buffers) suggests that, when there is no information of species’ dispersal, modelers may explore model performance across a series of training domains based on different sized spatial buffers. This method may help identify the buffer size that leads to the stratution performance among many different sized buffers. This 16 strategy is conceptually similar to other model tuning strategies, such as feature and regularization selection in Maxent (Muscarella et al., 2014; Cobos et al., 2019) and stepwise variable selection in multiple regression (Efroymson). If a rough estimate of dispersal distance across the time of interest is known, this information may also be used as the radius of the spatial buffer and to be further compared with buffers with different sizes. The conclusions here were based on cross-validations of the occurrence and dispersal data of a group of hummingbird species, and future studies can further investigate the role of dispersal ability in ecological niche modeling with other taxons that have adequate knowledge of dispersal as well as independent testing data. Barve, N., Barve, V., Jiménez-Valverde, A., Lira-Noriega, A., Maher, S.P., Peterson, A.T., References: Anderson, R.P. & Raza, A. 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DOI: https://doi.org/10.17161/bi.v2i0.4 Valavi, R., Elith, J., Lahoz-Monfort, J.J. & Guillera-Arroita, G. References: (2019) block CV : An r package for generating spatially or environmentally separated folds for k ‐fold cross‐validation of species distribution models. Methods in Ecology and Evolution, 10, 225–232. Data Accessibility Statement: The data supporting the results will be archived in a public repository and the data DOI will be included at the end of the article. 21 Figure 1. The hypothesized relationship between training domain and model performance. The black curve in each panel represents the unimodal, linear, or saturation relationship between increased size of training domain and model performance. The red dashed line represents the expected model performance based on a training domain that is delineated based on species’ mobility (bioM). Figure 1. The hypothesized relationship between training domain and model performance. The black curve in each panel represents the unimodal, linear, or saturation relationship between increased size of training domain and model performance. The red dashed line represents the expected model performance based on a training domain that is delineated based on species’ mobility (bioM). 22 Figure 2. Comparison of training domains based on species’ accessible aera (bioM; green polygon) and spatial buffers of occurrences (gray polygons). The green points are occurrences of Eutoxeres aquila. A total of 26 sized buffers are used in this study, and this figure only shows examples of 100, 500, 1000, 2000, 3000, 4000, 5000km buffers. Figure 2. Comparison of training domains based on species’ accessible aera (bioM; green polygon) and spatial buffers of occurrences (gray polygons). The green points are occurrences of Eutoxeres aquila. A total of 26 sized buffers are used in this study, and this figure only shows examples of 100, 500, 1000, 2000, 3000, 4000, 5000km buffers. Figure 2. Comparison of training domains based on species’ accessible aera (bioM; green polygon) and spatial buffers of occurrences (gray polygons). The green points are occurrences of Eutoxeres aquila. A total of 26 sized buffers are used in this study, and this figure only shows examples of 100, 500, 1000, 2000, 3000, 4000, 5000km buffers. 23 Figure 3. Model performance averaged across all species. Panel a) shows the sensitivity, specificity, and TSS based on background data from the largest spatial buffer, while those indices in panel b) are based on background data from bioM. Occurrence separation method is represented by different colors: red for environmental block and blue for random. References: The solid lines represent the fitted stepwise regressions of buffer size and model performance. The horizontal dashed lines represent model performance based on bioM. species. Panel a) shows the sensitivity, om the largest spatial buffer, while those indices oM. Occurrence separation method is Figure 3. Model performance averaged across al Figure 3. Model performance averaged across all species. Panel a) shows the sensitivity, specificity, and TSS based on background data from the largest spatial buffer, while those indices in panel b) are based on background data from bioM. Occurrence separation method is represented by different colors: red for environmental block and blue for random. The solid lines represent the fitted stepwise regressions of buffer size and model performance. The horizontal dashed lines represent model performance based on bioM. e 3. Model performance averaged across all species. Panel a) shows the sensitivity, Figure 3. Model performance averaged across all species. Panel a) shows the sensitivity, specificity, and TSS based on background data from the largest spatial buffer, while those indices in panel b) are based on background data from bioM. Occurrence separation method is represented by different colors: red for environmental block and blue for random. The solid lines represent the fitted stepwise regressions of buffer size and model performance. The horizontal dashed lines represent model performance based on bioM. 24 Table 1. Turning point of model performance along buffer size estimated from the two-segment stepwise regression. The turning point is estimated from the stepwise regression of mean model performances of all species. It is also shown as the median of the turning points estimated from model performances of individual species. ode pe o a ces o d v dua spec es. Estimated turning point, when background data from the largest buffer is used Estimated turning point, when background data from bioM is used random environmental block random environmental block Sensitivity mean: 65km median: 58km mean: 154km median: 124km mean: 65km median: 59km mean: 154km median: 130km Specificity mean: 44km median: 47km mean: 42km median: 42km mean: 45km median: 50km mean: 42km median: 42km TSS mean: 46km median: 46km mean: 108km median: 85km mean: 46km median: 50km mean: 108km median: 87km 25 Table 2. Number of cases (species) when a model calibrated with bioM is significantly different from the model based on a spatial buffer of similar size. References: Evaluation based on background data from the largest buffer Evaluation based on background data from bioM random environmental block random environmental block Sensitivity 0/87 0/87 0/87 0/87 Specificity 21/87 7/87 8/87 0/87 TSS 12/87 0/87 8/87 0/87 Table 3. Number of cases (species) when a model calibrated with bioM is significantly different from the model based on a spatial buffer that leads to the highest evaluation index. Evaluation based on background data from the largest buffer Evaluation based on background data from bioM random environmental block random environmental block Sensitivity 0/87 0/87 0/87 0/87 Specificity 61/87 16/87 10/87 0/87 TSS 40/87 1/87 10/87 12/87 Table 2. Number of cases (species) when a model calibrated with bioM is significantly different from the model based on a spatial buffer of similar size. Evaluation based on background data from the largest buffer Evaluation based on background data from bioM random environmental block random environmental block Sensitivity 0/87 0/87 0/87 0/87 Specificity 21/87 7/87 8/87 0/87 TSS 12/87 0/87 8/87 0/87 Table 3. Number of cases (species) when a model calibrated with bioM is significantly different from the model based on a spatial buffer that leads to the highest evaluation index. Evaluation based on background data from the largest buffer Evaluation based on background data from bioM random environmental block random environmental block Sensitivity 0/87 0/87 0/87 0/87 Specificity 61/87 16/87 10/87 0/87 TSS 40/87 1/87 10/87 12/87
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G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression
Neuropsychopharmacology
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1Department of Neuropsychiatry, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan. 2Center for Metabolic Regulation of Healthy Aging, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan. 3Division of Psychiatry and Neuroscience, Institute for Clinical Research, National Hospital Organization Kure Medical Center and Chugoku Cancer Center, Kure 737-0023, Japan. 4Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. 5Department of Pain Control Research, The Jikei University School of Medicine, Tokyo 105-8461, Japan. 6Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo 104-0045, Japan. 7Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan. 8Department of Molecular Brain Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan. 9Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan. 10These authors contributed equally: Naoto Kajitani, Mami Okada-Tsuchioka. ✉email: mtakebayashi@kumamoto-u.ac.jp ARTICLE OPEN G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for d i Naoto Kajitani 1,2,3,10, Mami Okada-Tsuchioka3,10, Asuka Inoue 4, Kanako Miyano5,6, Takeshi Masuda7, Shuken Boku1, Kazuya Iwamoto 8, Sumio Ohtsuki7, Yasuhito Uezono5, Junken Aoki 9 and Minoru Takebayashi1,3✉ Naoto Kajitani 1,2,3,10, Mami Okada-Tsuchioka3,10, Asuka Inoue 4, Kanako Miyano5,6, Takeshi Masuda7, Shuken Boku1, Kazuya Iwamoto 8, Sumio Ohtsuki7, Yasuhito Uezono5, Junken Aoki 9 and Minoru Takebayashi1,3✉ © The Author(s) 2023 Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on β-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant- like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA- seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants. Neuropsychopharmacology (2024) 49:561–572; https://doi.org/10.1038/s41386-023-01727-9 Neuropsychopharmacology (2024) 49:561–572; https://doi.org/10.1038/s41386-023-01727-9 TCAs is the lysophosphatidic acid receptor 1 (LPAR1), a G protein- coupled receptor (GPCR). LPAR1 is one of the six receptors (LPAR1–6) that are activated by lysophosphatidic acid (LPA), a bioactive phospholipid. The receptor is activated by TCAs [3–5] and contributes to emotional behaviors [6, 7]. LPAR1-deficient mice exhibited abnormalities in hippocampal functions and showed a phenotype with depressive and anxious features [7, 8]. However, such a depression-like phenotype was also observed when LPA was administered into rodent brains [9, 10], which seems to contradict our hypothesis. Received: 9 March 2023 Revised: 1 August 2023 Accepted: 24 August 2023 Published online: 6 September 2023 www.nature.com/npp INTRODUCTION A id TCAs is the lysophosphatidic acid receptor 1 (LPAR1), a G protein- coupled receptor (GPCR). LPAR1 is one of the six receptors (LPAR1–6) that are activated by lysophosphatidic acid (LPA), a bioactive phospholipid. The receptor is activated by TCAs [3–5] and contributes to emotional behaviors [6, 7]. LPAR1-deficient mice exhibited abnormalities in hippocampal functions and showed a phenotype with depressive and anxious features [7, 8]. However, such a depression-like phenotype was also observed when LPA was administered into rodent brains [9, 10], which seems to contradict our hypothesis. Antidepressants have been developed to increase monoamine selectivity, as evidenced by serotonin and noradrenaline reuptake inhibitors (SNRIs) and selective serotonin reuptake inhibitors (SSRIs). While SNRIs and SSRIs have reduced side effects and increased tolerability compared to prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), their therapeu- tic effects may be comparable or even lower than those of TCAs [1, 2]. As approximately 30% of depression is treatment-resistant, antidepressants with pharmacological actions other than increas- ing monoamine selectivity need to be developed. Tricyclic antidepressants have been reported to be more effective than SSRIs in treating severe depression [2]; however, the mechanisms underlying their therapeutic effects remain unclear. These reports have suggested that, in addition to modulating monoaminergic neurotransmission, TCAs may act on other therapeutic targets. In this study, we hypothesized that one of the potential targets of LPA binding to LPAR1 not only stimulates G protein signaling but also promotes receptor phosphorylation by G protein-coupled receptor kinases and subsequent binding of β-arrestins, which in turn mediates endocytosis and receptor desensitization [11, 12]. Although GPCRs typically activate both G protein signaling and β-arrestin-mediated endocytosis, some GPCR ligands preferentially activate either of the signaling pathways, a phenomenon referred N. Kajitani et al. 562 and/or Ki16425 (10 mg/kg/day, i.p.), and an SPT was performed subsequently. to as biased agonism [13]. For instance, fingolimod, a β-arrestin- biased agonist of sphingosine 1-phosphate receptor, is a functional antagonist, as it selectively induces desensitization via β-arrestin-mediated endocytosis [14]. LPA may cause functional antagonism of LPAR1 via endocytosis [15], potentially blocking LPAR1 signaling, as observed in LPAR1-deficient mice. Therefore, we sought to determine whether TCAs activate downstream signals of LPAR1 similar to LPA, or whether they activate selective signals distinct from LPA. Additionally, we examined whether LPAR1 may be involved in mediating the behavioral effects of TCAs. nsforming growth factor α (TGFα) shedding assay g g ( ) g y The TGFα shedding assay, which measures the activation of specific GPCR- dependent G protein signaling, was performed as described previously [16], with minor modifications (see supplementary methods). pp y For LPA treatment, we used 18:1 LPA (1-oleoyl-2-hydroxy-sn-glycero-3- phosphate (sodium salt); CAS No. 325465-93-8) throughout all experiments in this study. RNA-seq and data analysis Total RNA was extracted from frozen whole hippocampi using the AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany) according to the manufac- turer’s instructions. Library preparation and RNA-seq were outsourced to Macrogen Japan (Kyoto, Japan; see supplementary methods). For data analysis, raw reads were trimmed using Trimmomatic (v0.39) and then aligned to the mm10 reference genome using STAR (v2.7.9a). Gene expression was quantitated using RSEM (v1.3.3) and TCC-GUI [18] was used to determine the differentially expressed genes. Threshold-free genome- wide transcriptomic overlap analysis was conducted using rank-rank hypergeometric overlap (RRHO2, v1.0). Canonical pathways were gener- ated using Ingenuity Pathway Analysis (IPA, Release Date: 2021-10-22, Qiagen; see supplementary methods). Animals Male C57BL/6J mice and LPAR1-heterozygous mice (B6N(Cg)-Lpar1<tm1- b(EUCOMM)Wtsi>/J, Stock No.27468) were used in this study; the mice were 7–8 weeks old at the beginning of the experiments. Mice were obtained from The Jackson Laboratory Japan (Yokohama, Japan) and maintained with a 12 h light/dark cycle with food and water ad libitum at a controlled temperature (23–25 °C). All experimental procedures were performed in accordance with the Guideline for Animal Experiments in the National Hospital Organization Kure Medical Center and Chugoku Cancer Center (NHOKMCCCC). Protocols were approved by the Animal Research Ethics Committee, NHOKMCCCC (Approval No. 2019-01 and 2020-03). NanoBiT-based β-arrestin1 recruitment assay NanoBiT based β arrestin1 recruitment assay The NanoBiT protein-protein interaction assay-based β-arrestin1 recruit- ment assay was performed as described previously [17], with minor modifications (see supplementary methods). Affinity purification of LPAR1 with TCA-immobilized magnetic beads (TCA-beads) The affinity purification of LPAR1 from the LPAR1-overexpressing membrane lysates was performed using TCA-beads according to standard protocols (see supplementary methods). RESULTS TCAs directly bound to LPAR1 and competitively acted with LPA TCAs directly bound to LPAR1 and competitively acted with LPA p The forced swim test (FST), open field test (OFT) and sucrose preference test (SPT) were performed using standard protocols (see supplementary methods). LPA To investigate whether TCA binds to LPAR1 directly, affinity purification experiments [19] were performed with TCA-beads (schematic representation: Fig. 1A). We attempted to create TCA- beads with amitriptyline, a typical TCA. However, amitriptyline did not have the functional group required for the immobilization with the N-hydroxysuccimide (NHS)-magnetic beads (NHS-beads). Therefore, nortriptyline, which has the same structure as amitripty- line except for the secondary amine, was immobilized (Fig. S1). In this study, nortriptyline-immobilized beads are defined as “TCA- beads.” Unmodified forms of LPAR1 (41 kDa) as well as glycosylated forms of LPAR1 (50–75 kDa) were detected in the LPAR1-overexpressing cell lysates (Fig. 1B). TCA-beads eluted LPAR1 depending on the amount of immobilization. However, TCA-beads did not elute LPAR1 from negative control lysate (Fig. 1B). Next, we investigated whether the binding between TCA- beads and LPAR1 was competitively inhibited by using an Statistical analysis All data are presented as mean ± SEM. Statistical significance was determined using various methods depending on the experimental design. For parametric data sets, we employed one-way ANOVA. For post hoc analysis of one-way ANOVA, we employed Dunnett’s multiple comparisons test when comparing multiple groups against a control group. Tukey’s multiple comparisons test was used for situations where all groups were compared to each other. Sidak’s multiple comparisons test was employed when comparing only selected groups. For comparisons between two groups, the unpaired t-test was used. Two-way ANOVA was employed for experiments involving two independent variables. In cases where data did not meet parametric assumptions, the Kruskal-Wallis test was used, followed by post hoc Dunn’s multiple comparisons test. For experiments involving repeated measurements over time, we used mixed- effects models with post hoc Sidak’s multiple comparisons test. All statistical analyses were performed using GraphPad Prism 9 software. In the figures, significant effects are denoted by asterisks that indicate *P < 0.05, **P < 0.01, and ***P < 0.001. Neuropsychopharmacology (2024) 49:561 – 572 INTRODUCTION A id q y For continuous intrahippocampal injection, mice were anesthetized with isoflurane and surgically implanted with two subcutaneous osmotic minipumps (Alzet model 1004; Durect Corporation, Cupertino, CA, USA) and bilateral guide cannulae (Plastics One, Roanoke, VA, USA) targeting hippocampi. The minipumps were filled with LPA (15 nM in PBS), OMPT (15 nM in 40% DMSO and 60% PBS), PBS (vehicle for LPA), or 40% DMSO- PBS (vehicle for OMPT), and activated the evening before surgery by incubating them at 37 °C in saline to initiate a continuous delivery at 0.11 μL/h over 2 weeks. Bilateral cannulae were delivered into the hippocampus at –2.2 mm posterior to the bregma, ± 1.5 mm lateral to the midline, and –2.0 mm ventral to the surface of the skull. The antibiotic penicillin G (500 units/mouse, i.m.) and the analgesic carprofen (5 mg/kg, i.p.) were administered after surgery. Before surgery, an FST was performed to measure the basal immobility time. Mice were grouped to ensure that there was no bias in basal immobility time. Subsequently, an FST was performed at 1 and 2 weeks after the surgery, followed by an OFT. Immunoblotting I bl i Immunoblotting was performed using standard protocols (see supplemen- tary methods). Drug treatment timeline A Scheme of affinity purification experiment of LPAR1 with TCA-beads. B Immunoblots from affinity-purified lysates using TCA-beads or control beads. The amount of nortriptyline immobilized on the beads was 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpressed RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negative control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with the indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and then eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentration relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001 vs vehicle). Fig. 1 Tricyclic antidepressants directly bind to LPAR1. A Scheme of affinity purification experiment of LPAR1 with TCA-beads. B Immunoblots from affinity-purified lysates using TCA-beads or control beads. The amount of nortriptyline immobilized on the beads was 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpressed RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negative control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with the indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and then eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentration relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001 vs vehicle). and the TCA-beads in a concentration-dependent manner (Fig. 1C). Drug treatment timeline For subchronic amitriptyline treatment, amitriptyline was dissolved in tap water at a concentration of 160 mg/L. The average intake dose of amitriptyline per mouse during the experiment was 12.99 ± 0.19 mg/kg/ day based on the average amount of water consumed and the average weight of the mice used in this study. Before its administration, an FST was performed to measure the basal immobility time. Mice were then grouped to ensure that there was no bias in basal immobility time. Subsequently, an FST was performed at 1 and 2 weeks after administration. For chronic corticosterone (CORT) treatment, mice were treated with CORT (35 mg/L, Tokyo Chemical Industry) or 0.45% hydroxypropyl-β- cyclodextrin (Fujifilm Wako, Osaka, Japan), added to their drinking water, for 7 weeks. The average intake dose of CORT per mouse during the experiment was 7.29 ± 0.68 mg/kg/day. During the last 3 weeks of CORT treatment, mice were injected daily with amitriptyline (10 mg/kg/day, i.p.) Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. 563 Fig. 1 Tricyclic antidepressants directly bind to LPAR1. A Scheme of affinity purification experiment of LPAR1 with TCA-beads. B Immunoblots from affinity-purified lysates using TCA-beads or control beads. The amount of nortriptyline immobilized on the beads was 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpressed RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negative control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with the indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and then eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentration relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001 vs vehicle). 563 563 5 Fig. 1 Tricyclic antidepressants directly bind to LPAR1. A Scheme of affinity purification experiment of LPAR1 with TCA-bea B Immunoblots from affinity-purified lysates using TCA-beads or control beads. Drug treatment timeline The amount of nortriptyline immobilized on the beads w 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpress RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negat control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with t indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and th eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentrati relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 c lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statisti fi l l d h l l h l excessive amount of free ligand. The maximum concentration of competitive ligand was set to 10 times the amount of nortriptyline and the TCA-beads in a concentration-dependent manner (Fig. 1C) Moreover, free LPA, an endogenous agonist of LPAR1, also Fig. 1 Tricyclic antidepressants directly bind to LPAR1. A Scheme of affinity purification experiment of LPAR1 with TCA-beads B Immunoblots from affinity-purified lysates using TCA-beads or control beads. The amount of nortriptyline immobilized on the beads wa 19.3 nmol/mg (+) or 27.3 nmol/mg (++). Two cell lysates were used for affinity purification, namely lysates from human LPAR1-overexpressed RH7777 cells (left) and those from FLAG-tagged human LPAR1-transfected HeLa cells (right). Each parent cell lysate was used as a negative control lysate. C Competitive inhibition of LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 cell lysates were preincubated with the indicated concentrations of nortriptyline or LPA (shown as a ratio to the amount of nortriptyline immobilized on the TCA-beads) and then eluted with TCA-beads. Representative immunoblots are shown above the graph. D Amitriptyline and clomipramine (6.67-fold concentration relative to the amount of nortriptyline immobilized on the TCA-beads) inhibit LPAR1 binding to TCA-beads. LPAR1-overexpressed RH7777 ce lysates were used. A representative immunoblot is shown above the graph. N = 3–4. Data are presented as the means ± SEM. Statistica significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test (**P < 0.01, ***P < 0.001 vs vehicle). Fig. 1 Tricyclic antidepressants directly bind to LPAR1. Drug treatment timeline Moreover, free LPA, an endogenous agonist of LPAR1, also inhibited the binding at lower concentrations than free nortripty- line (Fig. 1C). It is likely that this assay detects the higher affinity of excessive amount of free ligand. The maximum concentration of competitive ligand was set to 10 times the amount of nortriptyline on the beads (0.75 mM). Free nortriptyline, the same ligand immobilized on the beads, inhibited the binding between LPAR1 Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. 564 To confirm the pharmacological results, we used genetically modified LPAR1 heterozygous mice in this study. C57BL/6 J background LPAR1-deficient mice were deemed unsuitable for behavioral experiments, as they exhibited neonatal mortality and weight loss (Fig. S6), which is consistent with a previous report [29]. The expression level of LPAR1 in the brain of these mice was found to be reduced by half compared to wild-type mice [30]. Like the results from Ki16425 treatment, LPAR1 heterozygous mice showed no effect of subchronic amitriptyline administration in the FST (Fig. 3C, D). To assess this with another behavioral test, we employed the SPT using a CORT-induced mouse model of depression [31]. Three weeks of amitriptyline treatment normalized the CORT-induced decrease in sucrose preference, an indicator of anhedonia, but had no effect in mice co-treated with Ki16425 (Fig. 3E, F). Administration of CORT decreased total water intake during the SPT, but neither amitriptyline nor Ki16425 affected the intake (Fig. 3G), suggesting that the changes in preference induced by amitriptyline and Ki16425 were not due to changes in intake. These results indicate that LPAR1 is involved in the behavioral changes induced by subchronic amitriptyline treatment. LPA for LPAR1 than nortriptyline. As the results of such a strategy have been shown previously [20], a competitive assay with free test ligands would likely reveal the direct binding of the ligand to LPAR1. Finally, other free TCAs, such as amitriptyline and clomipramine, also significantly inhibited the binding process (Fig. 1D), suggesting that TCAs directly bind to LPAR1. A previous study using an electrical impedance (ΔZ)-based biosensor reported that amitriptyline activated G protein (increase in ΔZ) via endogenous LPAR1 in label-free C6 cells [3]. LPA significantly increased ΔZ in a dose-dependent manner, which was saturated above 3 μM (Fig. S2A). Drug treatment timeline An additive effect by 25 μM of amitriptyline was observed in the low dose range (<1 μM) of LPA, but not above the saturated dose of LPA (≧3 μM) (Fig. S2A). DAMGO, a ligand for μ-opioid receptors (MOR), also increased ΔZ in a dose-dependent manner in stable MOR-expressing HEK293 cells (Fig. S2B). Moreover, 25 μM of amitriptyline showed a significant additive effect even at a saturated dose of DAMGO (≧1 μM) (Fig. S2B), suggesting that amitriptyline and DAMGO are mediated by distinct receptors. These findings suggest that amitriptyline directly interacts with endogenous LPAR1 in living cells. Characterization of LPAR1 downstream signaling pathways using various LPAR1 agonists g g We examined the effects of commercially available LPAR1 agonists, including VPC31143, phospho-anandamide (pAEA), and OMPT, on the downstream signaling of LPAR1. They showed agonistic activities in both TGFα shedding and β-arrestin recruitment assays (Fig. 4A). Among the three agonists, OMPT exhibited a greater decrease in β-arrestin signaling than in G protein signaling (Fig. 4B). p g g g LPA treatment decreased the cell surface expression of LPAR1 on HEK293 cells, whereas it did not decrease the expression of LPAR1 in β-arrestin1/2-deficient HEK293 cells (Fig. S8A, B). This suggests that LPA induces LPAR1 endocytosis in a β-arrestin-dependent manner. In contrast, OMPT and amitriptyline did not induce β- arrestin-dependent endocytosis (Fig. S8B). Long-term treatment with LPA or OMPT did not decrease the protein level of FLAG-LPAR1 in HEK293 cells or LPAR1 protein in the mouse hippocampus (Fig. S8C, D). These results suggest that LPA reduces the cell surface expression of LPAR1 while retaining LPAR1 intracellularly without degrading it, consistent with previously reported findings [32]; contrarily, OMPT does not reduce cell surface LPAR1 expression. Immunoprecipitation confirmed that recruitment of endogenous β- arrestins to FLAG-LPAR1 was increased in a concentration- dependent manner with LPA treatment, but not with OMPT treatment in HEK293 cells (Fig. S8E). Overall, these results suggest that OMPT is a potent G protein-biased LPAR1 agonist. TCAs, but not other types of antidepressants, acted as a G protein-biased LPAR1 agonist p ote b ased ago st To investigate whether antidepressants have a signaling bias downstream of LPAR1, we measured G protein signaling and β- arrestin response via TGFα shedding assay [16] (Fig. 2A) and NanoBiT-based β-arrestin recruitment assay [17] (Fig. 2B), respec- tively. LPA was used as a reference. Amitriptyline treatment showed LPAR1-specific G protein signaling activity (Emax = 60.9 ± 4.1%), but less β-arrestin recruitment to LPAR1 (Emax = 8.8 ± 1.9%) (Fig. 2C, D), indicating that amitriptyline is biased towards activating G protein signaling (Fig. 2E). Further, we examined whether different types of antidepressants could activate LPAR1 downstream pathways (Fig. S3). Considering that antidepressants can accumulate at concentrations of tens of micromolars in the brain [21–23], antidepressants with Emax > 25% and EC50 < 100 μM were defined as agonists in this study. To quantitatively evaluate the potency of individual signaling activities of each agonist, we used relative intrinsic activity (RAi) values [24, 25]. All tested TCAs showed agonist activity for G protein signaling, whereas the other tested antidepressant drugs, including SNRIs, SSRIs, ketamine, vortioxetine, or trazodone, did not (Fig. 2F). Mianserin and mirtazapine showed agonist activity for β-arrestin signaling but their G protein signaling activities were significantly higher or tended to be higher than their β-arrestin signaling activities (Fig. 2F). Results suggested that the G protein- biased agonism at LPAR1 is unique to TCAs. To investigate the effect of LPAR1 signaling on hippocampal monoamines, we analyzed hippocampal monoamine content. Heterozygous LPAR1 knockout mice exhibited no significant changes in hippocampal monoamine levels compared to wild- type mice, regardless of a 2-week amitriptyline treatment (Fig. S7). These findings suggest that the consistent decrease in LPAR1 signaling may not exert substantial influence on hippo- campal monoamine levels, regardless of the presence or absence of antidepressant treatment. LPAR1 mediated the behavioral effects of amitriptyline p y Next, we determined whether LPAR1 is required for the behavioral effects of amitriptyline using the FST, a commonly used method for evaluating the effects of antidepressants [26]. Acute treatment with amitriptyline decreased immobility in the FST, indicating an antidepressant effect, which was blocked by NAN-190, a 5HT1A antagonist, but not blocked by Ki16425, an LPAR1–3 antagonist (Fig. S4A). We then conducted repeated FSTs to assess the effects of antidepressants over time in the same animals [27, 28]. One- and two-week treatment with amitriptyline significantly reduced immobility time, which could be blocked by Ki16425 (Fig. 3A, B). These results suggest that LPARs may be involved in the long- term effects of amitriptyline. Continuous infusion of OMPT, but not LPA, into the hippocampus induced antidepressant-like effects To investigate whether OMPT induces an antidepressant-like effect, OMPT was continuously infused directly into bilateral hippocampi using osmotic minipumps (Fig. 4C). Two weeks of OMPT infusion significantly decreased immobility time in the FST (Fig. 4D). OFT revealed that OMPT had no effect on distance Continuous infusion of OMPT, but not LPA, into the hippocampus induced antidepressant-like effects y We also conducted the FST for fluoxetine, a typical SSRI. Similar to amitriptyline, acute administration of fluoxetine resulted in a decrease in immobility, which could be attributed to 5HT1A involvement rather than LPARs (Fig. S4B). In contrast, two weeks of fluoxetine treatment led to an increase in immobility, regardless of whether it was combined with Ki16425 (Fig. S5). To investigate whether OMPT induces an antidepressant-like effect, OMPT was continuously infused directly into bilateral hippocampi using osmotic minipumps (Fig. 4C). Two weeks of OMPT infusion significantly decreased immobility time in the FST (Fig. 4D). OFT revealed that OMPT had no effect on distance Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. Fig. 2 Tricyclic and tetracyclic antidepressants, but not other antidepressants, are G protein-biased LPAR1 agonists. A Schematic representation of TGFα shedding assay and B β-arrestin recruitment assay. Dose-response curves of LPA and amitriptyline for the LPAR1- specific C G protein activation and D β-arrestin recruitment. N = 3–4. Data are presented as means ± SEM. E Possible LPAR1 downstream pathways activated by amitriptyline. F Emax values (top; Emax of LPA = 100%) for each antidepressant calculated from dose-response curves and LogRAi values (bottom) for each antidepressant that showed agonist activity (Emax > 25% and EC50 < 100 μM). N = 3–4. Data are presented as the means ± SEM. *P < 0.05 (Unpaired t-test). 565 Fig. 2 Tricyclic and tetracyclic antidepressants, but not other antidepressants, are G protein-biased LPAR1 agonists. A Schematic representation of TGFα shedding assay and B β-arrestin recruitment assay. Dose-response curves of LPA and amitriptyline for the LPAR1- specific C G protein activation and D β-arrestin recruitment. N = 3–4. Data are presented as means ± SEM. E Possible LPAR1 downstream pathways activated by amitriptyline. F Emax values (top; Emax of LPA = 100%) for each antidepressant calculated from dose-response curves and LogRAi values (bottom) for each antidepressant that showed agonist activity (Emax > 25% and EC50 < 100 μM). N = 3–4. Data are presented as the means ± SEM. *P < 0.05 (Unpaired t-test). Fig. 2 Tricyclic and tetracyclic antidepressants, but not other antidepressants, are G protein-biased LPAR1 agonists. A Schematic representation of TGFα shedding assay and B β-arrestin recruitment assay. Dose-response curves of LPA and amitriptyline for the LPAR1- specific C G protein activation and D β-arrestin recruitment. N = 3–4. Data are presented as means ± SEM. Continuous infusion of OMPT, but not LPA, into the hippocampus induced antidepressant-like effects S9–13). OMPT was a potent G protein-biased agonist for all LPARs (Fig. S14). Therefore, the possibility that LPARs other than LPAR1 could be involved in antidepressant effects cannot be excluded. LPAR1 was remarkably expressed in the mouse brain (Fig. S15), suggesting that LPAR1 may be a major contributor to central LPA signaling. Neuropsychopharmacology (2024) 49:561 – 572 Characterization of LPAR2–6 downstream signaling pathways using various antidepressants and LPAR agonists We examined LPAR2–6 activities and found that most, though not all, TCAs have G protein-biased LPAR2–3 agonism; however, there was no effect on LPAR4–6 activities (Fig. S9–13). OMPT was a potent G protein-biased agonist for all LPARs (Fig. S14). Therefore, the possibility that LPARs other than LPAR1 could be involved in antidepressant effects cannot be excluded. LPAR1 was remarkably expressed in the mouse brain (Fig. S15), suggesting that LPAR1 may be a major contributor to central LPA signaling. Characterization of LPAR2 6 downstream signaling pathways using various antidepressants and LPAR agonists We examined LPAR2–6 activities and found that most, though not all, TCAs have G protein-biased LPAR2–3 agonism; however, there was no effect on LPAR4–6 activities (Fig. S9–13). OMPT was a We examined LPAR2–6 activities and found that most, though not all, TCAs have G protein-biased LPAR2–3 agonism; however, there was no effect on LPAR4–6 activities (Fig. S9–13). OMPT was a Neuropsychopharmacology (2024) 49:561 – 572 Neuropsychopharmacology (2024) 49:561 – 572 Continuous infusion of OMPT, but not LPA, into the hippocampus induced antidepressant-like effects E Possible LPAR1 downstream pathways activated by amitriptyline. F Emax values (top; Emax of LPA = 100%) for each antidepressant calculated from dose-response curves and LogRAi values (bottom) for each antidepressant that showed agonist activity (Emax > 25% and EC50 < 100 μM). N = 3–4. Data are presented as the means ± SEM. *P < 0.05 (Unpaired t-test). Fig. 2 Tricyclic and tetracyclic antidepressants, but not other antidepressants, are G protein-biased LPAR1 agonists. A Schematic representation of TGFα shedding assay and B β-arrestin recruitment assay. Dose-response curves of LPA and amitriptyline for the LPAR1- specific C G protein activation and D β-arrestin recruitment. N = 3–4. Data are presented as means ± SEM. E Possible LPAR1 downstream pathways activated by amitriptyline. F Emax values (top; Emax of LPA = 100%) for each antidepressant calculated from dose-response curves and LogRAi values (bottom) for each antidepressant that showed agonist activity (Emax > 25% and EC50 < 100 μM). N = 3–4. Data are presented as the means ± SEM. *P < 0.05 (Unpaired t-test). an anxious behavior. These results suggest that long-term administration of the G protein-biased LPAR1 agonist OMPT induces antidepressant-like effects, whereas mice treated with non-biased agonist LPA may exhibit anxious behavior rather than antidepressant-like effects. traveled (Fig. 4E). Thus, the antidepressant-like effect of OMPT shown in the FST was not due to changes in locomotor activities. Conforming with previous reports [9, 10], infusion of LPA instead tended to increase immobility time in the FST (Fig. 4D), and decreased time spent at the center in the OFT (Fig. 4E), indicating Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. 6 566 Characterization of LPAR2–6 downstream signaling pathways using various antidepressants and LPAR agonists We examined LPAR2–6 activities and found that most, though not all, TCAs have G protein-biased LPAR2–3 agonism; however, there was no effect on LPAR4–6 activities (Fig. S9–13). OMPT was a potent G protein-biased agonist for all LPARs (Fig. S14). Therefore, the possibility that LPARs other than LPAR1 could be involved in antidepressant effects cannot be excluded. LPAR1 was remarkably expressed in the mouse brain (Fig. S15), suggesting that LPAR1 may be a major contributor to central LPA signaling. Characterization of LPAR2–6 downstream signaling pathways using various antidepressants and LPAR agonists We examined LPAR2–6 activities and found that most, though not all, TCAs have G protein-biased LPAR2–3 agonism; however, there was no effect on LPAR4–6 activities (Fig. Long-term administration of LPA and OMPT showed different gene expression patterns antidepressants [39]. The action of LPAR1 may partly explain such high responsiveness of TCAs. Our findings provide new insights into the action mechanism of TCAs and suggest that targeting LPAR1 may be a potential strategy for developing more effective antidepressants. g p p RNA-seq was performed to analyze gene expression changes in the hippocampus after 2 weeks infusion with LPA or OMPT. Threshold-free rank-rank hypergeometric overlap (RRHO) analysis [33, 34] was used to characterize the relationship between gene expression patterns by LPA and OMPT. RRHO heatmaps allow to visualize the pattern and significance of the overlap between two independent datasets. The datasets on each axis were ordered transcripts by differential expression p-values and effect size direction, and colored by -log p-values for overlap between ranked transcripts. RRHO revealed a substantial overlap of discordantly regulated genes between LPA and OMPT (Fig. 5A). IPA [35] using the discordantly overlapping genes revealed four of the top five canonical pathways, predicted to be activated by OMPT, to be associated with downstream signals (Rho and MAPK) of LPAR1 [36] (Fig. 5B and Fig. S16). In contrast, the pathways predicted to be activated by LPA were negatively regulated by LPAR1 [37], suggesting that long-term infusion of OMPT may activate LPAR1 signaling, whereas LPA may suppress the same. p A typical SSRI, fluoxetine, was used in the present study as a comparison with amitriptyline. Both amitriptyline and fluoxetine acutely decreased immobility in the FST, which could be attributed to 5HT1A involvement rather than LPARs, suggesting that LPARs are not involved in the effects of acute antidepressants. In the subsequent experiments, subchronic treatment with amitriptyline significantly reduced immobility time, and this effect was blocked by Ki16425, indicating the potential involvement of LPARs in the long-term effects of amitriptyline. In contrast, two weeks of fluoxetine treatment resulted in an increase in immobility, regardless of whether it was combined with Ki16425. Previous studies have reported strain-dependent effects of long- term fluoxetine administration in the FST. For instance, treatment with fluoxetine for 12 days resulted in decreased immobility time in FST for BALB/cJ mice [40]. Conversely, studies on C57BL6/J mice reported no significant effect on immobility time with long-term fluoxetine administration [41, 42]. Moreover, Ihene et al. reported that chronic administration of fluoxetine increased immobility time in the FST [43]. In contrast, consistent reports indicate that amitriptyline reduces immobility time in the FST, irrespective of the mouse strain [44–47]. During the last 3 weeks of CORT treatment, mice were injected daily with amitriptyline (10 mg/kg/day, i.p.) and/or Ki16425 (10 mg/kg/day, i.p.). N = 15 (vehicle and CORT+Ki16425) and 17 (CORT, CORT+Ami, and CORT+Ami+Ki16425). Statistical significance was calculated using the Kruskal–Wallis test with Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). D Immobility time in the FST. Wild type (WT) and LPAR1 heterozygous (HET) mice were treated with Ami (160 mg/L) in drinking water for 14 days. Repeated FST was performed on days –1, 7, and 14. N = 22 (vehicle in WT), 19 (Ami in WT), 17 (vehicle in HET), and 16 (Ami in HET). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ns: not significant). E Timeline and group design of the corticosterone (CORT)-treated mice. F Percentage of sucrose preference and G total water intake. Mice were treated with CORT (35 mg/L) or vehicle (Veh), added to their drinking water, for 7 weeks. During the last 3 weeks of CORT treatment, mice were injected daily with amitriptyline (10 mg/kg/day, i.p.) and/or Ki16425 (10 mg/kg/day, i.p.). N = 15 (vehicle and CORT+Ki16425) and 17 (CORT, CORT+Ami, and CORT+Ami+Ki16425). Statistical significance was calculated using the Kruskal–Wallis test with Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). Fig. 3 LPAR1 mediates the behavioral effects of amitriptyline (Ami). A Timeline and group design of the experiment is shown in panel B. B Immobility time in the forced swim test (FST). Mice were treated with Ami (160 mg/L), added to their drinking water, and were injected daily with Ki16425 (10 mg/kg/day, i.p.) or vehicle for 14 days. Repeated FST was performed on Day –1, 7, and 14. N = 36. Data are presented as means ± SEM. Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). C Timeline and group design of the experiment shown in panel D. D Immobility time in the FST. Wild type (WT) and LPAR1 heterozygous (HET) mice were treated with Ami (160 mg/L) in drinking water for 14 days. Repeated FST was performed on days –1, 7, and 14. N = 22 (vehicle in WT), 19 (Ami in WT), 17 (vehicle in HET), and 16 (Ami in HET). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ns: not significant). E Timeline and group design of the corticosterone (CORT)-treated mice. F Percentage of sucrose preference and G total water intake. Mice were treated with CORT (35 mg/L) or vehicle (Veh), added to their drinking water, for 7 weeks. Long-term administration of LPA and OMPT showed different gene expression patterns Overall, these reports and our results suggest that long-term administration of amitriptyline may exert a stronger antidepressant effect than fluoxetine in the FST, possibly through the LPARs. g g, y pp We used RRHO analysis to compare the present RNA-seq dataset with the published RNA-seq dataset of the chronic social defeat stress model and TCA-treated mice [38]. Mice subjected to social defeat were divided into subpopulations that exhibited depression-like behaviors (susceptible) and those that did not (resilient). Depression-like behaviors in susceptible mice were ameliorated by the TCA imipramine [38]. The transcription pattern of LPA-injected mice visually showed a concordant overlap with that of susceptible mice, a discordant overlap with that of resilient mice inversely, and no directionally consistent overlap with that of imipramine-treated mice (Fig. 5C). In contrast, the transcription pattern of OMPT-injected mice seemed to show a reduced overlap with that of susceptible mice, and a concordant overlap with those of resilient and imipramine-treated mice (Fig. 5D). These results suggest that transcription pattern in the hippocampus of OMPT-treated mice may be similar to that of stress-resilient mice, while that of LPA-treated mice may be similar to that of depressed mice. g While our study yielded evidence that LPAR1 signaling does not directly affect hippocampal monoamine content, previous reports have explored the potential interactions between monoaminergic signaling and LPAR signaling. For instance, the exogenous LPAR ligand, Gintonin, has been found to stimulate serotonin release from enterochromaffin cells through LPARs [48]. Additionally, TCAs, such as amitriptyline, or LPA has been shown to transactivate fibroblast growth factor receptor (FGFR) and down- stream signaling in C6 glial cells [3]. Similarly, serotonin has been observed to transactivate FGFR signaling [49]. Considering these findings, it is plausible to hypothesize that TCAs, such as amitriptyline, may induce their antidepressant effects through synergistic interactions between LPAR1 and monoamine signaling pathways. Neuropsychopharmacology (2024) 49:561 – 572 Fig. 3 LPAR1 mediates the behavioral effects of amitriptyline (Ami). A Timeline and group design of the experiment is shown in panel B. B Immobility time in the forced swim test (FST). Mice were treated with Ami (160 mg/L), added to their drinking water, and were injected daily with Ki16425 (10 mg/kg/day, i.p.) or vehicle for 14 days. Repeated FST was performed on Day –1, 7, and 14. N = 36. Data are presented as means ± SEM. Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). C Timeline and group design of the experiment shown in panel D. D Immobility time in the FST. Wild type (WT) and LPAR1 heterozygous (HET) mice were treated with Ami (160 mg/L) in drinking water for 14 days. Repeated FST was performed on days –1, 7, and 14. N = 22 (vehicle in WT), 19 (Ami in WT), 17 (vehicle in HET), and 16 (Ami in HET). Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ns: not significant). E Timeline and group design of the corticosterone (CORT)-treated mice. F Percentage of sucrose preference and G total water intake. Mice were treated with CORT (35 mg/L) or vehicle (Veh), added to their drinking water, for 7 weeks. During the last 3 weeks of CORT treatment, mice were injected daily with amitriptyline (10 mg/kg/day, i.p.) and/or Ki16425 (10 mg/kg/day, i.p.). N = 15 (vehicle and CORT+Ki16425) and 17 (CORT, CORT+Ami, and CORT+Ami+Ki16425). Statistical significance was calculated using the Kruskal–Wallis test with Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). N. Kajitani et al. 5 N. Kajitani et al. 567 Fig. 3 LPAR1 mediates the behavioral effects of amitriptyline (Ami). A Timeline and group design of the experiment is shown in panel B. B Immobility time in the forced swim test (FST). Mice were treated with Ami (160 mg/L), added to their drinking water, and were injected daily with Ki16425 (10 mg/kg/day, i.p.) or vehicle for 14 days. Repeated FST was performed on Day –1, 7, and 14. N = 36. Data are presented as means ± SEM. Statistical significance was calculated using one-way ANOVA with Sidak’s multiple comparisons test (*P < 0.05, ***P < 0.001, ns: not significant). C Timeline and group design of the experiment shown in panel D. DISCUSSION In the present study, we demonstrated that TCAs directly interact with LPAR1 and exhibit G protein-biased LPAR1 agonism, which may contribute to their antidepressant effects. The agonist activity of LPAR1 was found to be unique to TCAs. Previous clinical findings have suggested that TCAs may be more effective than other types of antidepressants. For example, a network meta- analysis of 21 antidepressants for the acute phase treatment of major depressive disorder showed that amitriptyline was the most effective antidepressant [1]. Another meta-analysis showed that tricyclic antidepressants are more effective than SSRIs in patients with severe depression [2]. In addition, tricyclic antidepressants were more likely to cause a switch from depression to mania in patients with bipolar depression than other classes of In this study, we showed that TCAs bind to LPAR1 and act as G protein-biased agonists at micromolar concentrations; notably, these concentrations are higher than therapeutic blood concen- trations [50]. However, typical antidepressants accumulate in the brain, and are suggested to reach micromolar concentrations [21–23, 51, 52]. That is, antidepressants may act in the brain at concentrations more than 10 times higher than in the blood. Recently, evidence has indicated that higher concentrations of antidepressants may work in functionally meaningful ways. For example, it has been reported that the gradual increase in brain concentration of antidepressants to the micromolar levels Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. . 4 G protein-biased LPAR1 agonist induces antidepressant-like effects. A Dose-response curves and B logRAi values of LPAR1 agonists PA, VPC, pAEA, and OMPT) for the LPAR1-specific G protein activation and β-arrestin recruitment. N = 3. Data are presented as means ± SEM < 0.05, ***P < 0.001 (Unpaired t-test). C Timeline of the experiments using mice treated with LPA or OMPT intrahippocampally. D Immobility me in the forced swim test (FST) and E time in the center and distance traveled in the open field test (OFT). OMPT or LPA was continuously fused into the hippocampi for 2 weeks using osmotic pumps (concentration in pump: 15 nM, delivery rate: 0.11 μL/h). Repeated FST was rformed on days –1, 7, and 14, and the OFT was performed on Day 15. N = 24. *P < 0.05, ns: not significant (Unpaired t-test). N. Kajitani et al. 568 Fig. 4 G protein-biased LPAR1 agonist induces antidepressant-like effects. DISCUSSION A Dose-response curves and B logRAi values of LPAR1 agonists (LPA, VPC, pAEA, and OMPT) for the LPAR1-specific G protein activation and β-arrestin recruitment. N = 3. Data are presented as means ± SEM. *P < 0.05, ***P < 0.001 (Unpaired t-test). C Timeline of the experiments using mice treated with LPA or OMPT intrahippocampally. D Immobility time in the forced swim test (FST) and E time in the center and distance traveled in the open field test (OFT). OMPT or LPA was continuously infused into the hippocampi for 2 weeks using osmotic pumps (concentration in pump: 15 nM, delivery rate: 0.11 μL/h). Repeated FST was performed on days –1, 7, and 14, and the OFT was performed on Day 15. N = 24. *P < 0.05, ns: not significant (Unpaired t-test). Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. Fig. 5 Transcriptional characterization by continuous administration of LPA or OMPT. A Comparison of RRHO expression patterns in th hippocampus of mice intrahippocampally treated for 2 weeks with LPA or OMPT. B Canonical pathways enriched with discordant overlapping genes are presented in panel A. Pathways with positive z-scores indicate pathways that are predicted to be activated by OMP C Comparisons of RRHO expression patterns in the hippocampus of mice treated with LPA (Y-axis) and chronic social defeat stress mode (susceptible and resilient) treated with or without imipramine (X-axis). D Comparisons of RRHO expression patterns in the hippocampus o mice treated with OMPT (Y-axis) and chronic social defeat stress models treated with or without imipramine (X-axis). N. Kajitani et al. 569 Fig. 5 Transcriptional characterization by continuous administration of LPA or OMPT. A Comparison of RRHO expression patterns in th hippocampus of mice intrahippocampally treated for 2 weeks with LPA or OMPT. B Canonical pathways enriched with discordant 56 Fig. 5 Transcriptional characterization by continuous administration of LPA or OMPT. A Comparison of RRHO expression patterns in the hippocampus of mice intrahippocampally treated for 2 weeks with LPA or OMPT. B Canonical pathways enriched with discordantly overlapping genes are presented in panel A. Pathways with positive z-scores indicate pathways that are predicted to be activated by OMPT. C Comparisons of RRHO expression patterns in the hippocampus of mice treated with LPA (Y-axis) and chronic social defeat stress models (susceptible and resilient) treated with or without imipramine (X-axis). Neuropsychopharmacology (2024) 49:561 – 572 REFERENCES 1. Cipriani A, Furukawa TA, Salanti G, Chaimani A, Atkinson LZ, Ogawa Y, et al. Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis. Lancet. 2018;391:1357–66. 2. 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Identification of a primary target of thalidomide teratogenicity. Science. 2010;327:1345–50. 20. Lopez-Girona A, Mendy D, Ito T, Miller K, Gandhi AK, Kang J, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 2012;26:2326–35. 21. Miyake K, Fukuchi H, Kitaura T, Kimura M, Sarai K, Nakahara T. REFERENCES For example, G protein-biased agonists of MOR trigger conformational changes in the intracellular loop 1 and helix 8 domains of the MOR, thereby possibly impairing β-arrestin binding or signaling [62]. The findings could allow for structure-based design of biased ligands. Recently, agonist-bound structures of human LPAR1 have been identified [63, 64]. Therefore, future analysis of the conformational states of LPAR1 induced by OMPT and TCAs may yield important information for the discovery of novel antidepressant drugs. 7. 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A requirement for membrane cholesterol in the beta-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors. J Cell Sci. 2005;118:5291–304. 11. Urs NM, Jones KT, Salo PD, Severin JE, Trejo J, Radhakrishna H. A requirement for membrane cholesterol in the beta-arrestin- and clathrin-dependent endocytosis Our findings implied that MAPK and Rho-related signaling, which are downstream signals of LPAR1, mediate antidepressant- like effects in the hippocampus. Previous studies have reported that neuronal Rho/ROCK inhibition causes antidepressant-like effects via neuronal morphological alterations [65, 66]. Notably, LPAR1 has been reported to be heterogeneously expressed in the brain, with abundant expression in glial cells, such as oligoden- drocytes and astrocytes, rather than in neurons [30]. As the Rho/ ROCK pathway is important for glial cell proliferation [67], Rho signaling may play a distinct role in exhibiting antidepressant effects in LPAR1-expressing glial cells than in neurons. of LPA1 lysophosphatidic acid receptors. J Cell Sci. 2005;118:5291– 12. Zhou Y, Little PJ, Ta HT, Xu S, Kamato D. Lysophosphatidic acid and its receptors: pharmacology and therapeutic potential in atherosclerosis and vascular disease. Pharmacol Ther. 2019;204:107404. 13. DISCUSSION In contrast, G protein-biased LPAR1 agonists, OMPT and amitriptyline, did not induce β-arrestin- dependent endocytosis. Therefore, long-term administration of LPA might act as a functional antagonist and result in a behavior similar to that in LPAR1-deficient mice, whereas OMPT may be able to continue to activate LPAR1 and induce antidepressant-like effects. Thus, the concept of functional antagonism may provide a mechanism to resolve the previous inconsistent results in which LPAR1-deficient mice and LPA-treated mice both exhibited depression-like behaviors [7–10]. DISCUSSION D Comparisons of RRHO expression patterns in the hippocampus of mice treated with OMPT (Y-axis) and chronic social defeat stress models treated with or without imipramine (X-axis). required for interaction with low-affinity binding targets such as TrkB may be important for the onset of therapeutic effects [53]. LPAR1 may similarly be a target for TCAs at clinically meaningful concentrations. in the adult mouse brain. In addition, the behavioral change induced by amitriptyline in the FST was not observed in LPAR1 heterozygous mice. Therefore, the behavioral effects of amitripty- line are likely mediated, at least in part, by LPAR1 in the brain. Further clarification of the involvement of LPAR2–3 in antide- pressant effects will provide vital information for the development of new antidepressants that target mechanisms other than monoamines. In this study we used Ki16425, which has been reported to pharmacologically mimic the behaviors of LPAR1-deficient mice [54, 55]. Ki16425 inhibits LPAR1 (Ki value; 0.34 μM), but also inhibits LPAR2 and LPAR3 (Ki value; 6.5 μM and 0.93 μM, respectively) [56]. Moreover, some TCAs showed G protein- biased agonism in LPAR2–3 as well as LPAR1. Therefore, it is possible that LPAR2–3, in addition to LPAR1, may be involved in the antidepressant effects of TCAs. A previous review [57] and our data indicate that LPAR1 is the most abundantly expressed LPAR We observed an interesting phenomenon in which long-term treatment with LPAR agonists LPA and OMPT, which differ in their downstream signaling bias, induced conflicting emotional beha- viors and regulated distinct gene expression patterns in the hippocampus. LPA decreased the cell surface LPAR1 in a Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. 570 signaling of LPAR1 in the hippocampus can provide a basis for the development of novel antidepressants exhibiting activities other than monoamine restriction. β-arrestin-dependent manner, suggesting that it may act as a functional antagonist. In contrast, G protein-biased LPAR1 agonists, OMPT and amitriptyline, did not induce β-arrestin- dependent endocytosis. 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The depressogenic-like effect of acute and chronic treatment with dexamethasone and its influence on the activity of antidepressant drugs in the forced swim test in adult mice. Prog Neuropsycho- pharmacol Biol Psychiatry. 2014;54:243–8. ACKNOWLEDGEMENTS We thank K. Hashimoto (Chiba University) for providing (R, S)-ketamine; N. Maeda and K. Matsuura (Kumamoto University) for assisting with experiments; Y. Nakachi Neuropsychopharmacology (2024) 49:561 – 572 N. Kajitani et al. 572 Correspondence and requests for materials should be addressed to Minoru Takebayashi. Correspondence and requests for materials should be addressed to Minoru Takebayashi. (Kumamoto University) for helpful comments on this manuscript; and S Khanna from the Editage group for editing a draft of this manuscript. Reprints and permission information is available at http://www.nature.com/ reprints AUTHOR CONTRIBUTIONS Conceptualization: NK, MO-T, MT; Methodology: MO-T, AI, KI, SO; Investigation: NK, MO-T, KM, TM; Funding acquisition: NK, AI, JA, MT; Supervision: SB, KI, SO, YU, JA; Writing–original draft: NK, MO-T, MT; All authors reviewed, edited, and approved the manuscript. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. FUNDING This work was supported by Japan Society for the Promotion of Science KAKENHI (M.T., 18H02756; A.I., 21H04791, 21H051130; and N.K., 21K07501), Japan Science and Technology Agency grants (A.I., PMJFR215T and JPMJMS2023), AMED-LEAP grant 21gm0010004h9905 (J.A.), SENSHIN Medical Research Foundation (M.T.), and Takeda Science Foundation (N.K.). 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/. Neuropsychopharmacology (2024) 49:561 – 572 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/s41386-023-01727-9. © The Author(s) 2023 Neuropsychopharmacology (2024) 49:561 – 572
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Enhancing the Spin Hall Effect of Cylindrically Polarized Beams
Micromachines
2,024
cc-by
15,404
Citation: Kovalev, A.A.; Nalimov, A.G.; Kotlyar, V.V. Enhancing the Spin Hall Effect of Cylindrically Polarized Beams. Micromachines 2024, 15, 350. https://doi.org/10.3390/mi15030350 micromachines micromachines micromachines micromachines Alexey A. Kovalev 1,2,* , Anton G. Nalimov 1,2 and Victor V. Kotlyar 1,2 Alexey A. Kovalev 1,2,* , Anton G. Nalimov 1,2 and Victor V. Kotlyar 1,2 1 Image Processing Systems Institute, NRC “Kurchatov Institute”, 151 Molodogvardeyskaya Str., 443001 Samara, Russia; anton@ipsiras.ru (A.G.N.); kotlyar@ipsiras.ru (V.V.K.) 2 Samara National Research University, 34 Moskovskoye Shosse, 443086 Samara, Russia * Correspondence: alanko@ipsiras.ru 1 Image Processing Systems Institute, NRC “Kurchatov Institute”, 151 Molodogvardeyskaya Str., 443001 Samara, Russia; anton@ipsiras.ru (A.G.N.); kotlyar@ipsiras.ru (V.V.K.) 2 Samara National Research University 34 Moskovskoye Shosse 443086 Samara Russia 1 Image Processing Systems Institute, NRC “Kurchatov Institute”, 151 Molodogvardeyskaya Str., 443001 Samara, Russia; anton@ipsiras.ru (A.G.N.); kotlyar@ipsiras.ru (V.V.K.) 2 Samara National Research University, 34 Moskovskoye Shosse, 443086 Samara, Russia * Correspondence: alanko@ipsiras.ru * Correspondence: alanko@ipsiras.ru Abstract: Two linked gear wheels in a micromachine can be simultaneously rotated in opposite directions by using a laser beam that has in its section areas the spin angular momentum (SAM) of the opposite sign. However, for instance, a cylindrical vector beam has zero SAM in the focus. We alter a cylindrical vector beam so as to generate areas in its focus where the SAM is of opposite signs. The first alteration is adding to the cylindrical vector beam a linearly polarized beam. Thus, we study superposition of two rotationally symmetric beams: those with cylindrical and linear polarization. We obtain an expression for the SAM and prove two of its properties. The first property is that changing superposition coefficients does not change the shape of the SAM density distribution, whereas the intensity changes. The second property is that maximal SAM density is achieved when both beams in the superposition have the same energy. The second perturbation is adding a spatial carrier frequency. We study the SAM density of a cylindrical vector beam with a spatial carrier frequency. Due to periodic modulation, upon propagation in space, such a beam is split into two beams, having left and right elliptic polarization. Thus, in the beam transverse section, areas with the spin of different signs are separated in space, which is a manifestation of the spin Hall effect. We demonstrate that such light beams can be generated by metasurfaces, with the transmittance depending periodically on one coordinate. Keywords: spin Hall effect; spin angular momentum; cylindrical vector beam; beam energy; carrier frequency 1. Introduction The mechanism of the spin Hall effect is much similar in paraxial light beams and in focus, but in paraxial beams, the orbital Hall effect cannot arise since the longitudinal component of the electric field is negligibly small, whereas in a focus it becomes significant and, thus, transverse energy flows are generated, rotating in opposite directions (orbital Hall effect). In [12], we studied a Gaussian beam with several polarization singularities residing on a ring with a center on the optical axis. Such a beam can be constructed as a superposition of a radially polarized single-ringed Laguerre- Gaussian (LG) beam with a linearly polarized Gaussian beam. In [13], we investigated a coaxial superposition of two paraxial single-ringed LG beams with topological charges (TC) n and −n and linearly polarized along the horizontal axis, and of two LG beams with the TCs m and −m and linearly polarized along the vertical axis. y p g The work [12] shows that perturbation of a cylindrical vector beam by superposing a Gaussian beam, or other types of perturbations, can give rise to the spin Hall effect. The above-listed works do not consider the propagation of a paraxial vector beam with one-dimensional periodical modulation, and the longitudinal component of the SAM vector is not calculated for such a beam. In addition, these works do not study the generation of two vortex Gaussian beams with left and right circular polarization from a vector field with one-dimensional periodical modulation. In contrast to [10], generating light fields like those from [9,12,13] is more challenging since it requires modulating not only the phase distribution but polarization as well. To achieve this goal, metasurfaces can be employed. Separation of left-hand and right-hand elliptically polarized components of a light field by using a metasurface was implemented for the first time in work [14]. In this work, propagation of a near-infrared (1.2–1.7 µm) light field was studied through a metasurface composed of periodic gold V-shape nanoan- tennas. In the far field, the third component of the Stokes vector was measured. It was demonstrated that two areas were generated at the edges of the light beam, where the Stokes component had different signs. Thus, areas with left and right elliptic polarization are present in the beam. In [15,16], a metalens was used to enhance the transverse shift of beams with left and right circular polarizations. 1. Introduction Elements of micromachines can be driven by light, including structured light with phase and/or polarization singularities. Thus, of great interest are light fields with orbital and spin angular momentum (OAM and SAM). The SAM causes particles to rotate around their centers of mass [1], and engineering the SAM density can manipulate an ensemble of particles simultaneously. For generating a light field with a specific distribution of the SAM density, the initial field does not necessarily carry nonzero SAM. The optical spin Hall effect is manifested when a zero-SAM beam with complete linear polarization acquires non-zero SAM density as it propagates in space [2,3]. In addition to the spin Hall effect, the optical Hall effect can also be orbital [4,5] or spin-orbital [6]. Academic Editor: Yuankun Lin Received: 30 January 2024 Revised: 22 February 2024 Accepted: 28 February 2024 Published: 29 February 2024 The spin Hall effect can be radial or angular. The radial spin Hall effect was described in [7], where the authors showed that when a radially polarized optical vortex is focused, the longitudinal component of the SAM density vector has different signs at different distances from the optical axis in the focal plane. The angular spin Hall effect is demonstrated in many of our recent works, for instance in [8], where a high-order cylindrical vector beam is tightly focused and near the focal plane; areas with different signs of SAM density vector reside at different polar angles. Separation of left and right circular polarization in the tight focus was also shown for several other types of vector beams [9,10]. As was found in [10], the spin Hall effect arises in the tight focus of a linearly polarized optical vortex. Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/micromachines Micromachines 2024, 15, 350. https://doi.org/10.3390/mi15030350 Micromachines 2024, 15, 350 2 of 17 Such a beam can be easily generated without a metasurface [11] by employing a spatial light modulator. In our work [12,13], we have found that the spin Hall effect can also be achieved in paraxial vector beams that are not tightly focused. 1. Introduction For example, in [16], for enhancing the Hall effect, subwavelength diffraction grating was used. However, the beams with opposite spin were shifted along grating lines. In [17], for the wavelength of 633 nm and by using a q-plate, a vortex beam with linear polarization was split into a Gaussian beam with left circular polarization and a vortex beam with right circular polarization. In works [18–23], metasurface-based polarization converters were studied for millimeter-wave light. For instance, in [20], a metasurface composed of metallic stripes is illuminated by linearly po- larized millimeter-wave irradiation, and two beams with left and right circular polarization are reflected by angles of 30 degrees. In [21], a reflective metasurface was computed for the millimeter range, intended for converting a field with linear polarization into two fields with left-hand and two fields with right-hand circular polarization. A similar metasur- face, but for demonstrating the spin Hall effect at transmission, is considered in [22]. In works [24,25], metasurfaces are investigated for converting the polarization of an infrared radiance, whereas in [26–28]—for visible light. We note that the above-listed works do not study the theoretical propagation of light after passing the metalenses. The spin Hall effect in the above-mentioned works was investigated both in paraxial beams and in the focus, but it manifested itself poorly, i.e., in the beam section, areas with opposite-sign spins were generated, but the spin angular momentum density had a low magnitude, i.e., the polarization ellipses in these areas are strongly elongated. Therefore, a problem arises in finding such initial distributions of linear polarization that would enhance the Hall effect and make the polarization ellipse more circular. Vector light fields are usually generated by using SLM and q-plates, but it is more convenient to combine all the elements into one implemented as a metasurface. Micromachines 2024, 15, 350 3 of 17 3 of 17 In this paper, we investigate two perturbations of a cylindrical vector beam, i.e., we induce two different asymmetries into this beam, that can raise the spin Hall effect. At first, we study the superposition of general-shape cylindrically and linearly polarized beams. Thus, the first constituent beam can have radial, azimuthal, saddle [29], or another cylindrical polarization [30]. 1. Introduction The second beam should have linear polarization in an arbitrary direction, and both beams can have an arbitrary rotationally-symmetric shape, i.e., they can be Laguerre-Gaussian [31], Bessel-Gaussian [32], Hypergeometric-Gaussian beams [33], or some more exotic beams [34] with rotational symmetry. We prove that the spin Hall effect is the strongest, i.e., the spin angular momentum density is maximal, when both constituent beams in the superposition have the same energy. The second perturbation of a paraxial cylindrical vector beam is adding a spatial carrier frequency. This frequency splits the beam into two, and we implemented it with a metalens. An expression is obtained for the longitudinal component of the spin angular momentum density at an arbitrary distance from the waist plane. We also show that such a beam has linear inhomogeneous polarization in the initial plane, but, after propagation in space, two beams are generated, one with left circular polarization and another with right circular polarization, i.e., the spin Hall effect manifests itself almost fully. 2. Spin Angular Momentum of a Superposition of Rotationally Symmetric Beams with Cylindrical and Linear Polarization (5) (5) Micromachines 2024, 15, 350 4 of 17 Decomposing the cylindrically polarized field (2) into two optical vortices of the orders ±m and using the auxiliary integral (5), we obtain the following complex amplitude of the fields (2) and (3) at a distance z: EC(r, φ, z) = (−i)m+1 k z exp  ikr2 2z  × " ∞R 0 A(ρ) exp  ikρ2 2z  Jm  krρ z  ρdρ # cos(mφ + α) cos(mφ + β)  , (6) (6) EL(r, φ, z) = −i k z exp ikr2 2z   ∞ Z 0 B(ρ) exp ikρ2 2z  J0 krρ z  ρdρ   cos γ sin γ  . (7) (7) In a paraxial light beam, only the longitudinal component of the spin angular momen- tum vector is significant. In general, it is equal to In a paraxial light beam, only the longitudinal component of the spin angular momen- tum vector is significant. In general, it is equal to Sz = 2Im  E∗ xEy . (8) (8) For a sum of two fields, given by Equation (1), the SAM density is equal to Sz = 2Im (CCEC,x + CLEL,x)∗CCECy + CLELy  = C2 CSC,z + C2 LSL,z + 2CCCLIm n E∗ C,xEL,y + E∗ L,xEC,y o , (9) (9) where SC,z and SL,z are separate SAM densities of the cylindrically and of the elliptically polarized beams. They are zero, and thus only the third term remains in Equation (9). Substituting here the field components from Equations (6) and (7), we get Sz = 2CCCL  k z 2 [sin γ cos(mφ + α) −cos γ cos(mφ + β)] ×Im ( im " ∞R 0 A(ρ) exp  −ikρ2 2z  Jm  krρ z  ρdρ # × " ∞R 0 B(σ) exp  ikσ2 2z  J0  krσ z  σdσ #) . (10) (10) It is seen that the SAM density is, in general, nonzero. It is also seen that the transverse shape of the SAM density distribution depends on the shape of both constituent beams but is independent of their weights in the superposition, i.e., changing the weights changes the SAM density distribution only in magnitude but not in its shape. 2. Spin Angular Momentum of a Superposition of Rotationally Symmetric Beams with Cylindrical and Linear Polarization We consider here the superpositions of two paraxial vector rotationally symmetric light fields, one with cylindrical polarization and the other with linear polarization: E(r, φ, z) = CCEC(r, φ, z) + CLEL(r, φ, z), (1) (1) E(r, φ, z) = CCEC(r, φ, z) + CLEL(r, φ, z), with (r, φ, z) being the cylindrical coordinates, CC and CL being the superposition coeffi- cients, defining the contributions of the cylindrically and linearly polarized beams. i g y y y p In the initial plane z = 0, the complex amplitudes of both of these fields are given by EC(r, φ, 0) = A(r) cos(mφ + α) cos(mφ + β)  , (2) (2) EL(r, φ, 0) = B(r) cos γ sin γ  . (3) (3) In Equations (2) and (3), A(r) and B(r) are real-valued functions, m is the order of cylin- drical polarization, α and β define its azimuthal angle [30], and γ defines the orientation of linear polarization. p Paraxial propagation in free space is described by the well-known Fresnel transform, which in polar coordinates is given by E(r, φ, z) = −ik 2πz ∞ Z 0 2π Z 0 E(ρ, θ, 0) exp  ik 2z h r2 + ρ2 −2rρ cos(θ −φ) i ρdρdθ. (4) (4) For a scalar vortex field in the form EV(ρ, θ, 0) = G(ρ)exp(imθ), the integral over the angle θ is expressed via the Bessel function, and the Fresnel transform reads as For a scalar vortex field in the form EV(ρ, θ, 0) = G(ρ)exp(imθ), the integral over the angle θ is expressed via the Bessel function, and the Fresnel transform reads as EV(r, φ, z) = (−i)m+1 k z exp ikr2 2z + imφ  ∞ Z 0 G(ρ) exp ikρ2 2z  Jm krρ z  ρdρ. 2. Spin Angular Momentum of a Superposition of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Equation (10) also reveals that the SAM density becomes zero on rings when either the Ex or Ey component becomes zero, or the expression in the second line of Equation (10) becomes real. 3. Energies of Cylindrically and Linearly Polarized Beams for Maximizing the Spin Angular Momentum Density Thus, it is seen that, despite neither the cylindrically nor linearly polarized fields acquiring spin angular momentum upon propagation in free space, their superposition does. We aim to find the weights of the constituent beams in the superposition that yield the maximal SAM. It stands to reason that the SAM density in Equation (10) grows with both the coefficients CC and CL. But it follows that the energy of beam (1) grows as well. Thus, we should fix the energy at some value, e.g., W0. The energy of an arbitrary paraxial vector field can be obtained as W = ∞ Z 0 2π Z 0 h |Ex(r, φ, z)|2 + Ey(r, φ, z) 2i rdrdφ. (11) (11) Micromachines 2024, 15, 350 5 of 17 The energy of the cylindrically polarized beam (2) equals The energy of the cylindrically polarized beam (2) equals WC = ∞ Z 0 A2(r)rdr 2π Z 0 h cos2(mφ + α) + cos2(mφ + β) i dφ = 2π ∞ Z 0 A2(r)rdr, (12) (12) whereas the energy of the linearly polarized beam (3) equals WL = 2π ∞ Z 0 B2(r)rdr. (13) (13) The fields (2) and (3) consist of independent OAM components of the orders ±m and 0. Thus, the full energy of the beam (1) can be obtained as the sum: The fields (2) and (3) consist of independent OAM components of the orders ±m and 0. Thus, the full energy of the beam (1) can be obtained as the sum: W = C2 CWC + C2 LWL. (14) (14) Since the SAM density is proportional to the product CCCL, it can be maximized at an arbitrary point (r, φ, z) for all superpositions (1) with fixed energy W0 by solving the following optimization problem:  CCCL →max, C2 CWC + C2 LWL = W0. (15) (15) The Lagrangian function for this problem reads as The Lagrangian function for this problem reads as The Lagrangian function for this problem reads as L(CC, CL) = CCCL + λ0  C2 CWC + C2 LWL −W0  , (16) (16) where λ0 is the Lagrange multiplier. where λ0 is the Lagrange multiplier. ere λ0 is the Lagrange multiplier. 3. Energies of Cylindrically and Linearly Polarized Beams for Maximizing the Spin Angular Momentum Density g g p Differentiation by CC and CL yields two equations: Differentiation by CC and CL yields two equations: Differentiation by CC and CL yields two equations:  CL + 2λ0CCWC = 0, CC + 2λ0CLWL = 0. (17) (17) Multiplying the first and second equations by CC and CL, respectively, yields Multiplying the first and second equations by CC and CL, respectively, yields C2 CWC = C2 LWL = −CCCL 2λ0 . (18) (18) This means that the energies of both beams in the superposition should be equal to each other and to W0/2. We note that we obtained a similar result (18) earlier for a particular case [12] of a superposition of a cylindrically polarized single-ringed LG beam with a linearly polarized Gaussian beam. In the current work, we generalize the results of the work [12] for a similar superposition, but with arbitrary amplitudes A(r) and B(r) in Equations (2) and (3). 4. Intensity and Spin Density of a Gaussian Vector Field with One-Dimensional Periodical Modulation In this section, we analyze the propagation of a paraxial Gaussian vector beam with the following initial Jones vector: E(x, y) =  cos(αx) sin(αx)  exp  −x2 + y2 w2  , (19) (19) where (x,y) are the Cartesian coordinates in the beam cross-section, w is the waist radius of the Gaussian beam, α = 2π/d is the inverse period or spatial frequency of the field amplitude, and d is the period. The light field can be called the vector field with one- Micromachines 2024, 15, 350 6 of 17 dimensional periodic modulation. The field (19) can be represented as the superposition of a left circularly polarized wave and a right circularly polarized wave: dimensional periodic modulation. The field (19) can be represented as the superposition of a left circularly polarized wave and a right circularly polarized wave:  cos(αx) sin(αx)  e−(r/w)2 = 1 2  eiαx  1 −i  + e−iαx  1 i  e−(r/w)2, (20) (20) with r2 = x2 + y2. In the initial plane, the intensity of light field (19) is described by that of the Gaussian beam: 2 2 I0 = |Ex|2 + Ey 2 = exp  −2x2 + y2 w2  . (21) (21) The Jones vector of field (19) at an arbitrary propagation distance z from the initial plane is given by E(x, y, z) = −i  z0 2zq(z)  exp  ik r2 2z − z0y zw 2 1 q(z)  ×  exp  − 1 q(z) z0x zw −αw 2 2 1 −i  + exp  − 1 q(z) z0x zw + αw 2 2 1 i  , (22) (22) where q(z) = 1 −iz0/z and z0 = kw2/2 is the Rayleigh distance and k is the wavenumber of light. As seen from Equation (22), upon free-space propagation, the Gaussian beam (21) consists of two Gaussian beams, shifted along the x-axis and having left and right circular polarization. The centers of both beams are shifted from the optical axis, being located at points x± = ±(αz/k). If the shift is much larger than the diameter of the beams, then the intensity of vector field (22) is as follows: I(x, y, z) =  z0 z|q(z)| 2 exp  −2  z0r zw|q(z)| 2 − αw √ 2|q(z)| !2 cosh 2αxz0 z|q(z)|2 ! . 5. Cylindrical Vector Beam with Spatial Carrier Frequencyi The light field (19) can be generalized so that upon propagation in free space, it would generate not only the spin Hall effect but also the orbital one. To do this, we consider the initial field with the following Jones vector: En(x, y) =  cos(nφ + αx) sin(nφ + αx)  exp  −x2 + y2 w2  , (27) (27) where φ is the azimuthal angle in the beam transverse section, tan(φ) = y/x, and n is an integer number defining the order of the vector field. The field of (27) can be represented as a sum of two fields with right- and left-handed circular polarizations:  cos(nφ + αx) sin(nφ + αx)  e−(r/w)2 = 1 2  ei(nφ+αx)  1 −i  + e−i(nφ+αx)  1 i  e−(r/w)2. (28) (28) At α = 0, the field of (28) reduces to an nth-order cylindrical vector field [30]. Therefore, for a nonzero value of α, the field of (28) can be called a cylindrical vector field (beam) with a spatial carrier frequency. At an arbitrary propagation distance z from the waist plane, the field of (27) is given by En = En−  1 −i  + En+  1 i  , En±(x, y, z) = −in+1 2  z0 zq(z) q πQ± 2 exp  ∓inψ± + i kr2 2z −Q±  × × h I(n−1)/2(Q±) −I(n+1)/2(Q±) i , q(z) = 1 −i z0 z , tan ψ± = y x± αz k , Q± = (αw)2 8q(z) +  z0r √ 2zw 2 1 q(z) ± αz0x 2zq(z). (29) (29) The functions Iv(x) in Equation (29) are modified Bessel functions. According to Equation (29), the paraxial vector field is composed of two off-axis vortex beams with right- and left-handed circular polarizations, with centers of phase singularities (vortex centers) found at points x± = ±(αz/k). The phase singularity points and the intensity nulls of field (29) coincide. Optical vortices near these points have the opposite-sign topological charges, n and −n. Near the intensity nulls, each component of the light field is, respectively, defined by the amplitude (x + α −iy)n and (x −α + iy)n. We note that if n = 0, then field (29) reduces exactly to field (22). In the initial plane, field (27) has neither orbital angular momentum (OAM) nor spin density. 4. Intensity and Spin Density of a Gaussian Vector Field with One-Dimensional Periodical Modulation (23) (23) For a paraxial field, the SAM density has no transverse components. Thus, the longitudinal component of the spin angular momentum (SAM) [35] of field (22) can be derived in the form For a paraxial field, the SAM density has no transverse components. Thus, the longitudinal component of the spin angular momentum (SAM) [35] of field (22) can be derived in the form Sz = 2Im E∗ xEy  = −  z0 z|q(z)| 2 exp −2  z0r zw|q(z)| 2 −1 2  αw |q(z)| 2! sinh 2αxz0 z|q(z)|2 ! . (24) (24) The spin density normalized by the intensity is given by The spin density normalized by the intensity is given by Sz/I = −tanh 2αxz0 z|q(z)|2 ! . (25) (25) This indicates that the spin of the initial plane, field (19), is zero (Sz = 0), but as light field (22) propagates in free space, two areas of opposite-sign spin are generated, where the normalized spin density is given by (25). This is the simplest way to demonstrate the spin Hall effect and to obtain two opposite-handed circularly polarized focal regions from linearly polarized light. y p g Circular polarization is at points x where the condition Sz = ±I is fulfilled. This, however, can occur only when the argument of the hyperbolic tangent in Equation (25) tends to plus or minus infinity. With the normalized spin density in (25) depending on the propagation distance z from the waist, its maximum is achieved at the Rayleigh distance z = z0. Micromachines 2024, 15, 350 7 of 17 For generating the initial light field (19), a metasurface can be used, which is described by a polarization transformation matrix that rotates the linear polarization vector of the incident field by an angle of θ = αx, since the following identity takes place:  cos(αx) −sin(αx) sin(αx) cos(αx)  1 0  =  cos(αx) sin(αx)  . (26) (26) As seen from (26), such a metasurface rotates the linear polarization of the incident beam by an angle periodically depending on the horizontal coordinate x. As seen from (26), such a metasurface rotates the linear polarization of the incident beam by an angle periodically depending on the horizontal coordinate x. 5. Cylindrical Vector Beam with Spatial Carrier Frequency 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization The case when the cylindrically and linearly polarized beams are, respectively, a si gle-ringed Laguerre-Gaussian beam and a Gaussian beam has been investigated [12 Thus, here we may consider some other paraxial beams. For instance, we can construct cylindrically polarized beam with several light rings as the superposition of two Laguerr Gaussian beams of opposite orders with nonzero radial index and opposite circular p larizations, combining them with a linearly polarized Gaussian beam of different wai radius. The complex amplitude of the cylindrically polarized Laguerre-Gaussian beam the initial plane is given by The case when the cylindrically and linearly polarized beams are, respectively, a single- ringed Laguerre-Gaussian beam and a Gaussian beam has been investigated [12]. Thus, here we may consider some other paraxial beams. For instance, we can construct a cylindrically polarized beam with several light rings as the superposition of two Laguerre-Gaussian beams of opposite orders with nonzero radial index and opposite circular polarizations, combining them with a linearly polarized Gaussian beam of different waist radius. The complex amplitude of the cylindrically polarized Laguerre-Gaussian beam in the initial plane is given by p g y ( ) ( ) ( ) 2 2 1 2 C LG 2 2 0 0 0 cos 2 , , 0 exp sin m m p m r r r r z W L m w w w ϕ ϕ ϕ −         = = −                E , (3 EC(r, φ, z = 0) = W−1/2 LG  r w0 m Lm p 2r2 w2 0 ! 5. Cylindrical Vector Beam with Spatial Carrier Frequencyi On the left of Figure 1b, the pha distribution contains three screw dislocations of opposite sign, confirming that the l ring of the field Ex has a topological charge of n = −3. (a) (b) (a) (b) Figure 1. Intensity distribution of beam (27) with w = 1 mm, n = 3, and α = 0.001k at a distance of from the waist plane, shown by white-yellow rings (a), and polarization distribution over the bea transverse section, shown by ellipses (pink ellipses denote right-handed polarization Sz > 0 and cy ellipses denote left-handed polarization Sz < 0); phase distribution of one transverse component the light field Ex (b). The size of both figures is 30 × 30 mm. Figure 1. Intensity distribution of beam (27) with w = 1 mm, n = 3, and α = 0.001k at a distance of z0 from the waist plane, shown by white-yellow rings (a), and polarization distribution over the beam transverse section, shown by ellipses (pink ellipses denote right-handed polarization Sz > 0 and cyan ellipses denote left-handed polarization Sz < 0); phase distribution of one transverse component of the light field Ex (b). The size of both figures is 30 × 30 mm. (b) (a) Figure 1. Intensity distribution of beam (27) with w = 1 mm, n = 3, and α = 0.001k at a distance of from the waist plane, shown by white-yellow rings (a), and polarization distribution over the bea transverse section, shown by ellipses (pink ellipses denote right-handed polarization Sz > 0 and cy ellipses denote left-handed polarization Sz < 0); phase distribution of one transverse component the light field Ex (b). The size of both figures is 30 × 30 mm. Figure 1. Intensity distribution of beam (27) with w = 1 mm, n = 3, and α = 0.001k at a distance of z0 from the waist plane, shown by white-yellow rings (a), and polarization distribution over the beam transverse section, shown by ellipses (pink ellipses denote right-handed polarization Sz > 0 and cyan ellipses denote left-handed polarization Sz < 0); phase distribution of one transverse component of the light field Ex (b). The size of both figures is 30 × 30 mm. 5. Cylindrical Vector Beam with Spatial Carrier Frequencyi However, upon free-space propagation, the single field splits into two fields, both having the opposite-sign longitudinal SAM and the OAM. In the areas of negative spin (left-handed circular polarization), both the topological charge and the OAM are positive, and vice versa, where the spin is positive (right-handed circular polarization), both the OAM and the topological charge are negative. The total spin and OAM of the whole field remain equal to zero, as is the case for the initial field (27). Figure 1 illustrates the intensity distribution (white-yellow rings) (Figure 1a) and phase distribution of the Ex component (Figure 1b) of beam (27) with w = 1 mm, n = 3, and α = 0.001k at distance z0 from the waist plane. All distributions are obtained by using Micromachines 2024, 15, 350 8 of 17 e 1a) an n = 3 an 8 of 17 e 1a) an n = 3 an a Fresnel transform for the wavelength 532 nm. The polarization distribution pattern in Figure 1a is shown by pink (Sz > 0) and cyan (Sz < 0) ellipses. As seen in Figure 1a, the intensity distribution contains two bright rings, with near-circular polarization inside these rings. Besides, the left and right patterns show opposite-sign polarization (right-handed circular polarization near the left ring and left-handed circular polarization near the right ring). On the right of Figure 1b, three screw dislocations are seen, confirming that the topological charge of the right ring in the field Ex is equal to n = 3. On the left of Figure 1b, the phase distribution contains three screw dislocations of opposite sign, confirming that the left ring of the field Ex has a topological charge of n = −3. Fresnel transform for the wavelength 532 nm. The polarization distribution pattern in Fi ure 1a is shown by pink (Sz > 0) and cyan (Sz < 0) ellipses. As seen in Figure 1a, the intensi distribution contains two bright rings, with near-circular polarization inside these ring Besides, the left and right patterns show opposite-sign polarization (right-handed circul polarization near the left ring and left-handed circular polarization near the right rin On the right of Figure 1b, three screw dislocations are seen, confirming that the topologic charge of the right ring in the field Ex is equal to n = 3. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization exp −r2 w2 0 !cos(mφ) sin(mφ)  , (30) (3 (30)   where w0 is the waist radius, m and p are, respectively, the azimuthal and radial indic defining the topological charge and the number of rings, ( ) m p L x is the Laguerre polyn mial, and WLG is the normalizing factor equal to the energy of the Laguerre-Gaussian bea where w0 is the waist radius, m and p are, respectively, the azimuthal and radial indices defining the topological charge and the number of rings, Lm p (x) is the Laguerre polynomial, and WLG is the normalizing factor equal to the energy of the Laguerre-Gaussian beam and introduced to ensure that the energy of beam (30) is unit: he energy of beam (30) is unit: WLG = πw2 0 2m+1 (m + p)! p! . (31) (31) Micromachines 2024, 15, 350 9 of 17 We suppose that the linearly polarized Gaussian beam is horizontally polarized, and thus, it has the following complex amplitude: ! 2 p We suppose that the linearly polarized Gaussian beam is horizontally polarized, and h h h f ll l l d EL(r, φ, z = 0) = W−1/2 G exp −r2 w2 1 !1 0  , (32) ing complex amplitude: ( ) 2 1 2 L G 2 1 1 , , 0 exp 0 r r z W w ϕ −   = = −      E , (32) (32) (32) where w1 is the waist radius that should be greater than w0 so that the Gaussian beam overlaps the Laguerre-Gaussian beam, and WG is the normalizing factor for reducing the energy of beam (32) to a unit value: where w1 is the waist radius that should be greater than w0 so that the Gaussian beam overlaps the Laguerre-Gaussian beam, and WG is the normalizing factor for reducing the energy of beam (32) to a unit value: WLG = πw2 1 2 . (33) 2 1 LG 2 w W π = . (33) (33) (33) Figure 2 illustrates the intensity and SAM density distributions of several superposi- tions of light beams (30) and (32) with different weight coefficients. Figure 2 illustrates the intensity and SAM density distributions of several superposi- tions of light beams (30) and (32) with different weight coefficients. Figure 2. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Laguerre-Gaussian beams (30) and linearly polarized Gaussian beams (32) with different weight coefficients for the following parameters: wavelength λ = 532 nm, Gaussian beam waist radii w0 = 1 mm and w1 = 5 mm, radial and azimuthal orders of the cylindrically polarized Laguerre-Gaussian beam p = 2 and m = 3, propagation distance from the initial plane z = z0, super- position coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. Figure 2 confirms that the maximal SAM density is achieved when the energies of Figure 2. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Laguerre-Gaussian beams (30) and linearly polarized Gaussian beams (32) with different weight coefficients for the following parameters: wavelength λ = 532 nm, Gaussian beam waist radii w0 = 1 mm and w1 = 5 mm, radial and azimuthal orders of the cylindrically polarized Laguerre-Gaussian beam p = 2 and m = 3, propagation distance from the initial plane z = z0, super- position coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. Figure 2. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Laguerre-Gaussian beams (30) and linearly polarized Gaussian beams (32) with different weight coefficients for the following parameters: wavelength λ = 532 nm, Gaussian beam waist radii w0 = 1 mm and w1 = 5 mm, radial and azimuthal orders of the cylindrically polarized Laguerre-Gaussian beam p = 2 and m = 3, propagation distance from the initial plane z = z0, super- position coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Fi 2 fi th t th i l SAM d it i hi d h th i f Figure 2. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Laguerre-Gaussian beams (30) and linearly polarized Gaussian beams (32) with different weight coefficients for the following parameters: wavelength λ = 532 nm, Gaussian beam waist radii w0 = 1 mm and w1 = 5 mm, radial and azimuthal orders of the cylindrically polarized Laguerre-Gaussian beam p = 2 and m = 3, propagation distance from the initial plane z = z0, super- position coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. g y g both constituent beams are the same, i.e., CC = CL. It also confirms that the SAM density changes with the weight coefficients only by magnitude rather than shape. Another example of the composite light field in (1) is when a cylindrically polarized Figure 2 confirms that the maximal SAM density is achieved when the energies of both constituent beams are the same, i.e., CC = CL. It also confirms that the SAM density changes with the weight coefficients only by magnitude rather than shape. p p g ( ) y y p beam is constructed as the superposition of two Bessel-Gaussian beams [32] of the orders ±m and combined with a ring-shaped non-vortex beam. The complex amplitude of the cylindrically polarized Bessel-Gaussian beam in the initial plane is given by ( ) 2     g gi y y g p Another example of the composite light field in (1) is when a cylindrically polarized beam is constructed as the superposition of two Bessel-Gaussian beams [32] of the orders ±m and combined with a ring-shaped non-vortex beam. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Figure 3 depicts the intensity and SAM density distributions of several superpositions of light beams (34) and (36) with different weight coefficients. Figure 3 depicts the intensity and SAM density distributions of several superposi- tions of light beams (34) and (36) with different weight coefficients. Figure 3. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Bessel-Gaussian beams (34) and linearly polarized difference of two Gaussian beams (36) with different weight coefficients for the following parameters: wavelength λ = 532 nm, waist radius of the Gaussian envelope of the Bessel-Gaussian beam w0 = 1 mm, scaling factor α0 = k/1000, order of cylindrical polarization m = 5, waist radii of the subtracted linearly polarized Gauss- ian beams w01 = 5 mm and w02 = 7 mm (at these radii the light ring of the difference beam has the same radius as that of the Bessel-Gaussian beam), propagation distance from the initial plane z = z0, superposition coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. Figure 3 also confirms that in this case when both beams in superposition (1) are Figure 3. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylindri- cally polarized Bessel-Gaussian beams (34) and linearly polarized difference of two Gaussian beams (36) with different weight coefficients for the following parameters: wavelength λ = 532 nm, waist radius of the Gaussian envelope of the Bessel-Gaussian beam w0 = 1 mm, scaling factor α0 = k/1000, order of cylindrical polarization m = 5, waist radii of the subtracted linearly polarized Gaussian beams w01 = 5 mm and w02 = 7 mm (at these radii the light ring of the difference beam has the same radius as that of the Bessel-Gaussian beam), propagation distance from the initial plane z = z0, superposition coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. Figure 3. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylin- drically polarized Bessel-Gaussian beams (34) and linearly polarized difference of two Gaussian beams (36) with different weight coefficients for the following parameters: wavelength λ = 532 nm, waist radius of the Gaussian envelope of the Bessel-Gaussian beam w0 = 1 mm, scaling factor α0 = k/1000, order of cylindrical polarization m = 5, waist radii of the subtracted linearly polarized Gauss- ian beams w01 = 5 mm and w02 = 7 mm (at these radii the light ring of the difference beam has the same radius as that of the Bessel-Gaussian beam), propagation distance from the initial plane z = z0, superposition coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. Fi 3 l fi th t i thi h b th b i iti (1) Figure 3. Intensity (a–e) and SAM density (f–j) distributions of several superpositions of the cylindri- cally polarized Bessel-Gaussian beams (34) and linearly polarized difference of two Gaussian beams (36) with different weight coefficients for the following parameters: wavelength λ = 532 nm, waist radius of the Gaussian envelope of the Bessel-Gaussian beam w0 = 1 mm, scaling factor α0 = k/1000, order of cylindrical polarization m = 5, waist radii of the subtracted linearly polarized Gaussian beams w01 = 5 mm and w02 = 7 mm (at these radii the light ring of the difference beam has the same radius as that of the Bessel-Gaussian beam), propagation distance from the initial plane z = z0, superposition coefficients CC2 = 0.95, CL2 = 0.05 (a,f), CC2 = 0.70, CL2 = 0.30 (b,g), CC2 = CL2 = 0.50 (c,h), CC2 = 0.30, CL2 = 0.70 (d,i), and CC2 = 0.01, CL2 = 0.99 (e,j). The numbers near the color scales denote the minimal and maximal values. g p p ( ) different from those in Figure 2, the maximal SAM density is still achieved when the en- ergies of both constituent beams are the same, i.e., CC = CL. As shown in Figure 2, the SAM density changes with the weight coefficients only by magnitude rather than shape. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization In order to construct a non-vortex single-ringed light beam, we can subtract two G i b ith diff t i t dii d gi In order to construct a non-vortex single-ringed light beam, we can subtract two Gaussian beams with different waist radii w01 and w02: with Im(ξ) being the modified mth-order Bessel function. In order to construct a non-vortex single-ringed light beam, we can subtract two Gau ia bea ith diffe e t ai t adii a d EL(r, φ, z = 0) = W−1/2 DG " exp −r2 w2 01 ! −χ exp −r2 w2 02 !#1 0  , (36) eams with different waist radii w01 and w02: ( ) 2 2 1 2 L DG 2 2 01 02 1 , , 0 exp exp 0 r r r z W w w ϕ χ −       = = − − −               E , (36) (36) (36) where the coefficient χ is chosen so that the field has zero intensity on the optical axis at some distance z: χ = q2/q1 with qi = 1 + iλz/(πw20i), i = 1, 2, and WBG is the normalizing factor for reducing the energy of beam (36) to a unit value: where the coefficient χ is chosen so that the field has zero intensity on the optical axis at some distance z: χ = q2/q1 with qi = 1 + iλz/(πw20i), i = 1, 2, and WBG is the normalizing factor for reducing the energy of beam (36) to a unit value: WDG = πw2 01 2 + πw2 02 2 |χ|2 −2 πw2 01w2 02 w2 01 + w2 02 Reχ. (37) gy ( ) 2 2 2 2 2 01 02 01 02 DG 2 2 01 02 2 Re 2 2 w w w w W w w π π π χ χ = + − + . (37) (37) (37) Figure 3 depicts the intensity and SAM density distributions of several superpositions of light beams (34) and (36) with different weight coefficients. Figure 3 depicts the intensity and SAM density distributions of several superposi- tions of light beams (34) and (36) with different weight coefficients. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization The complex amplitude of the cylindrically polarized Bessel-Gaussian beam in the initial plane is given by ( ) ( ) ( ) ( ) 1 2 C BG 0 2 0 cos , , 0 exp sin m m r r z W J r m w ϕ ϕ α ϕ −     = = −       E , (34) EC(r, φ, z = 0) = W−1/2 BG Jm(α0r) exp −r2 w2 0 !cos(mφ) sin(mφ)  , (34) ( ) (34) where w0 is the waist radius, m is the order of cylindrical polarization, α0 is the scaling factor of the Bessel-Gaussian beam defining the radius of the light ring, Jm(x) is the mth- Micromachines 2024, 15, 350 10 of 17 10 of 17 order Bessel function of the first kind, and WBG is the normalizing factor for reducing the energy of beam (34) to a unit value: factor of the Bessel-Gaussian beam defining the radius of the light ring, Jm(x) is the mth- order Bessel function of the first kind, and WBG is the normalizing factor for reducing the energy of beam (34) to a unit value: WBG = πw2 0 2 exp  − α0w0 2 2 Im α0w0 2 2 , (35) 2 2 2 0 0 0 0 0 BG exp 2 2 2 m w w w W I π α α         = −                     , (35) (35) (35) with Im(ξ) being the modified mth-order Bessel function.     ith I (ξ) b i th difid th d B l f ti with Im(ξ) being the modified mth-order Bessel function.    ith I (ξ) b i th difid th d B l f ti gi In order to construct a non-vortex single-ringed light beam, we can subtract two Gaussian beams with different waist radii w01 and w02: with Im(ξ) being the modified mth-order Bessel function. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization The metasurface should have the following matrix describing the polarization transformation of the incident vector field: metasurface should have the following matrix describing the polarization transfo of the incident vector field: i   ˆR(x) =  cos αx −sin αx sin αx cos αx  , (38) cos sin ˆ( ) sin cos x x R x x x α α α α   =     , (38) where α is inversely proportional to the metasurface structure period along the x-axis. If the incident field is a plane wave linearly polarized along the x-axis, then the polarization direction of the outgoing beam depends on the coordinate x (26). where α is inversely proportional to the metasurface structure period along the x the incident field is a plane wave linearly polarized along the x-axis, then the pola direction of the outgoing beam depends on the coordinate x (26). Shown in Figure 4 is the polarization of the beam (19) at α = π/2 (in inverse microns, since x is measured in µm). Shown in Figure 4 is the polarization of the beam (19) at α = π/2 (in inverse m since x is measured in µm). Figure 4. Direction of linear polarization in the light field of (19). Figure 4. Direction of linear polarization in the light field of (19). Figure 4. Direction of linear polarization in the light field o Figure 4. Direction of linear polarization in the light field of (19). Figure 5 depicts a metasurface relief for implementing the transformation ˆ( ) R x of (26) at α = π/2 (in inverse microns) Figure 5 depicts a metasurface relief for implementing the transformation matrix ˆR(x) of (26) at α = π/2 (in inverse microns). ( ) R x of (26) at α = π/2 (in inverse microns). The metasurface in Figure 5 has a period of 4 µm. The size of the whole metasurface chosen was 8 × 8 µm. It was computed for the wavelength of the incident light, λ = 633 nm. The metasurface is composed of stripes with diffraction gratings with a subwavelength period of 220 nm (the groove width of 110 nm and the step width of 110 nm), rotated by the angle xα/2 + π/2 with respect to the axis x. Each period is split into 8 stripes, where the angle of the diffraction gratings is constant. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Figure 3 also confirms that in this case, when both beams in superposition (1) are different from those in Figure 2, the maximal SAM density is still achieved when the energies of both constituent beams are the same, i.e., CC = CL. As shown in Figure 2, the SAM density changes with the weight coefficients only by magnitude rather than shape. Micromachines 2024, 15, 350 11 of 17 Sign Sp 11 of 17 Sign Sp 7. Designing a Metalens for Generating Two Beams with the Opposite-Sign Spins , ( ) ( ) by using only one optical element. We designed this element using a method In this section, we demonstrate how the vector beams (19) and (27) can be generated by using only one optical element. We designed this element using a method described earlier in [11]. Such a metasurface [11] is composed of subwavelength binary gratings, each of which rotates the polarization vector by a given angle. We note that the spin Hall effect was observed in work [17] by generating a beam using an SLM and q-plates. Such a setup requires an exact adjustment. In addition, the opposite-sign spin in [17] was generated in the focus and in the ring that surrounds it. In the current work, we generate the opposite-sign spin in two similar off-axis foci or in two similar off-axis rings. This is more convenient for separate use of the generated beams. earlier in [11]. Such a metasurface [11] is composed of subwavelength binary g each of which rotates the polarization vector by a given angle. We note that the sp effect was observed in work [17] by generating a beam using an SLM and q-plates setup requires an exact adjustment. In addition, the opposite-sign spin in [17] wa ated in the focus and in the ring that surrounds it. In the current work, we gene opposite-sign spin in two similar off-axis foci or in two similar off-axis rings. This convenient for separate use of the generated beams. As we earlier noted, field (19) can be generated by using a metasurface in ( p g As we earlier noted, field (19) can be generated by using a metasurface in (26). 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization It was computed for the wa nm. The metasurface is composed of stripes with length period of 220 nm (the groove width of 110 tated by the angle xα/2 + π/2 with respect to the a where the angle of the diffraction gratings is const field rotates by an angle equal to the doubled ang seen from the comparison of Figures 4 and 5. The r index of the gratings is n = 4.352 + 0.486i (amorpho ngle equ rison of Figure 5. Binary metasurface relief. Figure 5. Binary metasurface relief. p g ndex of the gratings is n = 4.352 The metasurface in Figure 5 has a period of 4 µm. The size of the whole metasurface chosen was 8 × 8 µm. It was computed for the wavelength of the incident light, λ = 633 nm. The metasurface is composed of stripes with diffraction gratings with a subwave- length period of 220 nm (the groove width of 110 nm and the step width of 110 nm), ro- tated by the angle xα/2 + π/2 with respect to the axis x. Each period is split into 8 stripes, where the angle of the diffraction gratings is constant. The polarization vector of the light field rotates by an angle equal to the doubled angle of rotation of grating lines, which is seen from the comparison of Figures 4 and 5. The relief height is 140 nm, and the refractive For the simulation, we used the FDTD method and the Rayleigh-Somerfield transform. At first, we computed by the FDTD method how the light field propagates through the metasurface and obtained the fields Ex and Ey at a distance λ from the metasurface. This field was then the input field of the Rayleigh-Somerfield transform. Using this transform, the resulting light field at a distance of 50 µm was computed. Simulation by only the FDTD method at such a distance (nearly 50 µm) is impossible in 3D due to excessive computational complexity. Shown in Figure 6 is the intensity of the field, obtained at the distance λ beyond the metasurface. For the simulation, we used the FDTD method and the Rayleigh-Somerfield orm. At first, we computed by the FDTD method how the light field propagates thr he metasurface and obtained the fields Ex and Ey at a distance λ from the metasu This field was then the input field of the Rayleigh-Somerfield transform. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Using this orm, the resulting light field at a distance of 50 µm was computed. Simulation by he FDTD method at such a distance (nearly 50 µm) is impossible in 3D due to exce computational complexity. Shown in Figure 6 is the intensity of the field, obtained distance λ beyond the metasurface. index of the gratings is n = 4.352 + 0.486i (amorphous silicon). For the simulation, we used the FDTD method and the Rayleigh-Somerfield trans- form. At first, we computed by the FDTD method how the light field propagates through the metasurface and obtained the fields Ex and Ey at a distance λ from the metasurface. This field was then the input field of the Rayleigh-Somerfield transform. Using this trans- form, the resulting light field at a distance of 50 µm was computed. Simulation by only the FDTD method at such a distance (nearly 50 µm) is impossible in 3D due to excessive computational complexity. Shown in Figure 6 is the intensity of the field, obtained at the distance λ beyond the metasurface. (a) (b) Figure 6. Intensity (a) and polarization distribution (b) of the electric field at the distance λ fro metasurface. Figure 6. Intensity (a) and polarization distribution (b) of the electric field at the distance λ from the metasurface. index of the gratings is n 4.352 For the simulation, we use form. At first, we computed by the metasurface and obtained t This field was then the input fie form, the resulting light field a the FDTD method at such a dis computational complexity. Sho distance λ beyond the metasurf (a) 0.486i (amorphous silicon). he FDTD method and the Rayleigh-Somerfield t FDTD method how the light field propagates thr fields Ex and Ey at a distance λ from the metasur of the Rayleigh-Somerfield transform. Using this t distance of 50 µm was computed. Simulation by ce (nearly 50 µm) is impossible in 3D due to exce in Figure 6 is the intensity of the field, obtained a . (b) (b) (a) Figure 6. Intensity (a) and polarization distribution (b) of the electric field at the distance λ fro metasurface. Figure 6. Intensity (a) and polarization distribution (b) of the electric field at the distance λ from the metasurface. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization For the simulation, we used the FDTD method and the Rayleigh-Somerfield transform. At first, we computed by the FDTD method how the light field propagates through the metasurface and obtained the fields Ex and Ey at a distance λ from the metasurface. This field was then the input field of the Rayleigh-Somerfield transform. Using this transform, the resulting light field at a distance of 50 µm was computed. Simulation by only the FDTD method at such a distance (nearly 50 µm) is impossible in 3D due to excessive computational complexity. Shown in Figure 6 is the intensity of the field, obtained at the distance λ beyond the metasurface. Figure 5. Binary metasurface relief. The metasurface in Figure 5 has a period of 4 µm. The size of the whole metasu chosen was 8 × 8 µm. It was computed for the wavelength of the incident light, λ nm. The metasurface is composed of stripes with diffraction gratings with a sub length period of 220 nm (the groove width of 110 nm and the step width of 110 nm tated by the angle xα/2 + π/2 with respect to the axis x. Each period is split into 8 st where the angle of the diffraction gratings is constant. The polarization vector of the field rotates by an angle equal to the doubled angle of rotation of grating lines, wh seen from the comparison of Figures 4 and 5. The relief height is 140 nm, and the refr index of the gratings is n = 4.352 + 0.486i (amorphous silicon). For the simulation, we used the FDTD method and the Rayleigh-Somerfield form. At first, we computed by the FDTD method how the light field propagates th the metasurface and obtained the fields Ex and Ey at a distance λ from the metasu This field was then the input field of the Rayleigh-Somerfield transform. Using this form, the resulting light field at a distance of 50 µm was computed. Simulation by the FDTD method at such a distance (nearly 50 µm) is impossible in 3D due to exc computational complexity. Shown in Figure 6 is the intensity of the field, obtained distance λ beyond the metasurface. Figure 5. Binary metasurface relief. Figure 5. Binary metasurface relief. Figure 5. Binary metasurface relief. The metasurface in Figure 5 has a period of 4 chosen was 8 × 8 µm. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization The polarization vector of the light field rotates by an angle equal to the doubled angle of rotation of grating lines, which is seen from the comparison of Figures 4 and 5. The relief height is 140 nm, and the refractive index of the gratings is n = 4.352 + 0.486i (amorphous silicon). Micromachines 2024, 15, 350 Micromachines 2024, 15, x FO 12 of 17 12 of 17 Figure 5. Binary metasurface relief. Figure 5. Binary metasurface relief. Figure 5. Binary metasurface relief. The metasurface in Figure 5 has a period of 4 µm. The size of the whole metasu chosen was 8 × 8 µm. It was computed for the wavelength of the incident light, λ = nm. The metasurface is composed of stripes with diffraction gratings with a subw length period of 220 nm (the groove width of 110 nm and the step width of 110 nm tated by the angle xα/2 + π/2 with respect to the axis x. Each period is split into 8 str where the angle of the diffraction gratings is constant. The polarization vector of the field rotates by an angle equal to the doubled angle of rotation of grating lines, whi seen from the comparison of Figures 4 and 5. The relief height is 140 nm, and the refra index of the gratings is n = 4.352 + 0.486i (amorphous silicon). For the simulation we used the FDTD method and the Rayleigh Somerfield t Figure 5. Binary metasurface relief. The metasurface in Figure 5 has a period of 4 µm. The size of the whole metasurface chosen was 8 × 8 µm. It was computed for the wavelength of the incident light, λ = 633 nm. The metasurface is composed of stripes with diffraction gratings with a subwave- length period of 220 nm (the groove width of 110 nm and the step width of 110 nm), ro- tated by the angle xα/2 + π/2 with respect to the axis x. Each period is split into 8 stripes, where the angle of the diffraction gratings is constant. The polarization vector of the light field rotates by an angle equal to the doubled angle of rotation of grating lines, which is seen from the comparison of Figures 4 and 5. The relief height is 140 nm, and the refractive Figure 5. Binary metasurface relief. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization (a) (b) Fi 6 I i ( ) d l i i di ib i (b) f h l i fild h di λ f h It is seen that after passing through the diffraction gratings, the field has some inho- mogeneity, but nevertheless, the polarization direction is consistent with the distribution obtained by Equation (19) (Figure 4). Figure 7 illustrates the beam intensity at a distance of 50.633 µm from the metasurface, computed by the Rayleigh-Sommerfeld transform. Micromachines 2024, 15, 350 13 of 17 ribution distance Figure 7. Intensity of light at a distance of 50.633 µm from the metasurface as well as the polarization distribution. Arrows with circles indicate polarization direction in the center of each circle, and the arrow shows the rotation direction of the vector electric field with time. Figure 7. Intensity of light at a distance of 50.633 µm from the metasurface as well as the polarization distribution. Arrows with circles indicate polarization direction in the center of each circle, and the arrow shows the rotation direction of the vector electric field with time. igure 7. Intensity of light at a distance of 50.633 µm from the metasurface as well as the polariz istribution. Arrows with circles indicate polarization direction in the center of each circle, an rrow shows the rotation direction of the vector electric field with time. As seen in Figure 7, at a distance of 50.633 µm from the metasurface, two inten Figure 7. Intensity of light at a distance of 50.633 µm from the metasurfa A distance Figure 7. Intensity of light at a distance of 50.633 µm from the metasurface as well as the polarization distribution. Arrows with circles indicate polarization direction in the center of each circle, and the arrow shows the rotation direction of the vector electric field with time. Figure 7. Intensity of light at a distance of 50.633 µm from the metasurface as well as the polarization distribution. Arrows with circles indicate polarization direction in the center of each circle, and the arrow shows the rotation direction of the vector electric field with time. stribution. Arrows with circles indicate polarization direction in the center of each circle, an row shows the rotation direction of the vector electric field with time. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization As seen in Figure 7 at a distance of 50 633 µm from the metasurface two inte As seen in Figure 7, at a distance of 50.633 µm from the metasurface, two intensity maxima are generated on the axis x, with the distance between their centers being equal to 15.86 µm. In the left intensity maximum, dominating polarization is right-hand circular, while in the right maximum, it is left-hand circular. The distance between the maxima is defined by the period of the metasurface structure (Figure 5. When the metasurface period (Figure 5) doubles, up to 8 µm, the distance between the maxima decreases nearly two times, up to 7.64 µm. From the total energy of the beam coming out of the metasurface, nearly 78.5% goes to both intensity maxima. As seen in Figure 7, at a distance of 50.633 µm from the metasurface, two intensity maxima are generated on the axis x, with the distance between their centers being equal to 15.86 µm. In the left intensity maximum, dominating polarization is right-hand circular, while in the right maximum, it is left-hand circular. The distance between the maxima is defined by the period of the metasurface structure (Figure 5. When the metasurface period (Figure 5) doubles, up to 8 µm, the distance between the maxima decreases nearly two times, up to 7.64 µm. From the total energy of the beam coming out of the metasurface, nearly 78.5% goes to both intensity maxima. maxima are generated on the axis x, with the distance between their centers being e o 15.86 µm. In the left intensity maximum, dominating polarization is right-hand circ while in the right maximum, it is left-hand circular. The distance between the maxim efined by the period of the metasurface structure (Figure 5. When the metasurface pe Figure 5) doubles, up to 8 µm, the distance between the maxima decreases nearly mes, up to 7.64 µm. From the total energy of the beam coming out of the metasur early 78.5% goes to both intensity maxima. Sh i Fi 8 i th t l th t t th li ht fild (27) ith Shown in Figure 8a is the metalens that generates the light field (27) with parameters α = π/2 µm−1 and n = 1. In addition to the spatial frequency, which is present in the metasurface from Figure 5, the topological charge is added here. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Therefore, instead of the grating (Figure 5), fork grating was obtained, although only 2 periods were fitted in Figure 8a of such a fork grating (with an edge dislocation). In total, the metasurface with a size of 8 × 8 µm was split into 14 × 14 blocks, each 26 × 26 pixels (0.571 µm). Shown in Figure 8a is the metalens that generates the light field (27) with parameters α = π/2 µm−1 and n = 1. In addition to the spatial frequency, which is present in the metasurface from Figure 5, the topological charge is added here. Therefore, instead of the grating (Figure 5), fork grating was obtained, although only 2 periods were fitted in Figure 8a of such a fork grating (with an edge dislocation). In total, the metasurface with a size of 8 × 8 µm was split into 14 × 14 blocks, each 26 × 26 pixels (0.571 µm). Shown in Figure 8a is the metalens that generates the light field (27) with param = π/2 µm−1 and n = 1. In addition to the spatial frequency, which is present in metasurface from Figure 5, the topological charge is added here. Therefore, instead o rating (Figure 5), fork grating was obtained, although only 2 periods were fitted in Fi a of such a fork grating (with an edge dislocation). In total, the metasurface with a f 8 × 8 µm was split into 14 × 14 blocks, each 26 × 26 pixels (0.571 µm). (a) (b) Figure 8. Metasurface, generating the cylindrical vector beam (27) with spatial carrier frequency (a), and polarization of a plane linearly polarized wave passed through this metasurface at a distance λ from it (b). (a) (b) Figure 8. Metasurface, generating the cylindrical vector beam (27) with spatial carrier frequenc and polarization of a plane linearly polarized wave passed through this metasurface at a distan from it (b). Figure 8. Metasurface, generating the cylindrical vector beam (27) with spatial carrier frequency (a), and polarization of a plane linearly polarized wave passed through this metasurface at a distance λ from it (b). (b) cylindrical vector beam (27) with spatial carrier fre olarized wave passed through this metasurface at a (b) Figure and po (a) am (27 d throu (b) from it (b). gure 8. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Metasurface, generating the cylindrical vector beam (27) with spatial carrier frequenc nd polarization of a plane linearly polarized wave passed through this metasurface at a distan om it (b). Figure 8. Metasurface, generating the cylindrical vector beam (27) with spatial carrier frequency (a), and polarization of a plane linearly polarized wave passed through this metasurface at a distance λ from it (b). Shown in Figure 8b is the distribution of linear polarization immediately beyond the metasurface. It is seen that, due to the edge dislocation in the center of the pattern shown Micromachines 2024, 15, 350 14 of 17 14 of 17 in Figure 8b, the polarization singularity, or V-point, appears where linear polarization is indefinite. tasurface. It is seen that, due to the edge dislocation in the center of the pattern shown Figure 8b, the polarization singularity, or V-point, appears where linear polarization is definite. Figure 9 illustrates the simulation results of light propagation through the metasurface from Figure 8 at a distance of 150 µm from it. In this case, we also first computed the field at a distance of one wavelength from the metasurface by the FDTD method for an exact evaluation of the metasurface contribution. Then, using the Rayleigh-Sommerfeld integral, the field in the far diffraction zone was computed. Since in this case light rings are generated, the propagation distance, where both rings are generated and separated, is larger than in the case shown in Figure 7. definite. Figure 9 illustrates the simulation results of light propagation through the metasur- e from Figure 8 at a distance of 150 µm from it. In this case, we also first computed the d at a distance of one wavelength from the metasurface by the FDTD method for an act evaluation of the metasurface contribution. Then, using the Rayleigh-Sommerfeld egral, the field in the far diffraction zone was computed. Since in this case light rings generated, the propagation distance, where both rings are generated and separated, is ger than in the case shown in Figure 7 (a) (b) gure 9. Intensity of the cylindrical vector beam with the carrier frequency, generated by the talens, and polarization of this beam, depicted as ellipses with arrows (a), as well as the phase of Ey field component (b). Each ellipse (a) describes rotation of the electric field vector with time. Figure 9. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Intensity of the cylindrical vector beam with the carrier frequency, generated by th metalens, and polarization of this beam, depicted as ellipses with arrows (a), as well as the phase o the Ey field component (b). Each ellipse (a) describes rotation of the electric field vector with time. (a) (a) (a) ( ) (b) (b) ure 9. Intensity of the cylindrical vector beam with the carrier frequency, generated by the alens, and polarization of this beam, depicted as ellipses with arrows (a), as well as the phase of Ey field component (b). Each ellipse (a) describes rotation of the electric field vector with time. Figure 9. Intensity of the cylindrical vector beam with the carrier frequency, generated by the metalens, and polarization of this beam, depicted as ellipses with arrows (a), as well as the phase of the Ey field component (b). Each ellipse (a) describes rotation of the electric field vector with time. Shown in Figure 9a is an intensity distribution at a 150-µm distance behind the talens of Figure 8a. It is seen that three light beams are generated: a central one and o light rings to the left and right of the central beam. The intensity of the ring beams is ven since the metasurface is designed for only two diffraction orders of the grating. ure 9a also depicts the distribution of the polarization ellipses at a 150-µm distance m the metasurface of Figure 8a. Both rings generated in the far field (Figure 9a) have ptic and circular polarization—light with right-hand circular polarization generates a g in the negative part of the x-axis (i.e., to the left), whereas light with left-hand circular arization generates the right ring. In the phase distribution in Figure 9b, forks can be Shown in Figure 9a is an intensity distribution at a 150-µm distance behind the metalens of Figure 8a. It is seen that three light beams are generated: a central one and two light rings to the left and right of the central beam. The intensity of the ring beams is uneven since the metasurface is designed for only two diffraction orders of the grating. Figure 9a also depicts the distribution of the polarization ellipses at a 150-µm distance from the metasurface of Figure 8a. 8. Conclusions We have considered two perturbations of a cylindrical vector beam that generate a spin Hall effect, i.e., areas with a nonzero spin angular momentum density occur on propagation, despite a zero spin angular momentum in the initial plane. The first perturbation was introduced by adding a linearly polarized beam. Thus, we have analyzed the superposition of two rotationally symmetric paraxial vectorial light fields without a spin angular momentum, with one field being cylindrically polarized and the other linearly polarized. The radial distribution of these fields can be arbitrary. Upon separate propagation in free space, these fields do not acquire spin angular momentum and conserve their polarization. The superposition of these fields has inhomogeneous linear polarization in the initial plane, but on propagation in space, polarization becomes elliptic, and thus a nonzero spin angular momentum is generated. The distribution pattern of the spin angular momentum density consists of alternating spots with left- and right-handed elliptic polarization. Such a separation of the light field into areas with opposite-sign spin angular momentum is a manifestation of the optical spin Hall effect. In this work, we discovered and proved two properties of this phenomenon. The first property is that the transverse shape of the spin angular momentum density distribution does not depend on the weight coefficients of the superposition. The weights define only the magnitude of the spin angular momentum but not the distribution shape. The second property is that if each such superposition is normalized by its energy, then the greatest values of the spin angular momentum are achieved when both constituent beams have the same energy, i.e., one half of the whole beam energy is the energy of the cylindrically polarized field and the other half is the energy of the linearly polarized field. The second perturbation of a cylindrical vector beam was introduced by adding a spatial carrier frequency. Upon free-space propagation, such a beam has been shown to split The second property is that if each such superposition is normalized by its energy, then the greatest values of the spin angular momentum are achieved when both constituent beams have the same energy, i.e., one half of the whole beam energy is the energy of the cylindrically polarized field and the other half is the energy of the linearly polarized field. The second perturbation of a cylindrical vector beam was introduced by adding a spatial carrier frequency. 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams wit Cylindrical and Linear Polarization 6. Numerical Simulation of Superpositions of Rotationally Symmetric Beams with Cylindrical and Linear Polarization Both rings generated in the far field (Figure 9a) have elliptic and circular polarization—light with right-hand circular polarization generates a ring in the negative part of the x-axis (i.e., to the left), whereas light with left-hand circular polarization generates the right ring. In the phase distribution in Figure 9b, forks can be seen near the intensity minima of the two rings. These are optical vortices (screw dislocations) with the topological charges −1 (to the left) and +1 (to the right). Micromachines 2024, 15, 350 15 of 17 15 of 17 Author Contributions: Conceptualization, V.V.K. and A.A.K.; methodology, V.V.K. and A.A.K.; software, A.A.K. and A.G.N.; validation, V.V.K.; formal analysis, V.V.K.; investigation, V.V.K., A.A.K., and A.G.N.; resources, V.V.K.; data curation, V.V.K.; writing—original draft preparation, V.V.K. and A.A.K.; writing—review and editing, V.V.K. and A.A.K.; visualization, A.G.N. and A.A.K.; supervision, V.V.K.; project administration, V.V.K.; funding acquisition, V.V.K. and A.A.K. All authors have read and agreed to the published version of the manuscript. Funding: The work was supported by the RUSSIAN SCIENCE FOUNDATION (Project No. 23-12- 00236, theory). This work was also performed within the STATE ASSIGNMENT of NRC “Kurchatov Institute” (numerical simulation). 8. Conclusions The funders had no role in the design of the study, the collection, analysis, or interpretation of data, the writing of the manuscript, or the decision to publish the results. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study, the collection, analysis, or interpretation of data, the writing of the manuscript, or the decision to publish the results. References 1. Angelsky, O.V.; Bekshaev, A.Y.; Maksimyak, P.P.; Maksimyak, A.P.; Hanson, S.G.; Zenkova, C.Y. Orbital rotation without orbital angular momentum: Mechanical action of the spin part of the internal energy flow in light beams. Opt. Express 2012, 20, 3563–3571. [CrossRef] [PubMed] ] [ ] M.; Murakami, S.; Nagaosa, N. Hall effect of light. Phys. Rev. Lett. 2004, 93, 083901. [CrossRef] [PubMed] Onoda, M.; Murakami, S.; Nagaosa, N. Hall effect 3. Leyder, C.; Romanelli, M.; Karr, J.P.; Giacobino, E.; Liew, T.C.H.; Glazov, M.M.; Kavokin, A.V.; Malpuech, G.; Bramati, A. Observation of the optical spin Hall effect. Nat. Phys. 2007, 3, 628–631. [CrossRef] 4. Bliokh, K.Y. Geometrical optics of beams with vortices: Berry phase and orbital angular momentum Hall effect. Phys. Rev. Lett. 2006, 97, 043901. [CrossRef] [PubMed] 5. Zhang, J.; Zhou, X.X.; Ling, X.H.; Chen, S.Z.; Luo, H.L.; Wen, S.C. Orbit-orbit interaction and photonic orbital Hall effect in reflection of a light beam. Chin. Phys. B 2014, 23, 064215. [CrossRef] G.; Li, Y.; Yin, H.; Li, Z.; Chen, Z. Spin-orbit optical Hall effect. Phys. Rev. Lett. 2019, 123, 243904. [CrossRef] 6. Fu, S.; Guo, C.; Liu, G.; Li, Y.; Yin, H.; Li, Z.; Chen, Z. Spin-orbit optical Hall effect. Phys. Rev. Lett. [PubMed] 7. Li, H.; Ma, C.; Wang, J.; Tang, M.; Li, X. Spin-orbit Hall effect in the tight focusing of a radially polarized vortex beam. Opt. Express 2021, 29, 39419–39427. [CrossRef] p 8. Kotlyar, V.V.; Stafeev, S.S.; Kovalev, A.A.; Zaitsev, V.D. Spin Hall effect before and after the focus of a high-order cylindrical vector beam. Appl. Sci. 2022, 12, 12218. [CrossRef] pp 9. Kotlyar, V.V.; Stafeev, S.S.; Zaitsev, V.D.; Kovalev, A.A. Multiple optical spin-orbit Hall effect at the tight focus. Phys. Lett. A 2023, 458, 128596. [CrossRef] 10. Kotlyar, V.V.; Kovalev, A.A.; Kozlova, E.S.; Telegin, A.M. Hall effect at the focus of an optical vortex with linear polarization. Micromachines 2023, 14, 788. [CrossRef] 11. Kotlyar, V.; Nalimov, A.; Kovalev, A.; Stafeev, S. Optical Polarization Sensor Based on a Metalens. Sensors 2022, 22, 7870. [CrossRef] [PubMed] 11. Kotlyar, V.; Nalimov, A.; Kovalev, A.; Stafeev, S. Optical Polarization Sensor Based on a Metalens. Sensors 2022, 22, 7870. [CrossRef] [PubMed] 12. Kovalev, A.A.; Kotlyar, V.V.; Stafeev, S.S. Spin Hall effect in the paraxial light beams with multiple polarization singularities. 12. Kovalev, A.A.; Kotlyar, V.V.; Stafeev, S.S. Spin Hall effect in the paraxial light beams with multip Micromachines 2023, 14, 777. [CrossRef] [PubMed] 13. 8. Conclusions Upon free-space propagation, such a beam has been shown to split into two shifted off-axis vortex beams, one with right-hand and the other with left-hand circular polarization. This is also a manifestation of the spin Hall effect. Such a field has been generated by using a metasurface implemented in a thin amorphous-silicon film designed for a wavelength of 633 nm. The metasurface is composed of 14 × 14 blocks of binary subwavelength gratings with a period of 220 nm, which period- ically change their direction. A linearly polarized light field incident onto the metasurface was converted into two vortex beams diverging at a certain angle, with one beam being left-handed elliptically polarized and the other right-handed elliptically polarized. The diffractive efficiency of such a grating was found to be almost 80%. These are the simplest metasurfaces for efficiently generating the spin Hall effect in paraxial laser beams. A simi- lar splitting of light beams with opposite-sign spins was implemented by a polarization converter [36]. The discovered properties can be used to enhance the spin angular momentum of light fields. In optical trapping, their use should increase the efficiency of making trapped particles rotate around their centers of mass [37,38]. In optical data transmission, the results obtained can increase the signal power when the data are encoded in light beams by their polarization, and then the incoming beams are identified by polarizers [39,40]. In addition, the obtained results can be used for processing polarization-sensitive materials [41]. Author Contributions: Conceptualization, V.V.K. and A.A.K.; methodology, V.V.K. and A.A.K.; software, A.A.K. and A.G.N.; validation, V.V.K.; formal analysis, V.V.K.; investigation, V.V.K., A.A.K., and A.G.N.; resources, V.V.K.; data curation, V.V.K.; writing—original draft preparation, V.V.K. and A.A.K.; writing—review and editing, V.V.K. and A.A.K.; visualization, A.G.N. and A.A.K.; supervision, V.V.K.; project administration, V.V.K.; funding acquisition, V.V.K. and A.A.K. All authors have read and agreed to the published version of the manuscript. Funding: The work was supported by the RUSSIAN SCIENCE FOUNDATION (Project No. 23-12- 00236, theory). This work was also performed within the STATE ASSIGNMENT of NRC “Kurchatov Institute” (numerical simulation). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Micromachines 2024, 15, 350 16 of 17 16 of 17 Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. ta Availability Statement: Data are contained within the article. Conflicts of Interest: The authors declare no conflicts of interest. References Kotlyar, V.V.; Kovalev, A.A. Spin Hall effect of two-index paraxial vector propagation-invariant beams. Photonics 2023, 10, 1288. [CrossRef] 14. Yin, X.; Ye, Z.; Rho, J.; Wang, Y.; Zhang, X. Photonic spin Hall effect at metasurfaces. Science 2013, 339, 1405–1407. [CrossRef] [PubMed] 15. Kim, M.; Lee, D.; Yang, Y.; Kim, Y.; Rho, J. Reaching the highest efficiency of spin Hall effect of light in the near-infrared using all-dielectric metasurfaces. Nat. Commun. 2022, 13, 2036. [CrossRef] 16. Kim, M.; Lee, D.; Ko, B.; Rho, J. Diffraction-induced enhancement of optical spin Hall effect in a dielectric grating. APL Photonics 2020, 5, 066106. [CrossRef] 17. Li, Y.; Liu, Y.; Ling, X.; Yi, X.; Zhou, X.; Ke, Y.; Luo, H.; Wen, S.; Fan, D. Observation of photonic singularity at dielectric metasurfaces. Opt. 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Ausfallssymptome nach läsion des linken gyrus angularis in einem falle von Schädel-und gehirnverletzung
Zeitschrift für die Gesamte Neurologie und Psychiatrie
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public-domain
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Ausfallssymptome nach Liision des linken Gyrus angularis in einem Falle yon Sch~idel- und Gehirnverletzung. Von Dr. M. g e s n i k o w und Dr. S. D a w i d e n k o w (Charkow). Mit 2 T e x t f i g u r e n . (Eingegangen am 7. Mdrz 1911.) Die u n t e r e n P a r i e t a l w i n d u n g e n u n d besonders ihre hintere, u n t e r e Hglfte, die u n t e r d e m N a m e n G y r u s a n g u l a r i s b e k a n n t ist, b i l d e n ungef~hr d e n z e n t r a l e n Tell desjenigen W i n d u n g s s y s t e m s , welches in seiner G e s a m t h e i t d a s s o g e n a n n t e h i n t e r e A s s o z i a t i o n s z e n t r u m F l e c h = s i g s darstellen. W i r h a t t e n Gelegenheit, die d a u e r n d e n F o l g e n einer t r a u m a t i s e h e n Lgsion des u n t e r e n P a r i e t a l g e b i e t e s , h a u p t s ~ c h l i c h des G y r u s a n g u l a r i s zu b e o b a c h t e n . Dieser, fast einem physiologischen E x p e r i m e n t e gleichwertige, F a l l w a r d u r c h eine R e i h e von S y m p t o m e n ausgezeichnet, d i e uns veranla•ten, die S y m p t o m a t o l o g i e d e r E r k r a n k u n g e n des G y r u s a n g u l a r i s einer n e u e n P r i i f u n g zu u n t e r w e r f e n . 1) Taranow, Ephrem, ein Bauer des Gouvernements Charkow, Bezirks Bogodouehow, der 38 Jahre alt und verheiratet war, wurde am 7. April 1909 in Semstwos Hauptlandesirrenkrankenhans des Gouvernements Charkow untergebraeht. Die Fran des Kranken teilte mit, dab derselbe bis zu seiner Verwundung im letzten Kriege gesund war. Im Russisch-japanischen Kriege wurde er im Oktober 1904 in tier Schlaeht bei Sehaeh~ dureh ein Schrapnel| am Kopfe verwundet. Bei seiner Riickkehr nach tIause erz~hlte er, dal3 sein rechter Arm und sein reehtes Bein eine Zeitlang gel~hmt waren. Als er naeh Hause kam, hatte er aber keine L~hmung mehr. Seine Frau bemerkte auch in der ersten Zeit niemals Krampfanf~lle oder irgendwelche An@rung in seinem Charakter, welche unmittelbar dem Trauma gefolgt w~re. Anfangs sprach er sehr schleeht, jedoeh im Laufe der Jahre hat sich die Sprache allmghlieh wiederhergestellt. Er lebte im ttanse, ohne irgendeiner Besch~ftigung nachzugehen. In den letzten Monaten des Jahres 1908 ring die Frat~ an, eine gewisse Xnderung in seinem Charakter zu bemerken. Er versank 5ftem in Nachdenken, kam leicht in Erregung, schalt wiederholt seine Fran, wurde j~hzornig. Sobald ihm etwas nieht reeht war, wurde er auf einmal rot, geriet aul3er sich, ring an, wiist um sich zu schlagen. Er soll seine Frau in die H~nde gebissen haben. Auf dem Wege naeh dem Krankenhause war er sehr aufgeregt und schlug 1) Auf den gleichen Kranken bezieht sich unsere in dieser Zeitschrift (4, 129. 1911) erschienene Arbeit: Beitrage zur Pletysmographie des mensehliehen Gehirns. M. Resnikow und S. Dawidenkow: Ausfallssymptome usw. 651 u m sieh (er kam ins Krankenhaus mit kleinen Hauterosionen an Gesicht und H~ia~den). Verheiratet ist er seit ungef~hr 20 Jahren; Kinder hat er keine gehabt; seine Frau war nie 'gravide; weshalb ihre Ehe kinderlos ist, weil3 die Yrau nicht. Pat. best~itigt im allgemeinen diese anamnestischen Angaben. Seiner Meinung naeh geriet er ins Krankenhaus infolge eines Zankes mit seiner Frau und seinen Mietsleuten, die ihn schlugen. Im Krankenhause gewShnte er sich bald an die dortigen Verh~iltnisse, und es war ihm der Aufenthalt unter den Geisteskranken nicht besenders l~stig. Fig. 1. S t a t u s p r a e s e n s . Pat. ist grofl von Wuchs, normal gebaut und gut gens er hat das Aussehen eines riistigen und gesunden Mensehen, seinem Alter entsprechend. Betreffs der inneren Organe ist keine Abweichung von der Norm zu bemerken. Spuren einer iiberstandenen Syphilis sind nicht vorhanden. Auf der linken Seite seines Kopfes befindet sieh ein 7 x 4 cm grol3er Knochendefekt ; derselbe befindet sich im hinteren oberen Teile des Temporal- und im unteren Teile des Parietalbeins. Er hat die Form einer Schwertbohne, deren stumpfes Ende nach vorn und die konvexe Seite des L~ngendurchmessers nach unten und zugleich nach vorn gerichtet ist. Auf diese Weise hat der vordere obere Teil des Defektes eine gr613ere Fls ausdehnung als der hintere untere Teil. Die abgerundeten Rs des Knochens am Defekt sind eingedriickt, und zwar besonders am unteren und hinteren Teile des Defekts. Der untere Teil des Defekts ist mit einer Hautnarbe bedeckt, welehe 42* 652 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Lasion des oben yon einer horizontalen Linie begrenzt ist; der obere Teil des Defekts ist yon unver~nderter behaarter Haut bedeckt. Die Narbe ist 7 • 2 cm grol3. Der Grund des Defekts ist in seinem unteren narbigen Teile weieh und leicht eindriickbar; im oberen behaarten Teile ist die Resistenz des Grundes stark, aber gibt etwas dem Drucke des Fingers naeh. Die Narbe und' die Ris des Knochens schmerzen beim Druck, und ebenso schmerzhaft ist die Perkussion des Sch~dels an der vorderen I-I~lfto des Defektes; an der hinteren H~lfte ist dies nicht der Fall. Beim Sitzen oder Stehen des Kranken ist der Grund des Defekts eingezogen. Diese Eingezogenhei~ ist in den verschiedenen Durchmessern des Defekts ungleiehmi~l~ig; im vertikalen Durchmesser des Defekts ist sie am st~rksten ausgepr~gt, im horizontalen Durchmesser ist der Grund des Defekts jedoch nieht eingezogen, sondern wird dureh eino gerade Linie bezeichnet. Der Grund des Defekts zeigt eine deutliche Pulsation, die besonders im narbigen Teile bemerkbar ist. Beim Pressen, beim Husten und in horizontaler Lage wird die ganze Oberfl~che des Defekts convex. Bei genanerer Bestimmung der Lage des Knoehendefekts bekommen wir folgende Zahlenverh~ltnisse: der vordere Rand des Knochendefekts ist yon der yore ~ul~eren GehSrgang gezogenen Vertikallinie 2 cm, der untere Rand des Knochendefekts ist yon der dutch den ~ui~eren Gehiirgang gezogenen Horizontale 4 cm entfernt. Beim Aufzeichnen der Linien auf dem Seh~del nach C h i p a u l t {Fig. 1) bekommen wir folgende Verh~ltnisse: der obere Rand des Defekts beriihrt gerade die Linea Sylviana in der Entfernung von 12 cm von Tuberculum postorbitale; die Parallellinie kreuzt den Defekt ungefahr in der Mitre; auf dieser Linie ist der vordere Rand des Defekts 1089 cm von Tuberculum postorbitale entfernt. Die Fig. 2 zeigt folgende Linien: Linea Rolandica und Linea Sylviana, die Fig. 2. man beim Ausmessen nach einer anderen Methode (Godlee u. B e n n e t ) erhalten hat. Die Riehtung der Lineae Rolandicae stimmt mit der Riehtung derselben Linie nach Chi p a u 1t iiberein; was jedoch die Linea Sylviana betrifft, so haben wit zweeks Bestimmung ihrer Richtung in Betracht gezogen, da$ der Sehiidel des Kranken zum frontopetalen Typus gehSrte, tier das Auftragen der Orientierungslinie durch die obere Peripherie des Tuber parietale fordert. Hiermit erkliizt sieh die hShere Lage dieser Linie. Nach dieser Methode befindet sieh der Herd etwas tiefer und umfaBt haupts~iehlieh die hinteren Teile der ersten und zweiten Windung des Lobius temporalis und besonders die Umgebung des Suleus temporalis I. in seinem aufsteigenden Sehenkel, d.h. die Gegend des Gyrus angularis. Die Temperatur tier Narbe ist 33,4 ~ C. Auf der entsprechenden Stelle der anderen SeRe ist die Temperatur der Haut 33,2 ~ C. Die V e r i ~ n d e r u n g e n , die im I n n e r n des Sch~dels wi~hrend der Operation vorgefunden wurden: In Narkose wurde die Narbe ca. 7 cm lang und ca. 1~/2cm breit oberhalb des Defekts herausgeschnitten. Unter der Narbe, in der Gegend des unteren Teils des Defekts, befindet sich eine Cyste, aus weleher 5 cem klare Fliissigkeit mit darin linken Gyrus angularis in einem Falle yon Schi~del- und Gehirnverletzung. 653 sehwimmenden vereinzelten floekigen Gerinnungen ausgesogen wurden. I n der oberen H~lfte des Defekts ist die Haut mit den untenliegenden ttirnh~uten nnd mit Gehirnsubstanz verwachsen. Bei Incision der Cyste sind noch ca. 5--6 eem Fliissigkeit ausgeflossen. Im mittleren Teile der Narbe n~hert sieh die Cyvte der Oberfl~che; an den R~ndern sind die Wandungen der Cyste etwas dicker; weiter vertiefte sie sieh in die Sch~delhShle, indem vie die Richtung etwas mehr naeh vorn nahm. Die Tiefe der Cyste betr~gt ca. 2 ~ - - 3 cm. Die Deekung des Defekts (lurch einen Itautknochenlappen wurde yon Dr. Rose erfolgreieh vollzogen. Untersuchung des Nervensystems. Der Gang ist sieher und ohne Schwanken bei geschlossenen Augen; der Kranke steht auf jedem einzehmn FuBe fast. Die Bewegungen der Stirn und der Augenbrauen sind symmetrisch; die geschlossenen Augenlider lassen ein leises. Zittem bemerken. Das Blinzeln ist von normalem Umfange und syneh~onisch, ungefiihr 20 in einer Minute. Das SchlieBen der Augen!ider ist symmetriseh. B e i m Z ~ i h n e f l e t s e h e n u n d L i p p e n b e w e g e n m a c h t sich i m U m f a n g e der B e w e g u n g e n e i n e k l e i n e A s y m m e t r i e des G e s i e h t e s b e m e r k bar. Die l i n k e N a s o l a b i a l f a l t e ist t i e f e r als die r e c h t e , u n d die Bew e g u n g e n a u f der l i n k e n Seite des G e s i c h t e s h a b e n e i n e n e t w a s grSBeren U m f a n g . Die Bewegungen der Zunge und des weichen Gaumens sind normal. Artikulation und Phonation, Kauen und Sehlucken weisen keine StSrung auf. In der Gesichtsmuskulatur sind keine Mitbewegungen vorhanden. Die Beweglichkeit der Wirbels~iule ist normal, dag Beklopfen der Dornforts~itze nicht schmerzhaft. Die Fxtremit~tenmuskulatur ist sehr gut entwiekelt. Der Umfang der passiven Bewegungen und ihr Charakter weisen keine Ver~nderungen auf. I)er Umfang der aktiven Be~egungen ist aueh nicht ver~i~dert, die Kraft derselben ist iiberall sehr grog; jedoch beim Vergleich der Kraft beider Seiten l~Bt sieh konstatieren, da.l~ i n e i n i g e n M u s k e l g r u p p e n , n a m e n t l i e h i n d e n S c h u l t e r h e b e r n u n d U n t e r s c h e n k e l b e u g e r n , die K r a f t a u f der r e c h t e n Seite e t w a s g e r i n g e r ist als die a u f der l i n k e n , was a b e t d e n G r a d e i n e r a u s g e s p r o c h e n e n P a r e s e n i c h t e r r e i c h t . In allen iibrigen ~luskelgruppen ist die Kraft auf beiden Seiten gleich. Bei ausgestreckten H~nden bemerkt man ein leises Zittern der Finger. Alle aktiven Bewegungen ~erden rasch und mit geniigender Genauigkeit ausgefiihrt, Apraxie ist nicht vorhanden. B e ira B e r i i h r e n der N a s e n s p i t z e m i t d e m F i n g e r u n d ~ines K n i e s m i t der F e r s e de~ a n d c r e n Fui3es s i n d die B e w e g u n g e n der r e c h t e n H a n a u n d des r e e h t e n FuBes die e r s t e n Male e r s i c h t l i e h u n g e n a u e r ala die der l i n k e n . Allm~hlich aber gleieht sich dieser Unterschied aus, und diesr Bewegungen ~erden auf beiden Seiten mit derselben geniigenden Genauigkeit ausgeffihrt. Keine trophischen StSrtmgen in den Extremit~ten. Betreffs der vasomotorischen Erscheinungen ist zu bemerken, dab die t I a n d f l ~ c h e u n d die F i n g e r der r e c h t e n H a n d zeitweise k ~ l t e r s i n d als die der l i n k e n . Laut Angabe des Kranken soil dieser Temperaturunterschied friiher viel s t g r k e r gewesen vein. Der Geruchsinn ist auf beiden Seiten erhalten. Die zentrale Sehsch~rfe ist nicht herabgesetzt. Das Gesichtsfeld ist bedeutend ver~i~dert in Form einer H e m i a n o p s i a b i l a t e r a l i s h o m o n y m a d e x t r a . Der Kranke isv sich des Defektes in der reehten H~lfte seines Gesichtsfeldes nieht bewuBt. Der A u g e n g r u n d zeigt keine Ver~nderungen mit Ausnahme einer Bl~sve der b e i d e n l i n k e n P a p i l l e n h ~ l f t e n (Privatdozent Dr. B r a u n s t e i n ) . Die Beweglichkeit der Aug~ipfel ist normal, eine Ablenkung 654 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Litsion des der Augen und Nystagmus sind nicht vorhanden. Die Konvergenz und die Akkommodation sind erhalten. Die Pupillen sind rund, wobei die r e c h t e e t w a s w e l t e r i s t als die li~lke. Der Lichtreflex ist yon genSgender St~rke uud lebhaft, bei schwacher Beleuchtung aber r e a g i e r t die r e c h t e e r w e i t e r t e P u p i l ] e e t w a s t r~ g er , ttemiopische Reaktion ist nicht vorhanden. Es li~,t sich k e i n e G e h S r s t S r u n g bemerken. Die G e s c h m a e k e m p f i n d u n g e n s i n d n o r m a l . Die Tast.-, Schmerz-, Temperatur-Hautem[,findungen, so~ie ihre Lokali,~ation und das I.agegcfiihl der Extremit~ten sind nicht gestSrt. W~hrend der Muskel~,inn der Finger an beiden H~inden ~ollkommen symmetrisch nnd gleich erhalt~n ist, ist die s t e r e o g n o s t i s e h e E m p f i n d u n g in d er r e c h t e a H a n d d e u t l i c h h e r a b g e s e t z t . Als man in die reehte Hand des Kranken einen Schliissel legte, ~ar er der Meining, es sei ein Bleistift; es war ihm ein ZiindhSlzchen in die Hand gelegt, er g)aubte eine Feder zu halten; eine Stahlfeder hielt er fiir ein ZiindhSlzchen, eine Uhr fiir einen Geldbeutel. Einige Male gab er auch richtige Antworten, welche aber ebenso wie die falschen nicht sofort, sondern nach langem Nachdenken erfolgten. Bei einigen, und zwar bei wiederholten Ver~_uehen, konnte der Kranke seine Astereognosis etwas korrigieren und richtigere, wenn auch nieht genaue, Antworten geben. Wenn man z. B. ein Geldstiick in seine Hand legte, konnte er dessen Wert nicht bestimmen, dagegen verstand er beim Betasten gro6er und kleiner Gegenstgnde ihre GrSl~e ann~hernd zu sch~tzen. Beim Betasten verschiedenartiger Gegenstiinde mit der linken Hand nennt er dieselben sofort mit Sicherheit und bestimmt den Wert tier Miinzen fehlerlos. Die Raumorientierung in bezug auf die ihn umgebenden Gegensthnde ist vollkommen erhalten. B e i m V o r s c h l a g , e i n e L i n i e n a c h A u g e n m a l ~ in z w e i g l e i c h e T e i l e zu t e i l e n , macht der Kranke g r o b e F e h l e r , indem er die rechte tIglfte der zu teilenden Linie erheb!ich zu klein macht (Axenfeldsches Ph~nemen). Die T i e f e n w a h r n e h m u n g ist ziemlich erhalten. Der Kranke bestimmt genau die Veriinderungen in der Entfernung oder Ann~iherung zweier von ihm und voneinander in verschiedcnen Entfemungen angestellter Gegenst~nde. Jedoch bei der Aufforderung, mit der Fingerspitze der rechten Hand die Spitze eines vor ihm aufgestellten Bleistiftes zu beriihren, beriihrt der Kranke die Spitze genau, wenn sich der Bleistift an seiner linken Seite befindet, weniger genau, wenn derselbe an seiner rechten Seite steht. Wird derselbe Versuch mit der linken Hand gemacht, so ist dieser Unterschied kleiner, kaum bemerkbar. Die Conjunctival- und Cornealreflexe sind auf beiden Seiten erhalten. Der Rachenreflex ist abgeschwgeht. Die Plantar-, Baueh- und Cremasterreflexe zeigen keine Abweichung Con der Norm. Die Sehnenrefiexe des M. bieipitis brachii, tricipitis und periostale Reflexe (Radius) sind lebhaft und auf der linken und rechten Seite gleich stark; die Patellarreflexe sind gesteigert, der rechte grSl~er als der linke. Der Achillesreflex ist normal, aueh ist keine Muskelrigiditgt zu bemerken. Bab i n s k y s , O p p e n h e i m s und M e n d e l - B e c h t e r e w s Phi~nomene sind nicht ~,orhanden. Di e S prache. Die spontane Sprache ist sch~atzhaft und hastig (LogorrhSe). Der Kranke sucht miihselig naeh Worten, h~,lt im Sprechen an und sagt: .,,Ach Gott, ieh babe vergessen", oder ,,Wie soll ich sagen ?" Er ist sieh seines Wortmangels bewul~t, greift oft zur Beschreibung, um das fehlende Wort zu erkl~ren, l~l~t Worte aus und gebraucht ~alsche (Paraphasia verbalis). 0fters wendet er statt des passendenWortes dasjenige an, das er vorher bei einer anderen Gelegenheir gebraucht hat. So sagt er z. B. anstatt ,,vier Brfider" ,,vier Rubel", weft kurz vorher vom Gelde die Rede war (Perseveration). Viele Worte sprieht er linken Gyrus angularis in einem Falle yon Schlidel- und Gehirnverletzung. 655 falseh aus, indem er an Stelle der riehtigen Vokale oder Silben falsehe einsetzt (Paraphasia litteralis). Dieses m a c h t seine spontane Sprache so mangelhaft, da0 m a n ihn oft nicht verstehen k a n n (Kauderwelseh). Bei einer Aufregung iiberstiirzt er sieh, und seine Sprache wird noch unverst~ndlicher (Paraphasia affectiva). W e n n der Kranke ein fehlendes Wort durch ein falsches ersetzt, hSrt er es heraus u n d bemerkt es gewShnlich selbst (BewuI~tsein des Fehlers). Folgende Probe ist so gut wie mSglich iibersetzt: Frage: Was m a c h t m a n auf der Dreschtenne ? Antwort: ,,Nun, Menschen arbeiten." Frage: Brot ? Antwort: , , N e i n . . . m a n arbeitet, u m zu beenden, nicht in der Fabrik, wie soll ich s a g e n ? . . . Damit das Stroh beiseite gelegt wird u n d das Brot zuriickbleibt." l~rage: Was fiir Brot ? Semmeln ? Antwort: ,,Nein, n i c h t S e m m e l n . . . Ach, mein Gott, ich weil~ d o c h . . . Frage: Hirse? B u c h w e i z e n ? . . . K o r n ? Antwort: ,,Jawohl, Stroh separat u n d Korn separat." Bei der Erz~hlung yon seinen Feldarbeiten v e r g i l 3 t e r d i e B e n e n n u n g e n der Werkzeuge u n d b e s c h r e i b t sie, z. B. ,,zwei StScke". a n s t a t t ,,Dreschflegel" (Amnesia verbalis). Dieser Mangel einer ganzen Reihe yon Worten in seinem Ged~chtnisse t r i t t noch sch~rfer hervor, wenn m a n die F~ihigkeit, einzelne Gegenst~nde, Eigenschaften u n d Handlungen zu nennen, priift. E r wurde aufgefordert, ein ihm vorgelegtes Buch zu benennen. Antwort: ,,Ein B u c h . " - - Ein TintenfaB? - Antwort: ,,Ich weiI~ schon, das ist, es i s t . . , ein TintenfaI~." - - Tinte ? - - A n t w o r t : ,,Dieses ist ein Tintenfal~ u n d d i e s e s . . , ich habe es vergessen." - - Tinte? - Antwort: ,,Ja, Tinte." - - Eine Ziindholzschachtel? - - Antwort: ,,Das ist ein Seh~ichtelchen." - - Ein Zigarrenetui? - - ,,Ein Portemonnaie, ach, mein Gott, n e i n . . , ein Z i g a r r e n . . . " - - P o r t t a b a k ? - - ,,Ja, ja, P o r t t a b a k . " - - Schlfissel? --,,,Sehliissel." - - Ein Ring (Kolzo)? - - ,,Koletz, Koletschuk, Kolzo." - - Ein Perkussionshammer. - - ,,Bei uns heiBt es H~mmerchen, wie es bei 'Ihnen heil3t, well3 ich n i c h t . " - - Brille ? - - ,,Brille." - - Eine Nadel ? - - ,,Das ist eine S t e c k . . . Stecknadel." - - Swi~tscha (eine Kerze)? - - ,,Swjetschka." - - Podswietschnik (Leuchter) ? - - ,,Und d i e s e s . . . Ach, du mein G o t t . . . S w i e t s c h a . . . P o . . . Podswietschnik." - - Das Knie? - - ,,Die Hose, B e i n e . . . Die H~lfte d a v o n . . , es geht so u n d s o . . . " - - Ein Knie. - - ,,Ein Knie. Siebe, ein K n i e ! " - - Der Schenkel? - - ,,Kein Fleisch, keine S e h n e . . . ich weil~ es wirklich n i c h t . " - - Ein K e t t c h e n ? - .,Ein Giirtel. - - Nein, kein Giirtel, man muB besser sagen." - - Glasa (Augen) ? - ,,Glos, glost. " - - D e r Daumen ? - - , , D e r kleine F i n g e r . " - - V i e l e andere Gegenstgnde (Taschentuch, Uhr, Bleistift, Feder, Trinkglas) n e n n t er ganz richtig. Schon aus den oben angefiihrten Bruchstiicken seiner Sprache geht hervor, dab das Erkennen der Benennungen b e i i h m n i c h t g e s t S r t i s t ; wenn es ihm Miihe macht, beim Vorzeigen yon G e g e n s ~ n d e n ein passendes Wort zu finden u n d ihm d a n n ein falsches vorgesagt wird, so weist er dieses sogleich zuriick, oder, wenn er es zuweilen wieder holt, so erkennt er sofort seine Untauglichkeit. Wenn er aber das nStige Wort hSrt, so f~ngt er dasselbe sofort freudig auf u n d wiederholt es prompt. Das gleiche Erhaltensein der F~higkeit, die Benennung der Gegenstgnde riehtig zu erkennen, t r i t t auch bei folgender Versuchsanordnung klar hervor: wenn ihm vorgeschlagen wird, diesen oder jenen Gegenstand zu zeigen, so mac h t er es stets richtig, selbst wenn die Benennung der Gegenst~nde zu denen gehSrt, auf die er sich selbst nieht mehr erinnern kann. D a n k diesem U m s t a n d e v e r s t e h t der Kranke sehr gut alles, was zu ihm gesproehen wird und folgt 656 M. Resnikow and S. Dawidenkow: Ausfallssymptome nach Lasion des r i e h t i g e i n e m e i n f a e h e n B e f e h l ; wenn man ihm komplizierte und ungewShnliche Befehle erteilt, wie z. B. die Augen schliegen und mit geschlossenen Augen auf das Sofa steigen, wieder yore Sofa hinuntersteigen, zur Tilre gehen und zuriickkehren, so macht er es falsch, indem er die Augcn schlieBt und sich auf das Sofa legt. Wenn man jedoch den den Befehl enthaltenden Satz in einzeine Teile zerlegt, so fiihrt er die einzelnen Befehle befriedigend aus. SehheBen Sie die Augen! - - geschieht; - - Steigen Sie auf das Sofa! - - geschieht, usw. I r g e n d w e l c h e D a t e n aus seiner A n a m n e s e k a n n der K r a n k e n i c h t a n g e b e n ; er weiB nicht, wie alt er ist; er e r i n n e r t s i c h s c h w e r d e r g e o g r a p h i s c h e n B e n e n n u n g e n ( G e d ~ c h n i s s t S r u n g ) . Man muB erw~hnen, dub der Kranke diejenigen Worte, auf die er selbst nicht kommen kann, die er jedoch erkennt, wenn sie ibm gesagt werden, sofort wieder vergiBt, und wenn man ihn nach einiger Zeit fiber denselben Gegenstand fragen sollte, so ger~t er wieder in Verlegenheit. So erinnert er sich nicht, in welcher Schlacht er verwundet worden ist und, trotz best~ndiger Wiederholung, dab dies bei Schach6 geschehen ist, kann er dieselbe Frage das n~chste Mal nieht beantworten. Er kennt die Namen der/Xrzte, die sich mit ihm wiederholt und sogar l~ngere Zeit besch~ftigt haben, nicht mehr, obgleich diese Namen 6fters in seiner Gegenwart absichtlich und auch zufiillig wiederholt wurden. Die F i i h i g k e i t , v e r g e s s e n e W o r t e w i e d e r zu l e r n e n , i s t s e h r g e s c h w ~ c h t . So z. B. wurde dem Kranken ein Kalender gezeigt und seine ihm wohlbekannte Benennung mitgeteilt, die er aueh riehtig wiederholte; dann wurden ibm die Benennungen fiir Ring und Daumen gesagt. Beim Erlernen der letzten vergaB er sofort das Wort Kalender. Man muBte ihm die genannten Gegenst~nde mehrmals zeigen, ihn die Benennungen wiederholen lassen, damit er sie im Ged~chnis behalte, wenn auch nur fiir kurze Zeit - - einige Sekunden, ungeachtet des Weckens seiner Aufmerksamkeit, sowohl yon seiten des GehSrs ~ie auch des Gesichts. D a s L e r n e n u n d B e h a l t e n v o n W o r t e n war der E r l e r n u n g einer fremden Spraehe von einem unaufmerksamen und mit einem schlechten Ged~chtnis behafteten Schiller v o l l k o m m e n ~ h n l i c h . Anfangs vergaB er die Benennung vollst~ndig, dann behielt er sie in entstellter Form (Kondier start Kalender ~ Kalendar), und endlich behielt er sie richtig ein paar Sekunden im Ged~chtnis, um sie sofort wieder zu vergessen (StSrung der direkten Reproduktion ---- der Merkf~higkeit). Das W i e d e r h o l e n y o n Z a h l e n : Nur das Wiederholen von kleinen Zahlen ist mSglich, zweistellige Zahlen wiederholt er richtig; dreistellige nur mit Miihe. Die Wiederholung der Zahl 3275 geschieht in folgender Weise: ,,Dreitausend... zweitausend.., nein, schleeht gesagt." Einige St~dtenamen wiederholt er riehtig: z. B . P e t e r s b u r g - ,,Petersburg", andere mangelhaft; statt Jekaterinoslaw sagt er Jekiterislaw; statt Konstantinograd Konstograd, Konistoar, Konistograd usw. Einfache Worte wiederholt er besser als Zahlen und Stiidtenamen, aber die Wiederholung geschieht ebenfalls mangelhaft. Das a u s w e n d i g G e l e r n t e ist bedeutend gestSrt; vom ,,Vaterunser" kann er nur die ersten Worte wiedergeben. Da der Kranke e i n A n a l p h a b e t i s t , so k o n n t e m a n in b e z u g a u f L e s e n u n d S c h r e i b e n k e i n e F e s t s t e l l u n g e n maehen. Das B e n e n n e n u n d V e r s t e h e n d e r F a r h e n b e n e n n u n g e n nimmt eine etwas besondere Stellung ein; nicht nur das spontane Benennen der Farben ist gestSrt (dies bezieht sich auf alle Farben), s o n d e r n a u c h das V e r s t e h e n d i c s e r B e n e n n u n g e n i s t g e s t S r t . Die W a h r n e h m u n g u n d d a s Unters c h e i d e n d er F a r b e n i s t e r h a l t e n g e b l i e b e n . Wenn man dem Kranken verschiedene Farben zeigt und ihm auftr~gt, anzuzeigen, welche Farbe die BlOtter, das Gras, das Blut usw. haben, so sucht er dieselbe stets sicher und riehtig aus. linken Gyrus angularis in einem Falle yon Schadel- und Gehirnverletzung. 657 Wenn man aber ihn bitter diese Farbe zu nennen, so gibt er fast hie eine richtige Antwort oder versueht aufs Geradewohl metn'ere Farben zu neunen: ,,Rosa, rot, sehwarz." Wenn ihm farbige Gegenst~nde gezeigt und versehiedene Farben genannt werden, so kann er den riehtigen Farbennamen dei weitem nieht so sicher w~hlen, als die Benennung der Gegenst~nde. - - W e l e h e X~arbe hat alas Gras ? - ,,Rosa." Es werden ihm ffir die Farbe des Grases versehiedene Namen vorgelegt; sagt man ihm griin, so antwortet er: ,,So ahnlich, griin, vieneicht griin." Beim Vorzeigen eines gelben Gegenstandes und Aufz~hlen versehiedener Farbennamen wKhlt er aus den genannten Farbennamen ,,blau" und s~gt: ,,Es ist fast blau." - Gelb ? - - ,,Ja, gelb, so ist es." Beim Vorzeigen eines blauen Gegenstandes und beim Aufz~hlen des Farbennamen lautet die Antwort- ,,Gelb, vielleicht, es kann gelb sein." Wenn ihm die richtige Benennung der Farbe (blau) vorgesagt wird, seheint er sie nicht zu erkennen. Die Benennungen ,,schwarz" und ,,rot" erkennt er leichter. (Achromaoptisehe Aphasie nach Monakow oder amnestisehe Farbenblindheit nach Wilbrand.) Bei den Versuchen mit einfaehem Kopfrechnen und Geldz~hlen sind seine Antworten fast alle unbefriedigend. G e d ~ e h t n i s ffir Z i f f e r n u n d Z a h l e n i s t b e d e u t e n d g e s c h w ~ c h t. GewShnlieh wiederholt er blo$ die ihm gestellte Aufgabe und kann nichts weiter tun. Nur beim Operieren mit Zahlen unter 10 bekommt man richtige Antworten. Diese riehtigen Antworten werden erleiehtert, weun man ibm anstatt des Kopfreehnens Geldmfinzen zum Z~hlen vorlegt. Wieviel betr~gt 20 4- 15 ? - - ,,20 + 15 ? . . . " - - 5 -4- 3 ? - - ,,Ffinf und drei ? . . . ja, ja, aeht." - - 7 ~- 4 ? - - , , S e c h s . . . - - Wieviel sieben und vier ? - - Das kann ieh nieht verstehen." - - 5 + 2? - - ,,Wieviel macht f i i n f . . , und . . . zwei? Sieben!" - 3 § 1 0 ? - - , , F i i n f , n e i n . . , d r e i . . . , verstehe niehts!" Man zeigt ihm Miinzen von 20, 15 und 10 Kopeken. ,,20 -t- 15 A- 1 0 . . . zusammen 2 5 . . . nein, mehr, hier sind 25 (zeigt 10 und 15) und hier 20 und z u s a m m e n . . , weil~ ieh nieht." Man zeigt ihm zwei Miinzen zu drei Kopeken: ,,Seehs"; drei Kopeken und zwei Kopeken: ,,Fiinf"; drei Kopeken und drei Kopeken und zwei Kopeken: ,,Aeht !" In der psyehisehen Sphere sind irgendwelehe intensivere StSrungen nicht zu bemerken. Pat. fiihrt sich die ganze Zeit gut auf, orientiert sich geniigend fiber seine Umgebung. Die summarische als auch die sukzessive ~berlegung und die Kombinationsfahigkeit sind nicht merklieh gestSrt; weder Sinnest~uschung noch Wahnideen. Intellekt und Ged~ehtnis haben nur so welt gelitten, als aus obigen Beispielen hervorgeht. Was das Emotionsleben betrifft, so kann man auf die erhShte Erregbarkeit hindeuten. Schon bei einem gewShnhchen Gespr~ch kann man bei Pat. eine lebhafte Mimik und Gestikulation bemerken. Er errStet leicht; Weinen und Laehen treten leicht zum Vorsehein. Seine Stimmung ist meistens zuriiekhaltend und gutmiitig. Mit seiner Umgebung, mit den ihn behandeinden Xrzten, mit dem Abteilungspersonal ist er stets zufrieden und dankt ffir alles, sieh verbeugend und gutmiitig l~chelnd. Er wird leieht verlegen, es geniert ihn, vor den ~rrzten zu sitzen und diesen die Hand zu reichen. Wenn er etwas nieht versteht, oder bei der Untersuehung etwas nicht richtig maeht, so ist er betriibt, seufzt und sagt: ,,Mein Gott, mein Gott, Sie tun mir leid; Sie wollen fiir mich das Beste und ieh verstehe nicht." Er ist sehr offenherzig; beklagt sich fiber seine l~rau, die ilm schlecht behandelte und ihn in das Krankenhaus, in der Hoffnung, dal~ er sterben werde, gebraeht haben soil. ,,Bin Menseh g e w e s e n . . , und nun hat man reich ein wenig auf den Kopf g e s c h l a g e n . . . Jetzt sagt die Frau: ,Groschen verkaufen', sie wiinscht, Efrem Pawlowitsch sterbe." Nieht nur die Frau, sondern auch seine anderen Verwandten sollen dasselbe wiinschen: ,,Gott gebe, da$ er stirbt!" Er gewann ein kleines Bild, das in seiner Abteilung h~ngt, sehr lieb: ein junges M~id- 658 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Lasion des chen sitzt und liest ein groBes Buch mit Verschlu•. Pat. glaubt, es sei das Evangelium, und bithet, ihm das Bild zu schenken, wenn er nach Hause fahren wird. Er ist immer munter und hglt sich aufrecht. Seine Stimmung wechselt selten; Zornausbriiche wurden in der Abteilung nicht beobachtet. Analyse der Symptome. Indem wir die Symptome naeh den einzelnen Funktionen gruppieren, werden wir sie in aufsteigender Reihenfolge je naeh ihrer Ausgesprochenheit ordnen. Der Kranke zeigt folgende Symptome: 1. a) Kaum bemerkbare Spuren der iiberstandenen rechtsseitigen Hemiplegie; b) leiehte reehtsseitige Ataxie. 2. Erweiterung der rechten Pupille. 3. Homonyme bilaterale rechtsseitige Hemianopsie. 4. StSrung des stereognostischen Sinnes rechts. 5. StSrung der Tiefenwahrnehmung ? 6. SprachstSrung : a) Amnesia verbalis; b) Paraphasia litteralis; c) Paraphasia verbalis; d) Paraphasia affeetiva; e) Perseveration; f) Logorrhea. 7. Aehromaoptisehe Aphasie oder amnestische Farbenblindheit. 8. a) Ausgesproehene Abnahme der FS~higkeit, die frisehen Spuren von Eindriieken zu behalten (StSrung dez direkten ReproduktionsfS,higkeit) ; b) bedeutende Abnahme der Fi~higkeit, das friiher Erlebte zu reproduzieren (Stiirung des Gediiehtnisses im engeren Sinne). l a) Es ist nieht notwendig, bei den Motilitiitserscheinungen liinger zu verweilen, denn sie sind augenseheinlich die Spuren einer bis zu den motorisehen Zentren sieh erstreekenden StSrung, welche jedoch diese Gegend ohne stiirkere StSrungen gelassen hat. l b) Was die leieht ataktischen Erseheinungen betrifft, so stehen diese kaum in irgendweleher Beziehung zu den minimMen Motilit~tsstSrungen. Riehtiger ist es, sie als AssoziationsstSrung zwisehen motorisehen und sensiblen Zentren auf dem Gebiete ihrer funktionellen Gruppierung aufzufassen (Gyrus eentralis posterior). 2. Wenn aueh das Vorhandensein yon Pupillenzentren im Gyrus angularis der Allen von B e e h t e r e w 1) experimentell naehgewiesen ist, so ist es ihm doeh nieht gelungen, einen einseitigen Effekt weder von der Occipital- noeh v o n d e r Parietalrinde einer der Hemisphiiren her1) Grundziige der Lehre von den Funktionen des Gehirns, S. 1385--86 (russ.). linken Gyrus angularis in einem Falle yon Schadel- und Gehirnverletzung. 659 vorzurufen. Deshalb ist keine :Notwendigkeit vorhanden, die Pupillenerweiterung bei unserem Kranken als direktes lokales Symptom aufzufassen, das durch L~sion oder ZirkulationsstSrung in den Pupillenzentren der Parietal- und Occipitalwindungen hervorgerufen ist. Zwar hatte Patient keine hemiopisehe Reaktion, welche eine Differentialdiagnose zwischen der zentralen Hemianopsie und einer solehen, der durch L~sion der subcorticalen Ganglien und des in ihnen endenden Tractus opticus ( W i l b r a n d und W e r n i c k e ) hervorgerufen ist, ermSglicht. Folglich ist keine MSglichkeit vorhanden, die Erweiterung der rechten Pupille durch das Prevalieren der sekund~ren Degeneration in den subeorticalen Ganglien oder Fasern des linken Tractus optici zu erkl~ren. Aber unzweifelhaft l~l]t sich beim Kranken ein Abblassen der linken H~lfte beider Pupillen konstatieren. Die sekund~re Degeneration nach L~sion der Sehsph~re breitet sich auf den Tractus opticus nicht aus, aber eine einfache Atrophie seiner Fasern ist m5glich [Monakowl)]. Da die Einengung des rechten Gesiehtsfeldes bei unserem Kranken erheblich die des linken iibertraf, so ist es wahrscheinlich, daI~ die Abblassung der linken H~lften der Pupillen mit der langdauernden Inaktivit~t der sagittalen Sehstrahlung der linken Hemisphere in Verbindung steht. So kann man die Pupillenerweiterung dureh die st~rkere Verdunkelung (grSi~eren Ausfall) des Gesiehtsfeldes des rechten Auges erkl~ren; diese Verdunkelung fibte auf das reflektorisehe Zentrum dieser Pupille in den oberfli~chlichen und seitlichen Teilen der grauen Substanz des vorderen Zweihiigels einen negativen EinfluI~ aus. 3. Gehen wir jetzt zur Betraehtung desjenigen, sehr stabilen Symptoms fiber, das bei unserem Kranken und fiberhaupt bei Erkrankungen der Occipital- und hinteren Parietalwindungen sehr oft beobachtet wird. Dieses Symptom ist die H e m i a n o p s i a h o m o n y m a b i l a t e r a l i s d e x t r a . Die Erhaltung des zentralen SehvermSgens, das Fehlen der hemiopischen Pupillenreaktion, das Fehlen atrophischer Ver~nderungen am Augenhintergrunde und der Umstand, daI~ der Kranke vom Defekt der rechten H~lfte seines Gesiehtsfeldes keine Ahnung hat, das alles sprieht fiir eine zentrale (im Sinne der jenseits der subcorticalen Ganglien entstandenen) Amblyopie. Die Amblyopie kann sowohl yon der L~sion der Rindensubstanz der occipitalen Sehzentren, welche sich in den Gyri oeeipitales in der NiChe der L~sion befinden, abh~ngig sein, sowie auch vonder Li~sion der sagittalen Sehstrahlung (Radiatio optica), welche in dem tiefen Marklager unter und nach innen vom Gyrus angularis enthalten ist. Existieren genfigend zwingende Beweise im klinischen Bilde unseres Falles, um zwischen diesen MSgliehkeiten zu entscheiden ? Wir werden sp~ter noch zu dieser Frage zuriickkehren. 1) Gehirnpathologie l, 425. 660 M. Resnikow und S. Dawidenkow: Ausfallssymptomenach Liision des 4. Bei geniigendem Erhaltensein der Haut-Muskel-Gelenksensibilit~t (es wurden nur die Tastkreise nach W e b e r nicht untersucht) zeigte der Kranke eine starke StSrung des stereognostischen Sinnes. Soleh eine StSrung geht gewShnlieh den StSrungen anderer Qualit~ten der HautMuskelsensibilit~t parallel. Der Verlust des Muskelsinnes fiihrt zum Verlust der Stereognosis; die Unabhi~ngigkeit der letzteren vom erstgenannten ist keine h~ufige Erscheinung, besteht z. B. der Fall yon K u tnerl). In dem Falle von M o n a k o w ~) - - der unserem sehr ~hnlieh ist, bei welchem aueh fast keine motorisehen StSrungen vorhanden waxen, und eine alte h~morrhagisehe Cyste haupts~chlich im Gyrus angulaxis, teilweise aber aueh im Gyrus supramaxginalis saB - - war die stereognostisehe StSrung von einer MuskelsinnstSrung begleitet. Dagegen im Falle von Redlieha), wo bei einem epileptischen MKdehen die eorticalen Zentren des rechten Arms von ihm exzidiert wurden, konnte R e d l i c h konstatieren, dal~ die unmittelbar nach der Operation folgende totale Monoplegie des rechten Armes zwax gleichzeitig von dem Verlust des Muskel- und stereognostischen Sinnes begleitet wurde; nach drei Monaten aber, als v o n d e r L~hmung und der MuskelsinnstSrung nichts mehr zu bemerken war, dauerte die StSrung des stereognostischen Sinnes welter fort. Diese yon StSrungen anderer elementaxer Hautmuskelempfindungen unabh~ngige StSrung des stereognostisehen Sinnes ist unter dem Namen reine Astereognosis zum Untersehied v o n d e r sekundKren, abgeleiteten bekannt. W~hrend letztere natiirlieh als eine Folge einer L~sion der sensiblen Zentren und Leitungsbahnen angesehen werden kann, ist erstere am richtigsten als Resultat einer L~sion der Assoziationsbahnen zu betraehten, die die eortieale Hautmuskelfiihlsph~re mit anderen eortiealen, besonders der Sehsphere, verbinden (Wernieke). Die Stereognosis griindet sich haupts~chlieh auf die Erkenntnis der RaumverhKltnisse. Die zum Zwecke dieser Erkenntnis wiehtigsten Empfindungen werden von dem Lagegefiihl der einzelnen Glieder, von dem Muskelsinn der Finger und der Augenmuskeln und von den Gesichtsempfindungen gelierfert. Zwischen ihnen mul~ eine vollkommene Kongruit~t, welche durch die Art und individuelle Erfahrung erworben wurde, herrschen. In denjenigen F~llen, wo die prim~ren fiihlenden Tast- und anderen Zentren der Hantmuskelsensibilit~t nicht l~liert sind, geniigt schon eine L~sion der Assoziationsbahnen zwischen diesen und den Seh- bzw. motorischen Zentren derselben Seite, um eine Astereognosis zum Vorschein zu bringen. Ebenso wie die entsprechende 1) Monatsschr. f. Psych. 17 u. ~0, zit. nach Oppenheims Lehrbuch. 2) Opt. Zentren und Bahnen; Archiv f. Psych. 1891/92. a) StSrung des Muskelsinnesund des stereognostischen Sinnes. Wiener klin. Wochenschr. 1893, zit. nach Monakow, 1. e. 1905, 686. linken Gyrus angularis in einem Falle von Schiidel- und Gehirnverletzung. 661 Abart der sensorischen Aphasie kSnnte man die reine Astereognosis als transzentrale Astereognosis bezeichnen. Es ist nun leicht erkl~rlich, dal~ die Erseheinungen der Astereognosis bei LKsionen in versehiedenen Gebieten der tIirnsubstanz vorkommen kiinnen: wie bei L~sionen des motorischen Gebiets der Zentralwindungen, vorzugsweise der hinteren Zentralwindung ( W e r n i e k e u. a.), so auch bei L~sionen des Parietallappens, des Gyrus supramarginalis ( D e j e r i n e , O p p e n h e i m u. a.) und endlich such bei tterden im Gyrus angularis und seinen n~chstliegenden Verbindungen mit der occipitalen Sehrinde. In unserem Falle kann man die Astereognosis mit einiger Wahrscheinlichkeit als eine Folge der AssociationsstSrung mit der Sehsph~re auffassen, denn bier ist die dauerhafte Astereognosis bei sehr unbedeutenden Motiliti~tsstSrungen, sowie ohne irgendwelche Hautmuskelsensibilit~tsstSrungen, yon einer hartn~ckigen ttemianopsia homonyma bilateralis dextra begleitet. Die eben angeffihrten Betrachtungen fiber die StSrungen des stereognostisehen Sinnes in Begleitung yon anderen FunktionsstSrungen des senso-motorischen Gebietes gestatten uns die MSglichkeit, viele klinisehe und experimentelle Ergebnisse, welehe Physiologen und Kliniker erhalten haben, in Ubereinstimmung zu bringen (Ferrier, M u n k , Hitzig, Luciani, Horsley, Wernicke, Bechterewl), Muratow u.a.). Soweit unsere Erfahrung und unsere Literaturkenntnis reieht, ist das Vorkommen der StSrung des stereognostischen Sinnes, die ohne StSrung anderer Qualit~ten der Hautmuskelsensibilit~t existieren kann, doeh niemals ganz isoliert, ohne irgendwelehe anderen klinisehen Erseheinungen beobachtet wurden. Wenn D e j e r i n e die begleitenden StSrungen nieht allein durch Sensibiliti~tsstSrungen oder allgemeinen Sehwachsinn beschr~nkt h~tte, so kSnnte man seinen in der Soci6t6 de ~Neurologic ausgesprochenen Worten, die lauteten: ,,Je ne connais pas de cas d'asymbolie tactile, publi6 jusq'ici, dans lequel il n'ait pas 6t6 constat6 soit des alterations de la sensibilit6 peripherique, soit un deficit intellectuel", vollkommen beistimmen2). Die obengenannten Autoren und auch einer yon uns a) haben durch zahlreiche Beobachtungcn und Versuche festgestellt, da$ in den Parietalwindungen, die dutch die Temporal-, Occipital- und hinteren Zentralwindungen unmittelbar berfihrt und umgeben werden und mit ihnen in engster Verbindung stehen, sich jenes Territorium befindet, dureh dessen Erkrankung oder Entfernung eine StSrung des Itautmuskel1) Grundziige der Lehre yon den Funktionen des Gehirns, S. 786--832 (russ.). u) Siehe such Egger, M., Le r61e du faisceau sensitif dans le mecanisme de la reconnaissance des objects. Rev. neurol. 18, 116. 1910. s) Resnikow, M., Echinokokkus des Gehirns, Obosrenie Psychiatrii 1898 (russ.). 662 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Lasion des sinnes entsteht. Dieses Gebiet steht zu den obengenannten, benachbarten Gebieten in einer Assoziationsverbindung. Je n~her sich die Li~sion der Parietalwindungen zu einer yon den Nachbargebieten befinder, desto mehr wird die StSrung des stereognostischen Sinnes von der FunktionsstSrung dieser benachbarten Windung begleitet. Die Stereognosis, als psychisches Produkt einer komplizierten T~tigkeit der Nervenzentren und der Verbindungen zwischen ihnen, kann in ihrer Feinheit und Vollkommenheit auch bei geniigender Erhaltung der ad~quaten e l e m e n t a r e n Empfindungen angegriffen werden, entsprechend dem W e r n i c k e schen Begriff der reinen TastlKhmung. Aber unter allen Bedingungen i s t die A s t e r e o g n o s i s e i n Z e i c h e n d e r Erkrankung, sei es i n d e n P a r i e t a l w i n d u n g e n selbst, oder sei es a u f d e r G r e n z e z w i s c h e n i h n e n u n d d e n b e n a c h b a r t e n W i n d u n g e n . Mit Ausnahme derjenigen F~lle, wo, wie einer von uns beobachtet hat, die Astereognosis das erste und friiheste isolierte Symptom einer beginnenden Erweichung ist, w i r d sie f a s t i m m e r e n t w e d e r von residu~ren Motilit~tsstSrungen (Paresen oder Spasmen der distalen Extremit~tsenden) u n d t r a n s i t o r i s c h e n Sensibilit~tsstSrungen begleitet, falls der Herd n~her zu den Zentralw i n d u n g e n g e l e g e n ist~), o d e r y o n r e s i d u h r e r s c h a r f a u s gesprochener Hautmuskelan~sthesie und transitorischen Paresen bei einer mehr zentralen Lage des Herdes im Gyrus supramarginalis, oder von aphasischen StSrungen bei Erkrankungen auf der Grenze zwischen Gyrus supramarginalis und den Temporalwindungen, oder schliel~lich von Hemianopsie, Alexie und corticaler Ophthalmoplegie bei Erkrankungen d e s G y r u s a n g u l a r i s . Selbstverst~ndlich gibt es auch eine andere Symptomenordnung bei umfangreicher und ausgebreiteter Erkrankung dieser Gebiete mit nicht verschwindenden StSrungen der Sensibilit~t und Motilitgt. Zur illustration der Richtigkeit dieser Auseinandersetzungen scheint der Fall yon. Schaffere) mit beiderseitiger Erweichung der Gyr. supramargin, sehr geeignet zu sein: ,,Ein Jahr vor dem Tode erlitt die Patientin den ersten apoplektischen Insult, damals nahm sic Sprachunf~higkeit und L~hmung der rcchten K5rperseite wahr. Bei dieser Gelegenheit bemerkte sie die Unempfindlichkeit der rechten Hand. Ein halbes Jahr sp~ter traf sie der zweite Insult: Hemiplegie links und ausgepr~gte Unempfindlichkeit der linken Hand. Die klinische Untersuchung crgab folgendes: Beriihrungs- und Schmerzempfindung fehlen bei gewShnlicher Aufmerksamkeit. Bei gespannter Aufmerksamkeit aber reagicrt sie auf Beriihrung, ziemlich oft (nicht immer) und immer auf Stiche, doch mit den allergrSbsten Lokalisationsfehlern: Stichc in die Gesichtshaut werden bald in den Fu~, bald in den Arm projiziert. Thermischen Eindriicken gegeniiber verhglt sich die Patientin 1) Gcorges Guillain et G. Laroche, Astereognosis spasmodique juvenile. Revue neurol. 1910. 2) Monatsschr. f. Psych. u. Neurol. 2~', 53. 1910. linken Gyrus angularis in einem Falle yon Schadel- und Gehiraverletzung. 663 genau so, wie auf taktile und schmerzhafte Reize. Genau dasselbe Verhalten l~iBt sich beziiglich der tiefen Sensibilit/it feststellen. Put. hat yon den in den distalen Gelenken vorgenommenen passiven Beugungen keine Vorstellung. HSchstens dab bei vorheriger Anzeige des vorzunehmenden ManSvers die Kranke hier und da die Stellung der proximaleren Gelenke errKt. Vollko m me ne A s t e r e o g nose. AuBerdem l~Bt sich beim Krankea bemerken: Verlangsamtes und mehrmaliges Suchen nach Worten, Paraphasia litteralis, ausgeprs Unsicherheit bei Prs bewegungen, spastische Parese in den linken ExtremitEten und Ataxie der Hand. Die ]ortdauernden Sensibilitdtsst6rungen, die Asteriognose, welche yon Motilitgitsst6rungen links und yon Symptomen sensoriseher Aphasie rechts begleitet ist, itberhaupt das ganze Krankheitsbild im Einklang mit den oben ange]zihrten Betrachtungen er]ordern, daft der krankha]te Herd hauptsdchlich im Gyrus supramarginalis, vorn ndiher zur hinteren Zentralwindung, unten und hinten zur Temporalwindung gelegen ( lokalisiert) sei. Was zeigte nun die Obduktion? ,,Beiderseits in der unteren Parietalwindung befinden sich Erweichungsherde. Rechts erstreckt sich makroskopisch der offenbar malaeische Defekt auf die unteren zwei Drittel der hinteren Zentralwindung, wobei von letzterer am Winkel der Zentralfurche und des hinteren Astes der Sylvischen Furche ein unansehnlicher Rest yon der Erweichung verschont bheb. Ferner nimmt die Malacie einen Tell des Gyrus supramarginalis ein (klinisch: Sensibilits spastische Erscheinungen und Ataxie der Hs Die linke GroBhirnhemisphs weist eine etwas mehr riickwErts gelegene Grube auf, welche oben vonder Interparietalfurche, vorn oben yon der Postzentralfurche begrenzt wird, unten erscheint der hinterste Teil der ersten Temporalwindung in die Erweichung einbezogen zu sein. Demnach nimmt die Malacie genau den Gyrus supramarginahs ein (klinisch: die Hemiplegie hat sieh vollkommen zuriickgebildet, geblieben sind rechtsseitige Hemiataxie, SprachstSrung, Sensibihts und Astereognose). Mikroskopisch erstreckt sich die L/ision auf die benachbarten Windungen: der malakische Herd dringt besonders links in die weiBe Substanz der Temporal- und Occipitallappen (aber keine Hemianopsie) und auch in den Nuel. lenticularis und Insula Reilii ein. Das Corpus callosum war auf der HShe der Herde in den Parietalwindungen auf ein Drittel seines Volumens verkleinert und hauptss auf Kosten des zentralen Tells." Nach alledem k o m m t S c h a f f e r zu dem SchluB, dab 1. der Gyrus supramarginalis derjenige Hemisphiirenbezirk ist, welcher fiir den Muskelsinn, den Lokalisationssinn der H a u t u n d die Stereognosis dient. 2. der Gyrus supramarginalis entbehrt Projektionsverbindungen mit dem Sehhfigel im Sinne corticofugaler Fasern; er stellt ein Erinnerungsoder Vorstellungsfeld der tiefen, sowie oberfls cerebralen Sensibitits dar, seine ZerstSrung fiihrt zu assiativ sensiblen Defekten der oberfls wie tiefen Sensibilits welche als TopoanKsthesie und Astereognosis b e k a n n t sind. Das aufgefundene degenerierte Biindel rechts verls nach S c h a f f e r y o n der hinteren Zentralwindung zum hinteren lateralen Teil des Thalami optici (cortifugal), nicht v o m Thalamus opticus zum Gyrus supramarginalis (corticopetal), der naeh F l e c h s i g im Gegensatz zu M o n a k o w nicht unmittelbar projektionsweise mit dem Sehhiigel verbunden ist. 3. Unrichtige TiefenschStzung und Fixation. 664 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Lasion des Viele Autoren, unter ihnen von den neueren v. V a l k e n b u r g und Nie$1 v. M a y e n d o r f , halten die obengenannten StSrungen ffir charakteristisch bei Erkrankungen des Gyrus angularis. Sie sind nur in den Anschauungen fiber die Entstehung dieser StSrungen nicht einig. v. Val k e n b u r g macht die letzteren von der StSrung des Mechanismus, welcher die assoziative T~tigkeit der optischen Erregungen mit der Konvergenz des Blickes bedingt, abhi~ngig. NieI~l v. M a y e n d o r f ist anderer Meinung: indem dieser dem dorso-lateralen Teil der Radiation optica die Funktion des macul~ren Biindels zuschreibt, meint er, daf], wenn auch seine ZerstSrung keine gekreuzte Gesichtsfeldeinengung hervorrufen muB, sie doch eine Wortblindheit (Alexie), eine StSrung in dem Fixieren der Gegenst~nde, in der Tiefenwahrnehmung und Entfernungssch~tzung verursacht. Unser Kranker hatte scheinbar keine StSrung der Tiefenwahrnehmung und aueh des Fixierens der Gegenst~nde im Raume. Was als solche gelten konnte, ist eher eine Dissoziation zwisehen Konvergenz und Akkommodation einerseits und dem Umfange der Armbewegungen, um die fixierten Gegenst~nde zu beriihren, andererseits. Zwar hatte man bei der Untersuchung den Eindruck, als ob sich der Kranke in der Tiefenseh~tzung irre, weil der Finger welter oder n~her, als es nStig war, naeh dem Gegenstande suehte. Aber die wiederholte Untersuchung des stereoskopischen Sehens nach dem Lissauerschen Verfahren ergab, dab die von unserem Kranken gemachten Fehler anders zu deuten sind: die leichte rechtsseitige Hemiataxie einerseits und das A xenfeldsehe Ph~nomen andererseits bewirkten diese Fehler. Da in dem reehten Gesiehtsfelde im Fehlfiihren des Fingers beide Faktoren im Spiele sind, tritt die fehlerhafte Berfihrung sehr merklich auf; in dem linken Gesichtsfelde aber, wo die Fingerbewegung unter der Augenkontrolle sicherer und pr~ziser ist, wird der Fehler viel geringer. Nun ist es klar, dab die oben beschriebenen StSrungen nicht notwendig als eine StSrung ,,des Gefiihls des Grades der Konvergenz der Augen" (Munk) oder eine St5rung der Tiefenlokalisation (Pick) angesehen werden mfissen. Gehen wir jetzt zur Analyse der S p r a e h s t S r u n g e n fiber. Die oben angefiihrten Beispiele der Spraehst5rung lassen sich ihrer Kompliziertheit naeh in folgende Gruppen anordnen: a) S p o n t a n e S p r a c h e . a) A m n e s i a s y l l a b a r i s mit Ersetzung der vergessenen Silben durch andere (Paraphasia litteralis) oder ohne eine solche. Die Amnesie wird bald von einem Unsicherheitsgefiihl in der Richtigkeit des ausgesprochenen Wortes begleitet, bald ist der Kranke der Unrichtigkeit sich nicht bewuBt. fl) A m ne sia ve r b alis mit denselben Eigentiimliehkeiten : vSlliges linken Gyrus angularis in einem Falle yon Schi~del- und Gehimverletzung. 665 Vergessen des Wortes ohne Ersetzen desselben dutch ein neues oder mit Ersetzung durch ein ungeeignetes Wort. Z) P a r a p h a s i a a f f e c t i v a m i t L o g o r r h o e verbunden. Diese l~13t sich in affektivem Zustande beobachten. Die vergessenen Silben oder Worte werden durch neue ersetzt und dieses Ersetzen wird nieht yon einem Unsicherheitsgefiihl in der Richtigkeit des ausgesprochenen Wortes begleitet, was augenscheinlich in jenem Fall geschieht, wenn die Erfahrungsassoziationen infolge der vollkommenen Leitungsunterbreehung oder dem Untergang der zentralen Elemente vernichtet bzw. unterdriickt sind. 8) Die vergessenen Worte werden durch andere, unmittelbar friiher schon gebrauchte ersetzt; was wahrscheinlich dadurch bedingt sein kann, dal3 wegen der Erregungssehw~che in der Leitungsbahn yon dem in Szene tretenden komplizierten Begriffszentrum zu dem entsprechenden einfachen W.ortklangbildzentrum das Ergreifensein dieses letzteren durch die abklingende, doeh grSBere Erregung yon den unmittelbar vorher t~tigen Begriffszentren fortdauert. e) Partielle Erhaltung des Wortklangbildes mit haupts~chlicher Ver~nderung des W o r t e n d e s bei den gebrguchlichen Benennungen und der M i t t e des Wortes bei den selteneren und vielsilbigen. Im ersten Falle wird der labile Tail des Wortes ver~ndert oder deformiert, im letzteren - - der wenig akzentuierte und folglieh der weniger Aufmerkkeit in Anspruch nehmende Tell; im ersteren Falle haben w i r e s mit einem Verwischen der labilen Ged~chtnisspuren zu tun, im zweiten mit dem Niehtfesthalten der schwaehfixierten Abdrucke; im ersten Falle - - ein Defizit im Reservekapital des Ged~ehtnisses, im zweiten eine Stockung seines Wachstums. b) Das N a c h s p r e e h e n . oc) ist durch dieselben Eigentiimlichkeiten gekennzeichnet wie die spontane Sprache. Da aber die Erregung, die yore peripheren Organ zugefiihrt wird, sich durch die Kraft der unmittelbaren materiellen Ver~,nderung auszeichnet - - Umsetzung der Luftwellenschwingungen in GehSrnervenerregungen und GehSrempfindungen - - ist bier die expressive Reaktion des Kranken viel richtiger, aber unter der Bedingung, dal3 zwischen dem Anfange des nachgesprochenen Wortes und seinem Ende kein zu grol3er Zeitintervall vorhanden sei, der yon einer Fiille yon Silben ausgefiillt w~re. Oeshalb spricht der Kranke die kurzen Benennungen, ein- oder zweistellige Zahlen, richtig nach, die langen fehlerhaft. fl) Die StSrung, das friiher auswendig Gelernte zu rezitieren, ist demselben Grundprinzip unterworfen: das Erhalten des Anfangs des Rezitierten und das Entstellen oder das Vergessen der Mitte oder des Endes. Eigentlich ist diese Erscheinung, wenn auch in viel geringerem Z. f. d. g. Neut. u. P s y c h . O. IV. 43 666 M: Resnikow und S. Dawidenkow: Ausfallssymptomenach Liision des Mal~e, auch beim Ged~chtnis des normalen Menschen zu bemerken: wir behalten oft den Anfang eines Verses, w~hrend wir dessen Mitte oder Ende vergessen. Um uns das Wesentliche der SpraehstSrung bei unserem Kranken klar zu maehen, geniigt es, sieh der in der Krankengeschichte ~ngefiihrten Beispiele seiner Sprechf~higkeit, wie er ganz neue oder vergessene Worte wiedererlernt, wie er die ihm erteilten Befehle oder die von ihm verlangten Handlungen ausfiihrt, zu erinnern. Zwei GrundstSrungen beherrsehen alle oben aufgez~hlten Einzelheiten: t. StSrung der elemantaren nicht zerlegbaren FKhigkeit, frisch aufgenommerle E i n d r i i e k e ( O b j e k t b i l d e r ) f e s t z u h a l t e n , das, was Wern i e k e M e r k f ~ h i g k e i t nennt und was wir als d i r e k t e p e r i p h e r i s c h e R e p r o d u k t i o n zu bezeiehnen vorziehen; denn es handelt sieh hier um die Reproduktion yon Eindriicken, die unmit~elbar in dem n~chst vorgehenden Moment durch Erregungen eines Sinnesorganes, yon der Peripherie aus, eingewirkt haben. Das n o r m a l e F u n k t i o n i e r e n d i e s e r F ~ h i g k e i t ist die n o t w e n d i g s t e B e d i n g u n g j e d e s ges u n d e n G e d K e h t n i s s e s . 2. St Srung des W e c k e n s y o n E r i n n e r u n g s b i l d e r n v e r g a n g e n e r E r l e b n i s s e (GedKehtnis von aufbewahrten Eindriieken), was man gewShnlich ,,Ged~chtnis" nennt und das wit zum Unterschiede vom ersteren Vorgange als i n d i r e k t e zent r a l e R e p r o d u k t i o n beSraehr wollen, da die Wiedererzeugung auf assoziativem Wege und mehr zentral als im ersteren Falle ausbricht. Bei unserem Kranken erreieht die Wortklangerregung die Zentren der Wortbilder, ruft diese hervor, weckt entsprechende Seh- oder andere Sinnesbilder und pflanzt sieh assoziations- und projektionsweise fort, indem sie praxisehe und expressive Reaktionen bewirkt. Aber diese E r r e g u n g b e r u h i g t sieh u n g e h e u e r s c h n e l l , e r l i s c h t m o m e n t a n , ohne irgendw'elche S p u r e n zu h i n t e r l a s s e n u n d vers c h w i n d e t auf i m m e r aus s e i n e m B e w u l ~ t s e i n s f e l d e . D u r c h die T a t s a c h e der L ~ h m u n g d i e s e r e l e m e n t a r e n G r u n d f ~ h i g k e i t e n der h S h e r e n n e r v S s e n Z e n t r e n , der letzteren der Reihe n a e h - - N e u r o n e n - - ist d i e M S g l i e h k e i t g e g e b e n , n i e h t n u r die E i n z e l h e i t e n der S p r a e h s t S r u n g , s o n d e r n a u c h die StSr u n g a n d e r e r p s y c h i s c h e n Vorgi~nge, wie des Wiedererkennens, des Identifizierens, des Klassifizierens, des Urteilens zu erkl/~ren. Die hier von uns beschriebene SpraehstSrung ist als eine reine Form der amnestisehen Aphasic aufzufassen. Nach M o n a kow 1) ist sie ,,als ganz reine Form selten und nicht von allen Autoren anerkannt; sie kommt meist in Verbindung mit sensorischer Aphasie, meist als Vorstufe dieser (bei Trauma, Tumoren usw.) vor, meist transitorisch". Bei unserem Kranken zeiehnet sich die amnestische Aphasie durch 1) 1. c. 893. linken Gyrus angularis in einem Falle von Schadel- und Gehirnverletzung. 667 unver~nderliche Persistenz und Hartn~ekigkeit aus, und g e h S r t zu den fundamentalsten residu~ren StSrungen, die yon dem Trauma verursa~ht worden sind. Sie tritt in der reinsten isolierten Form hervor, ohne von sensorischer oder motorischer Aphasie begleitet zu sein. Die einzige sensorische SpraehstSrung, die uns beim Kranken noch auf zudecken gelungen ist, ist 7. die a c h r o m a o p t i s c h e A p h a s i e . o d e r die a m n e s t i s e h e Farbenblindheit. In der Krankengeschichte haben wir geniigend Beispiele, welehe diese StSrung eharakterisieren, angefiihrt. Wir sehen, dab die Assoziation zwischen der Vorstellung des Gegenstandes und seiner Farbe, wie aueh zwisehen dem Wortsymbol des Gegenstandes und seiner Vorstellung selbst erhalten blieb, es war n u r die A s s o z i a t i o n z w i s e h e n d e m W o r t s y m b o l der F a r b e u n d i h r e m o p t i s c h e n Bilde ges p r e n g t und offenbar nach beiden Richtungen hin, denn es bestand keine MSgliehkeit weder dureh die Farbenbenennung die Vorstellung (das Erinnerungsbild) v o n d e r Farbe, noch dureh das Vorzeigen derselben ihre Benennung hervorrufen. Der von L i s s a u e r publizierte Fall, sowie der von M o n a k o w 1) in seiner Gehirnpathologie erw~hnte ,,noeh in der Behandlung stehende" sind in vielen Hinsiehten unserem ~hnlich. M o n a k o w z~hlt sie mehr zur sprachlichen, als zur optisehen StSrung. ,,Es handelt sich ja eigentlieh noeh mehr als um eine optisehe, um eine sprachliche StSrung", ist sein wSrtlieher Ausdruek. Nach B e c h t e r e w ,,steht die aehromaoptisehe Aphasie der optischen sehr nahe und stellt gewissermaBen eine Abart der letzteren dar. Ihr Grund liegt seiner Ansieht naeh augenseheinlieh darin, da9 die Verbindung zwischen den optischen, resp. Farbenzentren und den HSrzentren in der Rinde gesprengt ist." Le w a n d o ws k y3) ~u~ert auf Grund einer sehr genauen und griindlichen Untersuehung eines Falles mit den besten Methoden seine auf diese Frage sieh beziehende Ansieht: ,,ob eine solehe direkte Bahn vom Farbensinnzentrum besteht, mul~ solange zweifelhaft bleiben, bis gut beobaehtete F~lle vorliegen, welehe diese SprachstSrung bzw. die Wilbrandsche amnestische Farbenblindheit rein ohne die von uns in unserem Falle aufgedeekten AssoziationsstSrungen zeigen." In der Tat bestand im Falle L e w a n d o w s k ys eine A b s p a l t u n g der Vor~ s t e l l u n g der F a r b e yon der V o r s t e l l u n g der F o r m und der Gestalt der Gegenst~nde, und darum ist seine Erkl~rung der Tatsaehe, dal~ der Kranke aui~erstande war, die Namen ihm gezeigter Farben anzugeben, dureh die Sprengung der Assoziation zwischen Form und Farbe sehr plausibel. Ganz anders in unserem Falle: der Kranke war nicht imstande, 1) 1. c. S. 762. 2) Ober Absprengung des Farbensinnes. l~Ionatsschr, f. Psych. u. Neurot. ~3, H. 6. 43* 668 M. Resnikow und S. Dawidenkow: Ausfallssymptomenach Li~sion des die genannte Farbe herauszusuchen und auch nicht die gezeigte Farbe zu benennen, aber er konnte sicher die dem genannten Gegenstande entsprechende Farbe heraussuchen und den gezeigten Farben entsprechende Gegenst~nde herausfinden (nennen). Das war yon uns wiederholt gepriift. Wir kSnnen also mit aller Bestimmtheit behaupten, dal3 die Assoziation zwischen der Vorstellung des Gegenstandes und der Vorstellung seiner Farbe unberiihrt blieb. Damit seheint uns der Zweifel an die Existenz dieser eigentiimlichen SpraehstSrung, die von W i l b r a n d als amnestisehe Farbenblindheit besehrieben und gedeutet wurde, endgiiltig gehoben. Wir wissen aueh, dab die optische Aphasie in denjenigen F~llen zu beobaehten ist, wo mit dem Gyrus angularis der einen Seite zugleich die Leitungsbahn, die vom Gyrus angularis der entgegengesetzten Seite durch das Corpus callosum zum Temporallappen zieht, zerstSrt ist [Beehterewl)]. So werden der Gyrus angularis und die von ihm durch das Corpus cullosum ziehenden Fasern ziemlich einstimmig fiir das anatomische Substrat dieser StSrung gehalten. Was aber jenes Assoziationsfasersystem zwischen den Seh- und HSrzentren betrifft, dessen L~sion durch die von uns beobachteten klinischen Erseheinungen postuliert wird, so sind hier nieht wenige Sehwierigkeiten zu iiberwinden. Friiher, als man M o n a k o w s Lehre zufolge den Fasciculus longitudinalis inferior (Stratum sagittale externum) als ein Assoziationsfasersystem, das seinen Ursprung in den Temporalwindungen nimmt und sein Ende in den Occipitalwindungen finder und auch umgekehrt auffaBte, konnte man ]eicht diese StSrung auf die LKsion dieses Biindels beziehen. Nachdem aber die Untersuchungen yon P. F l e c h s i g und M. Schlitz 2) der Lehre yon seiner Funktion eine ganz andere Richtung gaben, indem sie ihm die Bedeutung eines Projektionssystems gaben, wKre es geboten, ihn aus den anatomischen Faktoren, welche die StSrungen, um die es sich hier handelt, bedingen sollen, zu streichen. Andererseits haben in letzter Zeit T r o s c h i n und A g a d s c h a n j a n z in Bechterews Laboratorium bei ihren Untersuchungen der Assoziationssysteme des Gehirns ,,ein wohl abgrenzbares Bfindel aufgedeckt, das den Temporallappen mit der /iui~eren Flis des Occipitallappens, dem unteren L~ngsbiindel analog, verbindet." Bei dieser Gelegenheit /~uBert sieh B e e h t e r e w in folgenden Worten: ,,Wir halten die Meinung F l e c h s i g s fiber das Vorhandensein eines besonderen Projektionssystems an der Stelle des unteren L~ngsbiindels keineswegs ffir ausgeschlossen; wir sind aber der Meinung, dal~ es ein Assoziations2) Grundziige der Lehre yon den Gehirnfunktionen,S. 1492 (russ.). 2) Uber die Beziehungendes unteren L~ngsbiindels. Neurol. Centralbl. 1902, Nr. 19. linken Gyrus ang~laris in einem Falle von Schi~del-und Gehirnverletzung. 669 system zwischen dem Occipital- und Temporallappen gibt und aueh ein Projektionssystem an der Stelle des unteren L~ngsbiindels." Und so bringt die Ansehauung B e e h t e r e w s eine befriedigende Ausgleiehung zwischen den sich widersprechenden Lehren von M o n a k o w und Fle c h sig. Wenn wir unsere Meinung in dieser Frage ~uBern wollen, ohne eigene Erfahrungen in der Anatomic auf diesem Gebiete zu besitzen, so gesehieht es aus dem Grunde, weil die klinischen Tatsachen und die Ergebnisse der Untersuchungen mit der sekundi~ren Degenerationsmethode anderer Autoren uns veranlassen, eine bestimmte Stellung zu dieser Frage einzunehmen. ,,Nach isolierter Ausr~umung des Gyrus angularis (Mae a e u s) degenerieren nach M u n k und naeh M o n a k ow s eigenen Untersuehungen die Strahlung zum Pulvinar resp. die Stabkranzbiindel und die Assoziationsfasern im dorsalen Drittel der sagittalen Strahlungen (Ebenen kurz vor Beginn des Uberganges des Hinterhornes in den Seitenventrikel). Gleichzeitig mit den genannten Stabkranzbiindeln geht aueh das Pulvinar bis zum Corpus genieulatum und bis zu den ventr~len Kerngruppen des Sehhiigels zugrunde. Aueh bei alten Herden im Bereich des Gyrus angularis des Menschen degenerieren sekund~r Stabkranzfasern in der dorsalen Etage der sagittalen Strahlungen und im AnschluB daran das Pulvinar selbst in betr~chtlicher Weise [Dejerine]"l). Da aber isolierte Herde im Gyrus angularis nicht yon einer residu~ren Hemianopsie begleitet werden, die gerade bei unserem Kranken sieh jahrelang beobaehten lieB, so also ist es augenseheinlich, dab man bei ibm notwendigerweise die ZerstSrung der tiefer gelegenen Teile der sagittalen Strahlungen anerkennen muB. Wenn man nun in Betraeht zieht, daI~ neben der Hemianopsie sieh bei dem Kranken als residu~res Symptom noch die aehromaoptisehe Aphasie - - eine reine AssoziationsstSrung - - zeigte, so ist es verst~ndlieh, dab diese zwei physiologiseh so nahe stehenden Symptome in nahe aneinander liegenden, sogar sieh durehflechtenden Ganglienfasersystemen ihr anatomisches Substrat haben miissen, damit eine und dieselbe Herdl~sion beide pathologisehen Erscheinungen zutage treten lassen kann. Das eben sind die Griinde, die vom klinischen Standpunkte aus uns zwingen, die vermittelnde Ansicht B e c h t e r e w s anzunehmen und ~hnlieh, wie NieB1 y o n M e y e n d o r f , zu sehlieBen; wenn bei der L~sion des Gyrus angularis eine homonyme Hemianopsie beobaehtet wird, so ist das ~bergreifen des krankhaften Prozesses auf die ventrale Partie der Radiatio optica sehr wahrseheinlieh. 8. Die a m n e s t i s c h e A p h a s i e wird meistenteils in Begleitung yon Hemianopsie und Alexie beobaehtet und yon diesen drei Symptomen hat sich eben die Hemianopsie als das dauerhafteste und unkorrigierb~rste Symptom erwiesen. In unserem ]~alle fehlt die Alexie deswegen, well der 1) Monakow, Gehirnpathologie I, 427. 670 M. Resnikow und S. Dawidenkow: Ausfallssymptomenach Liision des Kranke analphabetisch war. Dagegen ist seine amnestische Amnesie durch eine nicht mindere Konstanz als die Hemianopsie gekennzeiehnet. Man muB sie also von der einzigen evidenten, lokalen L~sion abh~ngig machen. Wenn wir uns nun an die Autoren wenden, um die herrsehende Meinung fiber die Lokalisation dieses Symptomes festzustellen, so sehen wir, dab es noch keine so geniigend feste, fiber jeden Zweifel erhabene Tatsache gibt, um seine Abh~ngigkeit von der L~sion des Gyrus angularis im Sinne eines Zusammenhanges zwischen Organ und Funktion zu behaupten. Einerseits teilt M o n a k ow vier eigene Beobachtungen, wie auch die Beobaehtungen von Mills und anderen mit, in welchen die amnestische Aphasie eines der wichtigsten Symptome bei Tumoren in der weiBen Substanz des Lobus parietalis inferior war, andererseits zitiert er seine F~lle, wo h~morrhagisehe Cysten im Gyrus angularis aueh nicht die geringsten aphasisehen StSrungen verursaehten. Diese nicht fibereinstimmenden Beobachtungen veranlassen ihn, seine Meinung in der Weise zu ~uBern, dab eine sich auf die Oberfl~che des Gyrus angularis besehr~nkende Erkrankung ohne irgendwelche Symptome verlaufen kann; bei Erkrankungen aber des tiefen Markes des linken Parieto-Temporallappen, namentlieh bei tief im Gyrus angularis sitzenden Tumoren, bildet die amnestische Aphasie nach seinen Erfahrungen keine seltene Erscheinung und man kann daher der letzteren die Bedeutung eines Lokalsymptoms von gewissem Werte beimessen [Monakowl)]. Auf Grund zweier F~ille von B a n t i und je eines Falles von C o r n i l , K u B m a u l und B r o a d b e n t , meint Lueiani2), dab die reine verbale Amnesie ohne Worttaubheit als eine :Folge einer L~sion des linken unteren Parietallappens angesehen werden kann. Ha u n y n ffihrte in die Lehre fiber versehiedene Arten yon Aphasie die Gruppe yon Wortamnesie und Paraphasie ein und erkl~rte an vielen Beispielen seiner Erfahrung, dab sie yon den Erkrankungen im Gebiete des Gyrus angularis abhi~ngig seia). In einem reinen Falle von Amnesia verbalis fend H e n s c h e n 4) den vorderen unteren Teil des Gyrus angularis dureh einen Herd unterminiert. Es fehlt also nieht an Autoren, die den Gyrus angularis, seine l~inde und die unter ihr gelegene Marksubstanz als ein Gebiet betrachten, dessen L~sion oder Erkrankung eine amnestische Aphasie zur Folge hat. Es gibt aber auch Gegner dieser Ansieht. Auf Grund der Analyse von 30 Beobachtungen versehiedener Autoren und Berechnung des Verh~ltnisses der F~lle von amnestischer Aphasie, die bei der Erkrankung des Gyrus angularis ohne Beteiligung der angrenzenden Schl~fenwindungen beobachtet wurde und der F~lle 1) 1. c. S. 700 und 838. o.) Physiologie des Menschen ~, 627. 3) Verhandlungen des VI. Kongresses ffir innere Medizin. 4) Nagels Hundb. der Physiol. 4, 115. linken Gyrus angularis in einem Falle yon Schiidel- und Gehirnverletzung. 671 mit Heriibergreifen der Erkrankung auf den Schls zu allen iibrigen Fi~llen von Erkrankungen des Gyrus angularis gelangt NieB1 v o n M a y e n d o r f 1) zu dem Schlusse, ,,dal~ die hypothetischen von F l e c h s i g und P i t r e s vertretenen Anschauungen hinsichtlieh einer Sts der akustisehen Erinnerungsbilder der Worte im linken Gyrus angularis als durch die pathologisehe Anatomie nicht begriindet, ja mit diesen geradezu in Widerspruch stehend, abzulehnen seien." B e c h t e r e w 2) vertritt die Ansicht, ,,dal~ die L~sion in Fs yon amnestiseher Aphasie sich gewShnlieh im Gebiet des Sehls lokalisiert, da man sie bei Abseessen in diesem Lappen zu beobachten Gelegenheit Gelegenheit hat." Es ist nun klar, dal~ wir zur FeststeUung des gegenseitigen Verhs zwischen amnestischer Aphasie und L~sion des Gyrus angularis als einer FunktionsstSrung des letzteren andere festere Griinde suehen miissen. Bevor wir aber zur LSsung dieser Frage schreiten, wollen wir die yon uns gefundenen StSrungen bei unserem Kranken mit denjenigen vergleiehen, die zurzeit als am meisten charakteristisch und fiir eine Erkrankung in der Gegend des Gyrus angularis und der an seiner vorderen Seite grenzenden n~ehsten Teile pathognomiseh gelten. Naeh einer eingehenden Besprechung der beobachteten F~lle nimmt v. M o n a k o w folgende Symptome an, die gerade in dieser Gruppierung als ziemlieh sicheres Lokalzeichen einer a k u t e n Erkrankung des t i e f e n Markes des linken Gyrus angularis, eventuell auch des supramarginalis bezeiehnet werden kSnnen: a) die sogenannte reine Alexie; b) die rechtseitige Hemianopsie; c) eine sprachliche StSrung in Gestalt der amnestisehen resp. optischen Aphasie; d) Innervationsschwierigkeit im Gebrauch der reehten Hand (Apraxie) und e) StSrung der Tiefenlokalisation [M o n a k owa)]. Aus dieser Symptomenaufzs geht hervor, da~ M o n a k o w mit Ausnahme der StSrungen der Tiefenlokalisation keine andere StSrungen oder L~hmungen der Augenbewegungen unter die Symptome zs die fiir die Diagnose einer Erkrankung im Gebiete des Gyrus angularis unentbehrlieh sind. Niel~l y o n M a y e n d o r f kommt nach einer eingehenden kritisehen Sichtung von 25 aus der Faehliteratur genommenen Fiillen, sowie einem yon ihm selbst genau aueh mikroskopiseh untersuehten Fall zu folgenden diagnostischen S~tzen: 1) Die Diagnose auf Erkrankung des linken Gyrus angnlaris. Monatsschr. f. Psych. u. Neurol. 2~, 249. ~) h c. S. 1491. a) 1. c. S. 698. 67 2 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Litsion des ,,1. Eine Erkrankung des linken Gyr. angularis, welche sich fiber dessen Rinde und das unmittelbare darunter befindliche Marklager erstreckt, aber nur so welt vordringt, dab die Sehstrahlungen intakt bleiben, kann symptomlos verlaufen, vorausgesetzt, dab eine indirekte Sch~digung der Projektionsbahnen nicht vorhanden war (l~bereinstimmung mit M o n a k o w ) . 2. Werden die dorsalen Etagen der Sehstrahlungen mit ergriffen, tritt fast ausnahmslos Wortblindheit in Erscheinung, in seltenen F~llen gescllen sich Seelenblindheit, bier und da auch Gesichtshalluzinationen hinzu ( M o n a k o w s a). 3. Rechtseitige honmnyme Hemianopsie wird bei circumscripter Angularisl~sion in der Regel vermi]~t, sie zeigt sich jedoch bei umfangreichen Herden, welche ventralw~rts hinab und in die Schichten des Sagittalmarks hineinreichen (M o n a k o w s b). 4. Oculomotorische Symptome k S n n e n auftreten. Konjugierte Ablenkung des Augenpaares nach links infolge yon zentraler Li~hmung der Antagonisten. Bei Parese derselben Nystagmus bei der Blickbewegung nach rechts. Mangelnde Fi~higkeit des Fixierens, StSrungen im Absch~tzen yon Distanzen, Erweiterung dcr linken Pupille (Mon a k o w s e). 5. Agraphie ist ein h~ufiges Symptom, mu]~ aber, sobald seine Abh~ngigkeit yon gleichzeitig bestehender Alexie erwiesen ist, auf eine umfangreiche, im tiefen Marklager nach vorn sich erstreckende L~sion bezogen werden. Es wird bei Vorhandensein yon Agrapbie auf Motilitiit der Hand, Tastsinn, tiefe Sensibilit~t der :Fingergelenke und LokalisationsvermSgen in denselben zu priifen sein (Mona k o w spricht in diesem Falle eher yon Apraxie als yon Agraphie). 6. Verbale Amnesie und Paraphasie finden sich vorzfiglich bei Ausdehnung der Erkrankung auf die erste und zweite Schliifenwindung. Dieselben kSnnen jedoch auch durch A b k a p p u n g des h i n t e r e n B o g e n s des B u r d a c h s c h e n F a s c i c u l u s a r c u a t u s o h n e Mitb e t e i l i g u n g des S c h l g f e n l a p p e n s h e r v o r g e r u f e n w e r d e n " . Werm wir uns nun an die anderen Autoren wenden, so werden wir sehen, dab bereits C h a r c o t , was die Alexie anbetrifft, auf Grund klinischer Beobachtungen festgestellt hat, dab der Verlust der l~ihigkeit das Geschriebene oder Gedruckte zu lesen und zu verstehen, beim Erhaltensein des Sprech- und SchreibvermSgens, sowie des Wortverst~ndnisses yon Seh/idigung des linken Gyrus angularis abhiingig ist. B e c h t e r ew behauptet, dab die klinischen Erscheinungen keinen Zweifel daran lassen, dab bei der Beschiidigung des Gyrus angularis der linken Seite die Unfghigkeit zu lesen (Alexie) und mit Hilfe der Schriftzeichen die Wortbilder zu reproduzieren, zu den allergewShnlichsten krankhaften Erscheinungen gehSrt. linken Gyrus angularis in einem Falle yon Schiidel- und Gehirnverletzung. 673 F l e c h s i g schreibt der Rinde des Gyrus angularis speziell die Funktion zu, Buchstaben zu Worten zusammenzusetzen (vorwiegend Ged~chtnisleistung) und die Buchstabenbilder mit Lautvorstellungen zu verkniipfen. Wir diirfen demnaeh in der Gegend des Gyrus angularis mehr vorn und unten ein Rindenfeld des Ged~ehtnisses fiir Wortklangbilder, mehr hinten und oben ein Rindenfeld des Ged~chtnisses fiir Wortschriftbilder vermuten. Eine ganzeReihe vonAutoren, yon denen wir nur P a n i z z a , F e r r i e r , Gowers, A n g e l u c i , L u c i a n i , T a m b u r i n i et S e p i l l i , M o n a k o w und andere nennen wollen, erkennen an, da[~ auger einem Lesezentrum der Gyrus angularis in sieh noch ein Sehzentrum beherbergt. Allerdings wird diese Meinung nicht nur zurzeit yon Ni e 131v o n M a y e n d o r f, sondern bereits schon friiher von M u n k , H e n s c h e n , G a l l e m a e r t s , Seh~fer, H o r s l e y , B r o w n , T h o m s o n , T s c h e r m a k , H@don bestrittenl), die die bei Erkrankungen des Gyrus angularis oft zu beobachtende Hemianopsie durch die Mitl~sion der Gratioletschen Sehstrahlung zu erkl~ren suchen, und folglich stehe der Gyrus angularis in keiner direkten Beziehung zu den Sehfunktionen. ErbslSh~) hat einen Fall beschrieben, wo die am meisten konstante und augenf~lligste StSrung, die im Anfange der Erkrankung zum Vorscheine kam, sieh in der U n f ~ h i g k e i t des K r a n k e n , V o r s t e l l u n g e n , sogar e i n f a c h e r N a t u r , wie Dinge und n e u e Worte, im G e d ~ c h t n i s s e zu b e h a l t e n , ~ul3erte, ohne Riicksicht darauf, ob diese Vorstellungen das BewuI~tsein des Kranken mittels des Gesichts, des GehSrs oder des stereognostisehen Sinnes erreicht hatten. Diese StSrung wurde von einer Unfiihigkeit, sich im Raume zu orientieren, m i t m a s s e n h a f t e n G e s i c h t s - u n d w e n i g e n Geh S r s h a l l u z i n a t i o n e n , von einer rechtsseitigen Hemianopsie und einer reehtseitigen Parese des unteren Faeialis begleitet3), obwohl der Autor die L~sion in den Oceipitallappen verlegt, doch sprieht alles dafiir, daI3 der Gyrus angularis bei der Erkrankung in Mitleidensehaft gezogen war. K S n i g 4) publizierte einen Fall unter dem Titel ,,Subspiales Sarkom des reehten Gyrus angularis." Aul3er den Allgemeinerseheinungen, wie Kopfsehmerzen, Herabsetzung der Sehsch~rfe und Neuritis optica, war noch eine vollst~ndige linksseitige Paralyse des N. abducens vorhanden; natiirlieh kamen in diesem Falle, wie dies aueh zu erwarten war, keine anderen Symptome zum Vorsehein; Sehschw~che und Schl~frigkeit liel~en allerdings keine genaue Unter1) Nagels ttandb, der Physiol. 4, 78. 2) ~ber einen Fall yon Occipitaltumor. Monatsschr. f. Psych. u. Neurol. 1902, 31. 8) Zit. nach Bechtcrew, 1. c. S. 1437. 4) Neurol. Centralbl. 1910. 16 Ap. 674 M. Resnikow und S. Dawidenkow: Ausfallssymptome nach Liision des suchung der Funktion des auch sonst an Symptomen nicht reichen, rechten Gyrus angularis zu. Nichtsdestoweniger wurde die Diagnose bereits vor der Operation richtig gestellt und zwar auf "Grund einer bedeutend starker ausgepr~gten Stauungspapille auf der rechten Seite, als auf der linken, st~rkeren Kopfschmerz, Verkiirzung des PerkussionsschMls und Vermehrung der l~esistenz auf der rechten Seite; haupts~chlich aber auf Grund einer L~hmung des gekreuzten linken N. abducens. Mit Ausnahme der allgemeinen Gehirnerscheinungen, die den Gehirngeschwiilsten eigen sind, und sp~rlichen physikMischen Merkmalen von rein lokalem Charakter, diente als einziges Symptom, auf dem die Diagnose basierte, die L~hmung des linken N. abducens, d. h. eine einseitige (corticale?) StSrung der Bewegungen des Auges nach links (wenn wit den Autor recht verstanden haben), was wir gleich sehen werden, doch ganz ungewShnlich ist. Dies veranla6t uns, hier einige Ergebnisse der experimentellen Gehirnphysiologie zu zitieren, die zur Behauptung des Vorhandenseins yon Zentren der Augenbewegungen im Gyrus angularis fiihrten. Von den ~lteren Versuchen wollen wit die Experimente yon F e r r i e r erw~hnen, bei denen er mittels elektrischer Stromreizung des Gyrus angularis beim Affen, Augenund Kopfbewegungen nach der entgegengesetzten Seite beobachten konnte und die bald yon einer Verengerung, bald yon einer Erweiterung der Pupille begleitet wurden (zitiert nach B e c h t e r e w , 1. c. S. 1374). L u c i a n i und T a m b u r i n i erhielten bei der Reizung des Occipitallappens dieselben Erscheinungen. Mittels der Stromreizung der hinteren Teile des Parietallappens (Gyrus angularis) land B e c h t e r e w eine Reihe yon Zentren fiir Augenbewegungen. Bei st~rkeren Reizungen gesellten sich zu den Augen noch Kopfbewegungen hinzu. Sowohl die Augen als auch die Kopfbewegungen waren bei den horizontalen Bewegungen immer nach der entgegengesetzten Seite gerichtet, bei den vertikalen Augenbewegungen kamen noch Lidbewegungen nach oben und nach unten, je nach den Augenbewegungen hinzu. Die Augenbewegungen waren immer b e i d e r s e i t i g . Nach B e c h t e r e w s Meinung sind diese Bewegungen nicht willkiirlicher, sondern r e f l e k t o r i s c h e r Natur: sie werden nicht durch subjektive, durch die elektrische Reizung geweckte Empfindungen, sondern r e f l e k t o r i s c h durch a d e q u a t e R eiz eoder u n m i t t e l b a r durch den elektrischen Strom hervorgerufen, sie sind auch durch Reizung der tieferliegenden Marksubstanz zu erzeugen und werden durch entsprechende spezielle Leitungsbahnen welter auf die Augenmuskelkerne im Vierhiigel iibertragen. Durch einseitige Extirpation der Zone F (Gyrus angularis) beim Affen konnte M u n k eine StSrung in der Augeninnervation im Sinne einer langdauernden StSrung der assoziativen (konjugierten) Augenbewegungen hervorrufen, wie auch eine Herabsetzung der Sensibilit~t des Auges linken Gyrus angularis in einem Falle yon Schi~del-und Gehirnverletzung. 675 auf der anderen Seite bemerken. Bei doppelseitiger Exstirpation dieser Zone beobachtete M u n k auBerdem noch eine Erschwerung der Liderhebung bis zur normalen Grenze und Unfiihigkeit, die Gegenstande normal zu fixieren, ihre Lage im Raume richtig zu beurteilen (Physiologie der GroBhirnrinde 1902). U n d so e r k l ~ r t e M u n k den G y r u s a n g u l a r i s als F i i h l s p h s des Auges. Nach den Untersuchungen aber von K a l b e r l a h steht yon der Munkschen Augenfiihlsphs nur der l a t e r a l e v o r d e r e A b s c h n i t t , der zu dem von F r i t s c h und H i t z i g aufgefundenen Orbiculariszentrum gehSrt, zum Auge wirklich in B e zieh u n g, w~hrend der mediale Anteil ganz davon abzutrennen istl). Und so sehen wir a) dab viele Autoren die MSglichkeit zulassen, daB eine R i n d e n e r k r a n k u n g des G y r u s a n g u l a r i s o h n e i r g e n d welche S y m p t o m e v e r l a u f e n k a n n ; b) dab die Mehrzahl der Autoren die A l e x i e in d i r e k t e B e z i e h u n g z u r L s des G y r u s a n g u l a r i s s t e l l t ; und zwar bringen diejenigen, welche das Vorhandensein eines hSheren sekunds Sehzentrums in der Rinde des Gyrus angularis anerkennen, die Alexie in eine direkte Abh~ngigkeit von der Ls eben dieses Zentrums; diejenigen aber, die das nicht zugeben wollen, beziehen dieses Symptom auf die Beschs der sagittalen Strahlungen oder - - wie NieB1 v. M a y e n d o r f - - bloB des dorsalen Tells dieser Strahlungen, die, seiner Ansicht nach, zur Leitung der Erregungen des Macularbiindels dienen sollen; c) dab eine ganz is Stellung die Autoren gegeniiber der in der Symptomatologie des linken Gyrus angularis vorkommenden H e m ia n o p s i a b il a t e r a li s d e x t r a nehmen, indem sie diese zu den direkten Symptomen der L/ision der Rinde selbst zs oder als ihre Ursache die Beschs des ventralen Teils der Radianionis opticae beschuldigen, d) dab wir unter den sehr oft vorkommenden, fast konstanten und datum diagnostisch sehr wichtigen Symptomen bei a l l e n Autoren die amnestische Aphasie angezeigt finden, wenn auch einige yon ihnen sie als Folge der in die Erkrankung mit einbegriffenen Temporalwindungen aufzufassen geneigt sind. Was die Tiefenwahrnehmung und die Fixation anbetrifft, so h~ngen sie, weft sie abgeleitete Erscheinungen aus der Akkommodation und Konvergenz sind, vSllig yon der richtigen Funktion der Augenmuskeln ab, derel~ hShere Reflexzentren im Gyrus angularis vorhanden sind. Jedoch gehSren sowohl die sich isoliert s StSrungen wie in ihrem vollstis Komplex auftretende, d. h. Ablenkung der Augen nach der entgegengesetzten Seite, Erschwerung der Fixation, Itebrasenkung der Lider, fehlerhafte Tiefenschs und Erweiterung der 1) Handb. der Neurol. von Lewandowsky, Otto Kaliseher, Physiologie des GroBhirns, Berlin 1910, S. 390. 676 M. Resnlkow und S. Dawidenkow: Ausfallssymptomenach Lasion des gleichseitigen Pupille lange nicht zu den allt~glichen Symptomen bei Erkrankungen dieses Gebietes, wenn auch ihr Auftauchen ohne Zweifel fiir dasselbe zu den charakteristischen gereehnet werden mul~. Kehren wir zu unserem Falle zuriiek, so sehen wir, dal~ aus dem ganzen Symptomenkomplex eben dasjenige Symptom fehlt, dal~ am hs sten bei Erkrankungen des Gyrus angularis beobaehtet wird, ns die Alexie, was ja nur dureh den Analphabetismus unseres Kranken bedingt sein kann. Selbstversts hat dieses Symptom nur einen konventionellen Wert. Bei den Analphabeten ist der Gyrus angularis rs nicht weniger entwickelt als bei denen, die lesen und schreiben kSnnen, und phylogenetisch existierte er aueh vor der Schriftsprache in nicht kleinerer Ausdehnung. Wenn wir aueh mit Berlin1), H e n s c h e n , D e j e r i n e und F l e c h s i g das Vorhandensein eines besonderen speziellen Lesezentrums im Gyrus angularis zugeben wollen, so kSnnte sieh dieses allerdings doch nur auf einem urbar gemaehten Felde entwiekeln, und seine Verwiistung kann doch nicht ohne eine merkliche Spur im Leben des Individuums hinterlassen zu haben, gesehehen, wenn auch keine grSberen oder selbst feineren, dureh die Analyse mit den gew6hnlichen Untersuchungsmethoden aufzudeckenden Ver~nderungen zu konstatieren ws Es unterliegt in dieser Kichtung kaum einem Zweifel, dal~ der Gyrus angularis ein Knotenpunkt ist, wo die drei wiehtigsten, den Interessen der Erkenntnis der Aul~enwelt dienenden Fiihlsphs die Sehsph~re, die HSrsphs und die Hautmuskelfiihlsphs zusammenlaufen und sich gegenseitig beriihren. Alle diese drei Sinnesfunktionen waren bei unserem Kranken stark und dauerhaft gestSrt. Hemianopsie, amnestische Aphasie und Astereognosis - - das sind die wesentlichen Defekte, dureh welche sich der krankhafte Zustand seines Gefiihlsund Geisteslebens s Als ein pathologisches Verbindungsglied zwischen zwei sich beriihrenden Sphs tritt hier ein neues Symptom auf, das schon in seiner wissensehaftliehen Benennung den Stempel seiner zweifaehen Abstammung triigt, die a m n e s t i s c h e F a r b e n b l i n d h e i t . Wir wollen dieses Symptom als ein sehr w e s e n t l i c h e s fiir die E r k e n n u n g der E r k r a n k u n g des G y r u s a n g u l a r i s hier besonders betonen. Und s wie die reine Astereognosis von einem l~bergreifen der Erkrankung vom Gyrus angularis auf den Gyrus supramarginalis von uns abh~ngig gemacht wurde, so d e u t e t a u c h die a c h r o m a o p t i s e h e A p h a s i e a u f eine L ~ s i o n des G r e n z g e b i e t e s z w i s c h e n Gy~us a n g u l a r i s u n d G y r u s o e e i p i t a l i s , resp. auf ein (J-bergreifen yon dem ersten auf den zweiten. Im Mittelpunkt aber des Ganzen von uns beobachteten Symptomenkomplexes 1) Berl. klin. Woehensehr. 1886. 3) C6cR6 verbale pure Compt. send. d. 1. Soci6t6 biologique 1892. linken Gyrus angularis in einem Falle yon Schadel- und Gehirnverletzung. 677 steht die S c h w a c h e o d e r P a r e s e d e r d i r e k t e n p e r i p h e r i s c h e n R e p r o d u k t i o n (Merkfahigkeit) u n d d e r i n d i r e k t e n z e n t r a l e n (Gedachtnis). Die Produkte aller Fiihlspharen finden bei ihrer Wiedererzeugung ihren leichtesten, einfachsten und biindigsten Ausdruck imWortsymbol: jede andere Reproduktion beriihrt schon die Grenze der Kunst. Deshalb ist das W o r t ein f e i n e s R e a g e n s , u m die u n g e s t S r t e u n d t a d e l l o s e T a t i g k e i t u n s e r e r F i i h l s p h a r e n zu k o n s t a t i e r e n . Die a m n e s t i s c h e A p h a s i e , in welcher die Herabsetzung der Reproduktionsfahigkeit des Kranken am starksten zum Vorschein kommt, k a n n g l e i c h z e i t i g d a z u d i e n e n , zu e n t h i i l l e n , in w e l c h e r S i n n e s s p h a r e die R e p r o d u k t i o n s k r a f t a m m e i s t e n g e l i t t e n hat. U m eine E r k r a n k u n g des G y r u s a n g u l a r i s indenjenigen Fallen d i a g n o s t i z i e r e n zu k S n n e n , wo keine Hinweise darauf, weder seitens einer StSrung in den Vorrichtungen der in ihm gelegenen Zentren fiir die au•eren und inneren Augenbewegungen, noch seitens einer StSrung der sich mit ihm in Zusammenhang befindenden speziellen Einiibung im Lesen, vorhanden sind, miissen wir d a h e r die R e p r o d u k t i o n s k r a f t b e i d e r A r t e n (der d i r e k t e n peripherischenundindirektenzentralen) derdreiSinnesspharen e i n e r U n t e r s u c h u n g u n t e r w e r f e n , u n d je nach d e m G r a d e u n d d e m ~ b e r w i e g e n der S t S r u n g des W o r t e s , das die P r o dukte dieser oder jener Sinnessphare symbolisiert, sind wir i m s t a n d e , die g e n a u e r e Lage der L a s i o n im G y r u s a n g u laris s e l b s t f e s t z u s t e l l e n . In unserem Falle ist die Rel~roduktionskraft fiir Produkte aller drei Sinnesspharen vermindert, etwas weniger fiir Hautmuskel- und Gesichtseindriicke und mehr fiir GehSrseindriicke; deshalb wird es am richtigsten sein, die grSBte Lasion n~iher dem unteren Segment des Gyrus angularis zu lokalisieren. Abseits von allen analysierten Symptomen steht die BlutzirkulationsstSrung des rechten Armes. Dieselbo kommt darin zum Ausdruck, da~ der Arm oft kalt wird. Die MotilitatsstSrungen waren zu unbedeutend, als dal~ man ihnen eine derartige Erscheinung zuschreiben kSnnte. Inwiefern abet dabei die Vasomotoren der Zentralwindungen (Danilewsky, Richer, Francois-Frank) l~idiert sind oder inwiefern dieses Symptom von der Lasion der hypothetischen V~sodilatatorenzentren des L o b u s p a r i e t a l i s ( B o c h e f o n t a i n e , S t r i c k e r , B e c h t e r e w und M i s l a w s k y ) abhangig ist, ist selbstverstandlich nicht bestimmt zu sagen.
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A Clinical Perspective of Anti-Fibrotic Therapies for Cardiovascular Disease
Frontiers in pharmacology
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A Clinical Perspective of Anti-Fibrotic Therapies for Cardiovascular Disease Lu Fang 1*, Andrew J. Murphy 1 and Anthony M. Dart 1, 2 1 Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia, 2 Department of Cardiovascular Medicine, The Alfred Hospital, Melbourne, VIC, Australia Lu Fang 1*, Andrew J. Murphy 1 and Anthony M. Dart 1, 2 1 Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia, 2 Department of Cardiovascular Medicine, The Alfred Hospital, Melbourne, VIC, Australia Cardiac fibrosis are central to various cardiovascular diseases. Research on the mechanisms and therapeutic targets for cardiac fibrosis has advanced greatly in recent years. However, while many anti-fibrotic treatments have been studied in animal models and seem promising, translation of experimental findings into human patients has been rather limited. Thus, several potential new treatments which have shown to reduce cardiac fibrosis in animal models have either not been tested in humans or proved to be disappointing in clinical trials. A majority of clinical studies are of small size or have not been maintained for long enough periods. In addition, although some conventional therapies, such as renin-angiotensin-aldosterone system (RAAS) inhibitors, have been shown to reduce cardiac fibrosis in humans, cardiac fibrosis persists in patients with heart failure even when treated with these conventional therapies, indicating a need to develop novel and effective anti-fibrotic therapies in cardiovascular disease. In this review article, we summarize anti-fibrotic therapies for cardiovascular disease in humans, discuss the limitations of currently used therapies, along with possible reasons for the failure of so many anti-fibrotic drugs at the clinical level. We will then explore the future directions of anti-fibrotic therapies on cardiovascular disease, and this will include emerging anti-fibrotics that show promise, such as relaxin. A better understanding of the differences between animal models and human pathology, and improved insight into carefully designed trials on appropriate end-points and appropriate dosing need to be considered to identify more effective anti-fibrotics for treating cardiovascular fibrosis in human patients. Edited by: Chrishan S. Samuel, Monash University, Australia Reviewed by: Daniele Bani, University of Florence, Italy Thomas Bernd Dschietzig, Immundiagnostik AG, Germany *Correspondence: Lu Fang karenlu.fang@bakeridi.edu.au Edited by: Chrishan S. Samuel, Monash University, Australia Reviewed by: Daniele Bani, University of Florence, Italy Thomas Bernd Dschietzig, Immundiagnostik AG, Germany *Correspondence: Lu Fang karenlu.fang@bakeridi.edu.au *Correspondence: Lu Fang karenlu.fang@bakeridi.edu.au Specialty section: This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Received: 24 February 2017 Accepted: 22 March 2017 Published: 06 April 2017 Citation: Fang L, Murphy AJ and Dart AM (2017) A Clinical Perspective of Anti-Fibrotic Therapies for Cardiovascular Disease. Front. Pharmacol. 8:186. doi: 10.3389/fphar.2017.00186 Specialty section: This article was submitted to Cardiovascular and Smooth Muscle Pharmacology, a section of the journal Frontiers in Pharmacology Keywords: cardiac fibrosis, anti-fibrotic therapies, clinical trials, diffuse fibrosis, cardiac magnetic resonance imaging, collagen turnover markers, diastolic function MINI REVIEW published: 06 April 2017 doi: 10.3389/fphar.2017.00186 MINI REVIEW published: 06 April 2017 doi: 10.3389/fphar.2017.00186 INTRODUCTION Cardiac fibrosis is a hallmark of various cardiovascular disease such as hypertension, myocardial infarction (MI), and ischemic, dilated, and hypertrophic cardiomyopathies. Cardiac fibrosis not only leads to cardiac diastolic dysfunction, but is also a major determinant of malignant arrhythmias and end-stage systolic heart failure and consequently, increases the risk of cardiac death. There are two types of fibrosis: regional fibrosis (reparative fibrosis, scarring from MI) and diffuse fibrosis (reactive fibrosis, interstitial fibrosis in response to different stimuli). Cardiac fibrosis can be definitively diagnosed by endomyocardial biopsies, but they are an invasive evaluation only representative in diffuse fibrosis. Circulating biomarkers, particularly collagen turnover markers, are widely used to noninvasively assess cardiac fibrosis, however they are not reliable and unable to differentiate regional fibrosis from diffuse fibrosis. Cardiac magnetic Received: 24 February 2017 Accepted: 22 March 2017 Published: 06 April 2017 Citation: Fang L, Murphy AJ and Dart AM (2017) A Clinical Perspective of Anti-Fibrotic Therapies for Cardiovascular Disease. Front. Pharmacol. 8:186. doi: 10.3389/fphar.2017.00186 April 2017 | Volume 8 | Article 186 Frontiers in Pharmacology | www.frontiersin.org 1 Clinical Studies on Anti-Fibrotic Therapies Fang et al. decreased CVF (by endomyocardial biopsies) and LV chamber stiffness in patients with severe fibrosis (n = 7), but not in those with nonsevere fibrosis (n = 12) (Díez et al., 2002). In patients with end-stage renal disease, losartan (n = 13) more effectively suppressed cardiac fibrosis than did enalapril (n = 13) or amlodipine (n = 13) (Shibasaki et al., 2005). Another small study showed attenuation of progression of cardiac fibrosis with losartan in patients with nonobstructive hypertrophic cardiomyopathy (Shimada et al., 2013). Treatment with candesartan for 24 months also reduced the amino-terminal peptide of type III procollagen (PIIINP) in patients with atrial fibrillation (Kawamura et al., 2010). resonance imaging (CMR) is an emerging technique to accurately and noninvasively evaluate regional and diffuse cardiac fibrosis by late gadolinium enhancement and post-contrast myocardial longitudinal relaxation time (T1) mapping, respectively, but CMR is expensive and not easily accessible. Functional consequences of cardiac fibrosis, particularly impaired left ventricular (LV) relaxation and heart failure, are also potential, albeit nonspecific, markers of fibrosis. It is known that a complex interaction involving a network of growth factors/cytokines/hormones and fibroblasts and other cell types (such as cardiomyocytes, monocytes, lymphocytes) is responsible for initiating and maintaining fibrotic response (Kong et al., 2014). In addition to resident fibroblasts, fibroblasts originate from circulating pecursors, sometimes termed fibrocytes (Fang et al., 2013), endothelial cells or epithelial cells (Kong et al., 2014). Renin-angiotensin-aldosterone system (RAAS), growth factors [such as transforming growth factor (TGF)-β], endothelin, matricellular proteins (such as connective tissue growth factor (CTGF) and proinflammatory factors (such as tumor necrosis factor (TNF-α), interleukin (IL)-6 and IL-1) are some of the best studied mediators implicated in cardiac fibrosis (Kong et al., 2014). Recently, new mediators with therapeutic potential of cardiac fibrosis have been emerging such as cardiotrophin-1, galectin, and miRNAs etc. (Fang et al., 2015; Heymans et al., 2015). Although many anti-fibrotic therapies on cardiac fibrosis seem promising in experimental models, clinical data are limited and mixed. Most of new anti-fibrotic therapies have not been evaluated in patients. Citation: Some clinical data have demonstrated benefits on cardiac fibrosis mainly with RAAS inhibitors, but most clinical trials on anti-fibrotic drugs are disappointing. This review will summarize findings from clinical trials of anti-fibrotic therapies on cardiac fibrosis (Table 1) and discuss the discrepancy between animal research and clinical trials as well as future directions. The mineralo-corticoid receptor antagonists, spironolactone and eplerenone, also have anti-fibrotic effects in humans. Additional treatment of spironolactone for 6 months improved LV diastolic function and decreased PICP and PIIINP in 80 patients with metabolic syndrome treated with angiotensin II inhibition (Kosmala et al., 2011). In another study of 113 patients with obesity and mild LV diastolic dysfunction, spironolactone treatment for 6 months improved myocardial deformation and decreased PICP and PIIINP (Kosmala et al., 2013). In 44 patients with diastolic heart failure, eplerenone reduced PIIINP at 12 months after treatment, associated with modest improvement of diastolic function (Mak et al., 2009). Similar findings were made in another study showing that eplerenone reduced the amino-terminal peptide of type I procollagen (PINP) and PICP in 44 patients with heart failure with preserved ejection fraction (Deswal et al., 2011). Although the above clinical studies have shown that RAAS inhibitors reduces cardiac fibrosis in humans, the study population in these studies is rather small. Furthermore, inhibition of RAAS only modestly regresses cardiac fibrosis. Cardiac fibrosis persists in heart failure patients even when treated as recommended by the official guidelines (Querejeta et al., 2004). Thus, there is a compelling need to develop novel and effective anti-fibrotic therapies in cardiovascular disease. Frontiers in Pharmacology | www.frontiersin.org Inflammation Modulators Inflammatory modulation might have beneficial effects on cardiac fibrosis and heart failure since inflammation is involved in the formation and progression of cardiac fibrosis. TNF-α plays an important role in cardiac fibrosis. However, the RENEWAL study (Mann et al., 2004) which examined the effect of TNF-α antagonist etanercept in patients with heart failure was negative. Additionally, the ATTACH trial was stopped prematurely as the high dose of the TNF-α antagonist infliximab increased all- cause mortality in patients with moderate-to-severe chronic heart failure (Chung et al., 2003). The finding that TNF receptor 1 and 2 exert opposing effects on cardiac remodeling may partly explain that direct blockade of one inflammatory actor could cause these unexpected clinical results (Hamid et al., 2009). RAAS Inhibitors The first family of anti-fibrotic drugs are inhibitors of angiotensin II. Angiotensin II interacts with angiotensin II type I receptors, which stimulates fibroblast proliferation, and increases collagen synthesis (Kong et al., 2014). Several clinical studies have shown that both angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers reduce cardiac fibrosis in patients independent of their antihypertensive effects. In hypertensive patients, endomyocardial biopsies at baseline and 6 months revealed a decrease of collagen volume fraction (CVF) only in the group treated with lisinopril (n = 18), but not with hydrochlorothiazide (n = 17) (Brilla et al., 2000). A comparison between losartan (n = 21) and amlodipine (n = 16) given for 1 year in hypertensive patients revealed that only losartan significant decreased both CVF (by endomyocardial biopsies) and the carboxy-terminal peptide of procollagen type I (PICP) (López et al., 2001). Another study demonstrated that in patients with hypertensive heart disease, losartan treatment for 12 months Statins possess potent anti-inflammatory effects and are widely used in cardiovascular disease. Rosuvastatin attenuated cardiac fibrosis in animal models (Chang et al., 2009), which is supported by a small clinical study showing that statin therapy for 6 months reduced PIIINP in heart failure population (n = 56) (Abulhul et al., 2012). However, two large-scale clinical Frontiers in Pharmacology | www.frontiersin.org April 2017 | Volume 8 | Article 186 2 Clinical Studies on Anti-Fibrotic Therapies Fang et al. TABLE 1 | Anti-fibrotic therapies on cardiac fibrosis in clinical trials. Study Agent Length of treatment Patient included (n) Main findings RAAS INHIBITORS Brilla et al., 2000 Lisinopril 6 months 35 Lisinopril but not hydrochlorothiazide decreased CVF in hypertensive patients. López et al., 2001 Losartan 12 months 37 Losartan but not amlodipine decreased CVF and PICP in hypertensive patients. Díez et al., 2002 Losartan 12 months 19 Losartan decreased CVF and LV chamber stiffness in hypertensive patients with severe fibrosis, but not in those with nonsevere fibrosis. Shibasaki et al., 2005 Losartan 6 months 39 Losartan more effectively suppressed cardiac fibrosis than enalapril or amlodipine in patients with end-stage renal disease. Shimada et al., 2013 Losartan 12 months 20 Losartan attenuated the progression of cardiac fibrosis in patients with nonobstructive hypertrophic cardiomyopathy. Kawamura et al., 2010 Candesartan 24 months 153 Candesartan reduced PIIINP in patients with atrial fibrillation. Kosmala et al., 2011 Spironolactone 6 months 80 Additional spironolactone decreased PICP and PIIINP in patients with metabolic syndrome. RAAS Inhibitors Kosmala et al., 2013 Spironolactone 6 months 113 Spironolactone improved myocardial deformation and decreased PICP and PIIINP in patients with obesity and mild LV diastolic dysfunction. Mak et al., 2009 Eplerenone 12 months 44 Eplerenone reduced PIIINP and modestly improved diastolic function in patients with diastolic heart failure. Deswal et al., 2011 Eplerenone 6 months 44 Eplerenone reduced PINP and PICP in patients with heart failure with preserved ejection fraction. INFLAMMATION MODULATORS RENEWAL Etanercept 24 weeks 2,048 The study ruled out a clinically relevant benefit of etanercept on the rate of death or hospitalization due to chronic heart failure in patients with heart failure. ATTACH Infliximab At 0, 2, 6 weeks 150 High dose of infliximab increased all-cause mortality in patients with moderate-severe heart failure. Abulhul et al., 2012 Atorvastatin 6 months 56 Atorvastatin reduced PIIINP in heart failure patients. CORONA Rosuvastatin 32.8 months 5,011 Rosuvastatin did not reduce the primary outcome or the number of deaths from any cause in older patients with systolic heart failure. GISSIF-HF Rosuvastatin 3.9 years 4,574 Rosuvastatin daily did not affect clinical outcomes in patients with chronic heart failure of any cause. UNIVERSAL Rosuvastatin 6 months 86 Rosuvastatin did not beneficially alter parameters of LV remodeling in patients with chronic systolic heart failure. TGF-β INHIBITORS PRESTO Tranilast 1, or 3 months 11,484 Tranilast did not improve the quantitative measures of restenosis (angiographic and intravascular ultrasound) or its clinical sequelae in patients receiving successful percutaneous coronary intervention. ENDOTHELIN INHIBITORS Sütsch et al., 1998 Bosentan 2 weeks 36 Bosentan improved systemic and pulmonary hemodynamics in heart failure patients who were symptomatic with standard triple-drug therapy. EARTH Darusentan 24 weeks 642 Darusentan did not improve cardiac remodeling or clinical outcomes in patients with chronic heart failure. Prasad et al., 2006 Enrasentan 6 months 72 In asymptomatic patients with LV dysfunction, LVEDVI increased over 6 months with enrasentan compared with enalapril treatment. SELECTIVE HEART RATE-REDUCING DRUG SHIFT Ivabradine 22.9 month follow up 6,558 Ivabradine improved clinical outcomes in patients with symptomatic heart failure. SHIFT substudy Ivabradine 8 month follow up 411 Ivabradine reversed cardiac remodeling in patients with heart failure. LOOP DIURETICS López et al., 2004 Torsemide 8 months 36 Torsemide but not furosemide reduced PICP and CVF in hypertensive patients with symptomatic heart failure. López et al., 2007 Torsemide 8 months 22 Torsemide but not furosemide decreased PCP in patients with chronic heart failure. RAAS Inhibitors arboxy-terminal peptide of procollagen type I; LV, left ventricular; PIIINP, the amino-terminal peptide of type III procollagen; PINP, the amino- P); PCP, procollagen type I carboxy-terminal proteinase; TGF-β, transforming growth factor-β; MCP-1, monocyte chemoattractant protein-1; trials, the CORONA(Kjekshus et al., 2007) and GISSIF-HF (Tavazzi et al., 2008) observed a neutral effect of rosuvastatin compared to placebo on major clinical outcomes in heart failure. Beneficial effects of statin on cardiac remodeling were not observed in universal trial either (Krum et al., 2007). A sub- study of universal trial actually showed reduced coenzyme-10 and increased serum collagen markers in the statin-treated group (Ashton et al., 2011). So, statin’s effect on cardiac fibrosis in human are generally disappointing. Peroxisome proliferator- activated receptor (PPAR) agonists have anti-inflammation properties. Preclinical data showed that PPAR-α agonist inhibited cardiac fibrosis and improved cardiac function (Ogata et al., 2004). However, considerable controversy exists on the cardiac safety profile of PPAR agonists (Sarma, 2012). Overall, there is lack of effective inflammatory modulators to inhibit cardiac fibrosis in patients. However, the negative results of inflammatory modulators do not necessarily mean the end of inflammatory modulators in cardiac fibrosis. Future studies should identify the crucial actors and their mechanisms of action in the immunopathogenesis of cardiac fibrosis, which is a prerequisite for the development of new inflammatory modulators in patients with cardiac fibrosis. Selective p38 MAPK inhibitors blocking the secretion of TNF-α and decreasing cardiac fibrosis in mice (Westermann et al., 2006) may be a new treatment modality in humans. canonical TGF-β signaling pathway might not be applicable clinically. While TGF-β promotes fibrogenesis, it also inhibits inflammation, suggesting that broad targeting of TGF-β may be problematic. Alternatively, targeting TAK or NOX4 downstream of TGF-β might be viable anti-fibrotic approaches. Clinically, two agents, pirfenidone and tranilast, which inhibit TGF-β and other growth factors (Edgley et al., 2012), have been available. Both pirfenidone and tranilast have been shown to reduce cardiac fibrosis in animal studies (Edgley et al., 2012). However, tranilast was disappointing in the PRESTO study for post-percutaneous transluminal coronary angioplasty restenosis prevention (Holmes et al., 2002). The use of pirfenidone and tranilast also have adverse effects such as liver dysfunction. Now research is being conducted to search for new compounds that could overcome these potential safety concerns. RAAS Inhibitors López et al., 2009 Torsemide 8 months 24 Torsemide corrected both lysyl oxidase overexpression and enhanced collagen cross-linking leading to normalization of LV chamber stiffness in patients with TABLE 1 | Anti-fibrotic therapies on cardiac fibrosis in clinical trials. SELECTIVE HEART RATE-REDUCING DRUG (Continued) Frontiers in Pharmacology | www.frontiersin.org April 2017 | Volume 8 | Article 186 3 Clinical Studies on Anti-Fibrotic Therapies Fang et al. TABLE 1 | Continued Study Agent Length of treatment Patient included (n) Main findings TORAFIC Torsemide 8 months 155 In hypertensive patients with chronic heart failure randomized to torsemide or furosemide, there were no difference in PICP levels between the two groups. CYCLIC GMP-SPECIFIC PHOSPHODIESTERASE TYPE-5A INHIBITOR Giannetta et al., 2012 Sildenafil 3 months 59 Sildenafil improved LV contraction parameters and reduced TGF-β and MCP-1 in patients with diabetic cardiomyopathy. Redfield et al., 2013 Sildenafil 24 weeks 216 Sildenafil did not improve exercise activity in patients with heart failure with preserved ejection fraction. MATRIX METALLOPROTEINASE INHIBITOR PREMIER PG-116800 90 days 253 PG-11680 did not prevent LV remodeling or improve clinical outcomes 90 days after myocardial infarction. RELAXIN Pre-RELAX-AHF Relaxin 48 h 234 Relaxin improved dyspnoea and lowered cardiovascular deaths or readmissions due to heart or renal failure at day 60 in patients with acute heart failure. RELAX-AHF Serelaxin 48 h 1,161 Serelaxin improved dyspnoea and reduced cardiovascular deaths and all-cause mortality through day 180 in patients with acute heart failure. CVF, collagen volume fraction; PICP, the carboxy-terminal peptide of procollagen type I; LV, left ventricular; PIIINP, the amino-terminal peptide of type III procollagen; PINP, the amino- terminal peptide of type I procollagen (PINP); PCP, procollagen type I carboxy-terminal proteinase; TGF-β, transforming growth factor-β; MCP-1, monocyte chemoattractant protein-1; LVEDVI, LV end diastolic volume index. MATRIX METALLOPROTEINASE INHIBITOR CVF, collagen volume fraction; PICP, the carboxy-terminal peptide of procollagen type I; LV, left ventricular; PIIINP, the amino-terminal peptide of type III procollagen; PINP, the amino- terminal peptide of type I procollagen (PINP); PCP, procollagen type I carboxy-terminal proteinase; TGF-β, transforming growth factor-β; MCP-1, monocyte chemoattractant protein-1; LVEDVI, LV end diastolic volume index. Endothelin Inhibitors Endothelin is another important contributor of fibrotic process and bosentan, a dual endothelin receptor subtype A and B antagonist, prevents fibrosis of various organs in animal models (Clozel and Salloukh, 2005). Dual endothelin subtype A and B inhibitors bosentan and macitentan and the ETA inhibitor ambrisentan are approved in the U.S. for the treatment of pulmonary hypertension. An initial small study in human showed that additional administration of bosentan improved systemic and pulmonary hemodynamics in severe heart failure patients receiving conventional treatments including ACE inhibitors (Sütsch et al., 1998). However, most of subsequent clinical trials of endothelin receptor antagonists were negative or neutral (Anand et al., 2004; Prasad et al., 2006). The harmful effects of endothelin receptor antagonists were generally RAAS Inhibitors A new compound called FT011 displays improved activity and reduced toxicity compared to tranilast (Zammit et al., 2009), which needs to be investigated in clinical studies. Sildenafil Sildenafil inhibits cyclic GMP-specific phosphodiesterase type- 5A and it has been used to treat idiopathic pulmonary fibrosis. In the first proof-of-concept human study, 59 patients with isolated diabetic cardiomyopathy randomly treated 3 months with sildenafil showed improved LV contraction parameters and reduced TGF-β and monocyte chemoattractant protein-1, when compared with controls (Giannetta et al., 2012). However, among patients with heart failure with preserved ejection fraction sildenafil for 24 weeks (n = 113), compared with placebo (n = 103), did not improve exercise capacity or clinical status (Redfield et al., 2013). Notably, fibrosis parameters were not measured in these two studies. The discordant results indicate that the same treatment does not exert similar effects in various cardiovascular disease. TGF-β inhibitors TGF-β plays a central role in activating cardiac fibrosis and it activates both canonical (ALK/Smad2/3/Smad4) and noncanonical (TAK/p-38/JNK and NOX4/ROS) signaling pathways. Anti-TGF-β antibodies and ALK5 inhibitors attenuated cardiac fibrosis in animal models, but they were associated with adverse cardiovascular effects (Frantz et al., 2008; Engebretsen et al., 2014), suggesting that targeting April 2017 | Volume 8 | Article 186 Frontiers in Pharmacology | www.frontiersin.org 4 Clinical Studies on Anti-Fibrotic Therapies Fang et al. attributable to enhanced fluid retention, which could be alleviated by early diuretic therapy. However, in general, additional blockade of endothelin may not be beneficial in patients with heart failure or cardiac fibrosis receiving angiotensin inhibitors. or furosemide, investigators did not find significant differences between the two groups in changes of PICP (Group, 2011). The TORAFIC study’s patient population had less severe heart failure and lower baseline serum PICP compared to those in the studies by Lopez and colleagues, which possibly explains the divergent results among these studies. So, it is important to select patients who may benefit from torsemide treatment. Relaxin Relaxin is an intriguing endogenous hormone that is a potent vasodilator with a number of pleiotropic effects. Relaxin inhibits fibrosis through various mechanisms including inhibiting TGF- β and Smad, regulating the balance between MMPs and tissue inhibitors of metalloproteinases, and inhibiting inflammatory response (Samuel et al., 2016). Relaxin has been shown to have anti-fibrotic effects in a range of experimental models of cardiovascular disease including MI (Samuel et al., 2011), fibrotic cardiomyopathy (Samuel et al., 2014), hypertension (Lekgabe et al., 2005), diabetes (Samuel et al., 2008), and atrial fibrillation (Henry et al., 2016). Furthermore, relaxin more effectively ameliorated cardiac fibrosis than enalapril in an experimental model of fibrotic cardiomyopathy and relaxin in combination with enalapril augmented the anti-fibrotic efficacy of enalapril (Samuel et al., 2014). However, relaxin is not universally beneficial in cardiac fibrosis since relaxin did not affect pressure overload-induced cardiac fibrosis that was associated with biochemical wall stress rather than elevated TGF- β1 levels (Xu et al., 2008). The beneficial effects of a single 48-h infusion of relaxin in the acute heart failure trials (Teerlink et al., 2009, 2013) has led to great interest in its clinical application in in human disease. Although anti-fibrotic effects of relaxin Selective Heart Rate-Reducing Treatment: Ivabradine Ivabradine is an oral medication that provides selective heart rate reduction by inhibiting the f-channel. A large trial SHIFT showed that over a median follow-up of 22.9 months, ivabradine significantly reduced cardiovascular death or hospital admission for worsening heart failure in patients with symptomatic heart failure with an LV ejection fraction ≤35%, and in sinus rhythm with a heart rate of ≥70 bpm (Swedberg et al., 2010). An echocardiographic sub-study of SHIFT further found that ivabradine improved both LV end-systolic and end-diastolic volume indexes compared with placebo from baseline to the 8-month follow-up (Tardif et al., 2011). Thus, ivabradine has been introduced in the treatment guidelines for chronic heart failure in patients (McMurray et al., 2012). However, evidence on whether ivabradine attenuates cardiac fibrosis in patients with heart failure is still lacking although ivabradine effectively reduced fibrosis and circulating angiotensin II and aldosterone levels in animal models (Busseuil et al., 2010). Ivabradine could also reduce fibrosis through its inhibitory effects on inflammatory responses and cardiac apoptosis (Becher et al., 2012). Matrix Metalloproteinase (MMP) Inhibitors Cardiac fibrosis is associated with activation of MMPs. It has been shown that MMP inhibition attenuates cardiac fibrosis and LV remodeling in experimental models (Heymans et al., 2005; Matsusaka et al., 2006). However, the PREMIER study of an orally active MMP inhibitor, PG-116800, in 253 patients after M failed to prevent LV remodeling or improve clinical outcomes 90 days after MI (Hudson et al., 2006), although it should be noted that no fibrosis parameters were measured in this study. β-Blockers β-blockers have been demonstrated to prevent cardiac fibrosis and improve survival in a rat model (Kobayashi et al., 2004). A meta-analysis showed that the β-blockers treatment for the patients with heart failure with preserved ejection fraction was associated with a lower risk of all-cause mortality (Liu et al., 2014). However, the mechanisms of β-blockers’ benefit on mortality have not been precisely clarified and whether β- blockers attenuate cardiac fibrosis in human remains unknown. Frontiers in Pharmacology | www.frontiersin.org Loop Diuretics: Torsemide There are three loop diuretics utilized in heart failure patients: furosemide, torsemide, and bumetanide. López et al. reported that torasemide (n = 19), but not furosemide (n = 17), reduced circulating PICP and myocardial collagen in hypertensive patients with symptomatic heart failure (López et al., 2004). They then found that activation of the enzyme responsible for the cleavage of PICP, procollagen type I carboxy-terminal proteinase (PCP), was also decreased in 22 patients with chronic heart failure taking torasemide (López et al., 2007). They further reported the ability of torsemide to correct both lysyl oxidase overexpression and enhanced collagen cross-linking leading to normalization of LV chamber stiffness in patients with heart failure (López et al., 2009). This was supported by preclinical data showing torsemide’s effect on RAAS inhibition including decreasing aldosterone secretion, inhibiting aldosterone receptor and Ang II effects (Buggey et al., 2015). However, in the TORAFIC study, a multi-center study of 155 hypertensive patients with chronic heart failure randomized to torsemide Frontiers in Pharmacology | www.frontiersin.org April 2017 | Volume 8 | Article 186 5 Clinical Studies on Anti-Fibrotic Therapies Fang et al. is well characterized in various experimental models, clinical trials have failed in patients with other fibrotic conditions such as scleroderma (Khanna et al., 2009). The negative results of clinical trials of relaxin have pointed to the challenges previously underscored. First, relaxin has short in vivo half-life and it is costly to produce. The relatively short duration of relaxin treatment have partly contributed to the failed clinical trials since fibrosis is a slow process in human. Second, it is important to know the expression of relaxin receptors in different tissues and organs and to understand tissue competence to respond to relaxin along with signaling pathways. Nevertheless, relaxin still holds great potential as a therapy for cardiac fibrosis associated with various cardiovascular disease. new treatments with relatively small group sizes since it allows accurate assessment of regional and diffuse fibrosis. Second, anti-fibrotic therapies targeting downstream signaling pathways may improve safety and efficacy of current treatments such as TGF-β inhibitors. In addition to their role in fibrosis, many proteins are involved in other biological processes. Thus, developing more specific agents targeting fibrotic signaling pathways is likely to be beneficial to minimize potential side effects. Third, combined anti-fibrotic therapies seem more effective than single drug treatment. CONCLUSION The failure of many clinical trials on anti-fibrotic drugs indicates that extrapolating research data from animal models to human requires caution since there are significant species differences in physiology and genetics between animals and human. Compared to mice, fibrosis is a slower condition in humans, which takes decades to develop and require long- term treatment to diminish its progression. Furthermore, many animal models of diseases do not mimic various clinical settings, thus controversial results are likely to be obtained since there are different signaling pathways and mechanisms in cardiac fibrotic processes in various diseases. In addition, the animals used are normally young while patients with cardiac fibrosis are at a more advanced age. In order to improve clinical translation, it is important to design, conduct and analyse animal experiments properly and to summarize data from animal research adequately before conducting clinical trials. Moreover, the failure of previous trials also emphasizes the need for optimal design of future clinical trials including a selection of suitable patients, appropriate dose, and route and timing and length of administration. Although many fibrotic therapies on cardiac fibrosis are promising in preclinical models, clinical translation is limited. There is still a lack of effective treatments to regress cardiac fibrosis in patients with various cardiovascular disease. Future optimally designed clinical studies are required to test new potential treatments and currently available drugs with improved safety and efficacy after adequate analysis of evidence from animal research. Loop Diuretics: Torsemide It is shown that spironolactone or relaxin in combination with angiotensin II inhibitors augmented anti-fibrotic efficacy (Kosmala et al., 2011; Samuel et al., 2014). Combined anti-fibrotic agents with different mechanisms of actions is likely to exert better effects on cardiac fibrosis. ACKNOWLEDGMENTS This study was supported in part by the Victorian Government’s Operational Infrastructure Support Program. AD is an NHMRC fellow. AM is an NHMRC Career Development Fellow and a National Heart Foundation Future Leader Fellow. There are some important areas for future research in this field. First, although CMR is too expensive to be used in large populations, it should be used to investigate potential AUTHOR CONTRIBUTIONS LF was responsible for assembling and drafting of the manuscript. AM and AD contributed to the drafting of the manuscript. REFERENCES 50, 859–867. doi: 10.1016/j.jacc.2007.04.080 Group, T. I. (2011). Effects of prolonged-release torasemide versus furosemide on myocardial fibrosis in hypertensive patients with chronic heart failure: a randomized, blinded-end point, active-controlled study. Clin. Ther. 33, 1204–1213.e1203. doi: 10.1016/j.clinthera.2011.08.006 López, B., Querejeta, R., González, A., Beaumont, J., Larman, M., and Díez, J. (2009). Impact of treatment on myocardial lysyl oxidase expression and collagen cross-linking in patients with heart failure. 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Impact of the angiotensin II receptor antagonist, losartan, on myocardial fibrosis in patients with end-stage renal disease: assessment by ultrasonic integrated backscatter and biochemical markers. Hypertens. Res. 28, 787–795. doi: 10.1291/hypres.28.787 Copyright © 2017 Fang, Murphy and Dart. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Shimada, Y. J., Passeri, J. J., Baggish, A. L., O’Callaghan, C., Lowry, P. A., Yannekis, G., et al. (2013). Effects of losartan on left ventricular hypertrophy and fibrosis in patients with nonobstructive hypertrophic cardiomyopathy. JACC Heart Fail. 1, 480–487. doi: 10.1016/j.jchf.2013.09.001 April 2017 | Volume 8 | Article 186 Frontiers in Pharmacology | www.frontiersin.org 8
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Impact Assessment Framework for Grid Integration of Energy Storage Systems and Renewable Energy Sources Toward Clean Energy Transition
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2023 The Authors. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Received 29 September 2023, accepted 18 November 2023, date of publication 27 November 2023, date of current version 5 December 2023. Digital Object Identifier 10.1109/ACCESS.2023.3337133 Digital Object Identifier 10.1109/ACCESS.2023.3337133 FARHAD ANGIZEH 1, (Member, IEEE), JINWOO BAE1, (Student Member, IEEE), JOYCE CHEN2, ALEXANDER KLEBNIKOV 2, AND MOHSEN A. JAFARI1, (Member, IEEE) 1Department of Industrial and Systems Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA 2Atlantic Shores Offshore Wind LLC, Brooklyn, NY 11205, USA C di th F h d A i h (f h d i h@ t d ) responding author: Farhad Angizeh (farhad.angizeh@rutgers.edu This work was supported by Atlantic Shores Offshore Wind, a 50:50 Joint Venture Partnership Between Shell New Energies and EDF Renewables. This work was supported by Atlantic Shores Offshore Wind, a 50:50 Joint Venture Partnership Between Shell New Energies and EDF R bl ABSTRACT This paper proposes a two-stage decision-making tool to assess the impacts of energy storage systems (ESSs) and offshore wind farms (OSW) integration in the power grid. To quantify the potential impacts, various key performance indicators (KPIs) are incorporated. These KPIs gage the environmental, technical, and economical attributes of the integrated system. The proposed framework uses a unit commitment (UC) mixed-integer linear programming (MILP) model. Two case studies (one for New Jersey and one for New York) are examined with clean power transition targets. The uncertainties of the net-load and power generation from renewable energy sources (RESs) are characterized by Gaussian Process Regression (GPR) models. These models are fine-tuned using the base forecasts generated using our in-house load forecasting tool and the National Renewable Energy Laboratory’s (NREL) publicly available generation calculator. The results show that ESS installations almost always improve the performance of the grid, regardless of the location and configuration. Furthermore, the unequally distributed ESS installations show better impacts than standalone centralized ESSs; and the mixed ESS technologies outperform single-type ESS deployments. INDEX TERMS Clean energy transition, net-zero energy system, 100% renewable energy, sustainability, offshore wind (OSW), energy storage system (ESS), planning and operation optimization. INDEX TERMS Clean energy transition, net-zero energy system, 100% renewable energy, sustainability, offshore wind (OSW), energy storage system (ESS), planning and operation optimization. FARHAD ANGIZEH 1, (Member, IEEE), JINWOO BAE1, (Student Member, IEEE), JOYCE CHEN2, ALEXANDER KLEBNIKOV 2, AND MOHSEN A. JAFARI1, (Member, IEEE) 1Department of Industrial and Systems Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA 2Atlantic Shores Offshore Wind LLC, Brooklyn, NY 11205, USA Corresponding author: Farhad Angizeh (farhad.angizeh@rutgers.edu) FARHAD ANGIZEH 1, (Member, IEEE), JINWOO BAE1, (Student Member, IEEE), JOYCE CHEN2, ALEXANDER KLEBNIKOV 2, AND MOHSEN A. JAFARI1, (Member, IEEE) 1Department of Industrial and Systems Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA 2Atlantic Shores Offshore Wind LLC, Brooklyn, NY 11205, USA I. INTRODUCTION The developed benchmarks unlock further investigations, such as energy policy and regulations, market mechanisms, and pricing/incentive programs. • A benchmark test and evaluation system for both New Jersey and New York States using publicly available databases. The developed benchmarks unlock further investigations, such as energy policy and regulations, market mechanisms, and pricing/incentive programs. p g p g The remainder of this paper is organized as follows: The proposed UC-based impact-assessment framework and uncertainty characterization of RESs are elucidated in Section II. The mathematical formulation is detailed in the Appendix. Section III represents the numerical experiments for the New Jersey and New York case studies. Finally, the conclusions are drawn and discussed in Section IV. II. UC-BASED IMPACT-ASSESSMENT FRAMEWORK Here a two-stage UC-based approach is employed to assess the impacts of different integration scenarios using a set of KPIs. But prior to delving into the specifics of the proposed two-stage approach, let us first lay the groundwork by discussing the generic UC model and further clarify the KPIs selection and measurement. g In that context, this paper proposes a novel integrated impact-assessment framework to quantify the potential impacts of ESSs and RESs integration scenarios. The pro- posed framework, which is developed on a Unit Commitment (UC)-based optimization model, encompasses a heuristic two-stage process to assess various impacts of ESSs and RESs integration using a set of key performance indicators (KPIs). The employed KPIs measure grid characteristics, including technical/engineering, economical, and environ- mental aspects. The built-in UC-based model co-optimizes the day-ahead hourly schedules of conventional/renewable generating units and candidate ESSs with their corresponding dispatch/injection and charging/discharging levels to balance supply and projected demand without contravening the transmission network constraints. ESS sizing and siting are also decided. To characterize the uncertainty of the RESs and projected demand, a set of fine-tuned Gaussian Process Regression (GPR) models are utilized. The value metrics can be measured from both the grid operator’s and individual developer’s perspectives, though this paper only taps into the grid values and leaves the latter for future work. Two case studies are demonstrated to reveal the practicality of the proposed impact-assessment framework. One case study is for the state of New Jersey (NJ), where the Energy Master Plan (EMP) of 2019 calls for a 100% clean power grid by 2050. The second case study is for the state of New York (NY) with its Climate Act of 2019 that involves ESSs and OSWs integration targets and sets the state to pursue a net-zero clean power grid by 2040. I. INTRODUCTION friendly scenarios aligned with the planned interim phases?’’ To address this problem, a set of tools to assess the merits of the potential strategies are essential. These tools must be capable of incorporating the unique geo- graphical and network-related features and targeted plans which vary from one region to another region in the country. Increasing the deployment of renewable energy sources (RESs) integrated with energy storage systems (ESSs) has become one of the widely accepted practices to decarbonize the supply-side of power grid by gradually substituting conventional generating units [1]. The transi- tion from current fossil-fueled-dominated systems to 100% clean power grids is quite a challenging task. This paper aims to answer the question of ‘‘How to strategically implement techno-economically viable and environmentally There have been some research works in the literature addressing the challenges and opportunities of the clean energy transition planning problem. The National Renewable Energy Laboratory’s (NREL) study in [2] uses its publicly available Regional Energy Deployment System capacity expansion model to investigate supply-side scenarios to a The associate editor coordinating the review of this manuscript and approving it for publication was Chenghong Gu . 134995 134995 VOLUME 11, 2023 VOLUME 11, 2023 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs net-zero power grid by 2035, considering least-cost options. In [3], the authors propose a two-factor learning curve model to analyze the impact of innovation and deployment policies on the cost of energy storage technologies over time from an empirical dataset. Reference [4] reviews Southeast Asia’s energy sector trends, focusing on electricity supply and demand, highlighting the crucial role governments and public policy can play in accelerating the region’s clean energy transition. Khan et al. generate and measure a principal components index in [5] as an independent variable to capture the effects of the clean energy transition on crucial trade-offs between economic growth and environmental sustainability. An integrated method to explore the environmental impacts of robust energy policy mixes towards clean energy transitions is proposed in [6]. The literature, however, lacks sufficient evaluation tools to guide policymakers and developers to evaluate and validate various potential scenarios for maximum value generation. ation fleet expansion/deactivation and RESs generation and net-load forecasts to carry out reliable day-ahead supply-demand balance plans, and • A benchmark test and evaluation system for both New Jersey and New York States using publicly available databases. A. PROBLEM FORMULATION AND KPI SELECTION The generic form of the UC model that aims to minimize the total operation cost of a system is formulated as: min G,I F(G,I) (1) s.t. f(G,I) = 0 (333) (2) h(G,I) ≤0 (5 5 5) (3) G ∈ (4) min G,I F(G,I) (1) s.t. f(G,I) = 0 (333) (2) h(G,I) ≤0 (5 5 5) (3) G ∈ (4) (1) (2) (3) (4) (3) (4) (4) where F(·) is the total operation cost with G and I to be generating units’ dispatch and commitment variables, respectively. f(·) and h(·) are equality, e.g., power balance equations, and inequality, e.g., generation limits, constraints with respectively corresponding Lagrange multiplier matri- ces 333 and 5 5 5. Also,  denotes the feasible region of the generating units. In our proposed framework, the generic model presented in (1)-(4) is reconstructed by incorporating RESs availability and ESSs operation models, transmission network constraints, and the system’s reserve requirements, which are detailed in the Appendix. The built-in UC-based model co-optimizes the most economical set of power generating units, dispatches the available RESs, i.e., wind and solar farms, and schedules the charging/discharging of the ESSs to meet the day-ahead demand forecast. It also sets aside sufficient capacities to supply the required system reserves. In summary, the core contributions of this paper include: • A set of KPIs, including economic, environmental, and technical/engineering value factors, to quantify the potential impacts of integrating ESSs and RESs in the power grid. These KPIs are intended to provide insights for decision-makers to strategically implement the validated scenarios, As discussed in Section I, a set of KPIs that measure grid characteristics is essential to quantify the potential impacts of RESs and ESSs integration scenarios. With this in mind, five KPIs are selected and measured, including (i) Total operation cost, (ii) Locational marginal prices (LMPs), (iii) Transmission lines congestion/loading, (iv) Renewable • An hourly operation scheduling tool on a UC-based optimization model that incorporates the planned gener- 134996 VOLUME 11, 2023 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs energy curtailment, and (v) Carbon footprint. Total operation cost and LMP directly measure the economical aspect of the grid where the former is the optimal value of the objective function (see (5) in the Appendix) and the latter is the Lagrange multiplier of the power balance equation—that is formulated in (23) in the Appendix. III. CASE STUDY AND NUMERICAL EXPERIMENT Two case studies are investigated to delineate the efficacy of the proposed impact assessment framework: Case I for New Jersey State and Case II for New York State. The New Jersey Energy Master Plan (EMP) [11] follows a path to 100% clean energy by 2050. The New Climate Leadership and Community Protection Act (CLCPA) [12] sets the New York State on a path to reaching net-zero greenhouse gas (GHG) emissions by 2040. For both cases, our analysis includes all the expected targeted changes in the network (e.g., expansion of renewable sources and retirement assets with GHG footprints). The projected changes in electricity load due to the increasing penetration of Electric Vehicles are also included. For ESS configuration, multiple scenarios are considered, including centralized and distributed, with equal and unequal distributed capacities. For distributed configurations, different sets of candidate ESS locations and capacities are explored. Regarding the ESS technologies, three types are considered: Li-ion battery, vanadium flow (VF) battery, and hydrogen fuel cell (FC). Additionally, various mixes of these technologies are analyzed. It should be noted that the three ESS technologies modeled were selected as most likely to be deployed varieties, after comprehensively evaluating sixteen total ESS technologies. The characteristics of the suggested top three ESS technologies are presented in Table 1. Next, the measured KPIs are analyzed, and based on the LMPs, congested transmission zones, and renewable curtail- ments, a set of candidate sweet spots are offered to install new ESSs. In the second stage, the model is rerun multiple times with different ESS deployment scenarios considering various configurations obtained from the feasible permutations of the candidate locations given the aggregate planned capacities. Finally, the KPIs are measured again to observe the impacts and compared with Baseline measurements to assess the validated scenarios. Fig. 1 illustrates the two-stage procedure to quantify the potential impacts of the ESSs and OSWs integration. The ESSs deployment scenarios includes cases with a single centralized ESSs in each candidate location and cases with multiple distributed ESSs given the planned aggregate capacities. In the distributed ESSs, both equal and unequal capacities in different locations are analyzed. This allows a quantified comparison between different types of ESS technology. Furthermore, the impacts of mixes of different technologies are analyzed to investigate the effectiveness of diversifying the new ESS installations. A. PROBLEM FORMULATION AND KPI SELECTION Transmission loading and renewable energy curtailment, which both measure the technical/engineering aspect of the grid, are directly calculated from the decision variables designated for the transmission line flows Plt and the RESs’ deployments, i.e., Pwt and Pst, in the Appendix. Lastly, the carbon footprint is selected to measure the environmental aspect of the grid, which is also calculated from the decision variable modeling the optimal dispatches of the conventional generating units, i.e., Pgt in the Appendix, using their corresponding emission rates adopted from the U.S. Energy Information Administration (EIA) database [15]. target years using our in-house high-resolution demand forecasting tool [7]. The NREL’s PVWatts Calculator [8] is utilized to generate a set of preliminary hourly profiles for on-land wind and solar photovoltaic (PV) powers in different locations across the two states. To estimate hourly OSW power productions over the target years, a 3-day raw data is extracted for each season from the publicly available wind speed database in [9]. To inject more variability, the forecast- ing horizon is expanded to span over a 7-day representative period; the 7-day data is randomly generated from the 3- day raw data by random sampling through Gaussian Process Regression (GPR) [10]. The GPR model captures the mean of the data in the same hours and the covariance of each hourly data in a day. Then, random sampling is conducted to generate the hourly 7-day data based on the functions with 95% prediction interval. To investigate the effectiveness of the proposed model, numerical experiments are conducted next. Now that the built-in UC-based model is elaborated and the KPIs selection and measurement are clarified, the proposed two-stage impact-assessment approach is detailed. In the first stage, an hourly UC-based optimization model is simulated. The simulation takes for input the target year’s infrastructure data, including the techno-economic data of the projected generation fleet with planned expansions/deactivations, transmission network data, and forecasted demand, including the massive impact of planned offshore wind farms off the coast of New York and New Jersey, but not assuming any new ESS installations. This constructs the Baseline analysis to measure the five KPIs. TABLE 1. Technical characteristics of ESS technologies. TABLE 1. Technical characteristics of ESS technologies. B. UNCERTAINTY CHARACTERIZATION The projected demand and RESs generations are two main input parameters with inherent stochasticity. The nodal hourly demand profiles are forecasted over the planning The optimization models for the two case studies have slight differences in formulation and input data. Both models were implemented and solved using CPLEX 12 solver under 134997 134997 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs FIGURE 1. Proposed two-stage UC-based framework with the 5 KPIs. FIGURE 1. Proposed two-stage UC-based framework with the 5 KPIs. FIGURE 2. NJ’s 30-bus representative transmission network with its planned OSWs at A–G expected to be operational between 2025 to 2035. TABLE 2. NJ’s planned OSW Farm installation with rated capacity, POI, and expected COD. GAMS [13] on a desktop computer with a Core i7-11800H TABLE 2. NJ’s planned OSW Farm installation with rated capacity, POI, and expected COD. TABLE 2. NJ’s planned OSW Farm installation with rated capacity, POI, and expected COD. GAMS [13] on a desktop computer with a Core i7-11800H processor at 2.30 GHz and 16 GB of RAM. GAMS [13] on a desktop computer with a Core i7-11800H processor at 2.30 GHz and 16 GB of RAM. A. CASE STUDY I: NEW JERSEY POWER TRANSMISSION GRID 1) TEST SYSTEM AND INPUT DATA 1) TEST SYSTEM AND INPUT DATA New Jersey is committed to building 7,500 MW of OSW, 17,000 MW of solar energy, and 2,500 MW of ESS by 2035, with ambitious interim targets of 3,500 MW of OSW and 2,000 MW of ESS by 2030. Moreover, the State’s EMP plans to fulfill a set of demand-side electrification activities, including 330K EVs on the road by 2025 and 2M by 2035 and 400 MW per year of behind-the-meter solar PV through 2030. The above-mentioned demand-side parameters are fed into the net-load forecasting tool to estimate the hourly nodal demand profiles over the planning years, which is elaborated in [7]. FIGURE 2. NJ’s 30-bus representative transmission network with its planned OSWs at A–G expected to be operational between 2025 to 2035. The NJ’s power grid is part of the PJM interconnection, and to assure sufficient accuracy in the analyses, a county- level representative transmission network is selected to mimic the state’s power grid. Fig. 2 illustrates NJ’s representative 30-bus power transmission grid with the planned OSW interconnections. The state’s planned OSW farms with their corresponding rated capacities, point-of-interconnections (POIs), and expected commercial operation date (COD) are implemented as presented in Table 2. (b26–b30) states that are notably important to balance supply and demand. Finally, the generation fleet data, including techno-economic data for each power plant and the state’s expansion/deactivation plans, are adopted from PJM [14] and the U.S. Energy Information Administration (EIA) [15] databases. The in-state county-level aggregate generation capacities, primarily natural gas and nuclear power, are shown in Fig. 3 for the year 2018. As can be seen from Fig. 2, nine interconnections are considered between NJ and the neighboring states, i.e., Pennsylvania (b22–b24), Delaware (b25), and New York The analyses are carried out over four target planning years, 2025, 2028, 2030, and 2035. In each planning year, the state’s target aggregate capacity is used with VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs TABLE 4. KPIs of the baseline scenarios. FIGURE 3. New Jersey’s county-level aggregated generation mix and capacities in MW in 2018. TABLE 4. KPIs of the baseline scenarios. and (iii) renewable curtailments, or high-loss buses, yields eight candidate buses that are presented in Table 3. 1) TEST SYSTEM AND INPUT DATA Note that the candidate buses are consistent with the findings reported in our previous work in [16], where a UC-based optimization model was reconstructed with additional decision variables to find the optimal siting for an aggregate 600 MW of ESSs with 4-hr discharge duration. Table 4 provides the average KPIs of the baseline scenarios in each planning year. The LMP and the transmission loading are divided into daily peak and off-peak times to demonstrate the impact of the ESS more effectively. The ESS is a sink node drawing power during off-peak times, demanding more generating units and transmission lines capacity utilization. However, the ESS turns out to be a source node releasing its stored energy during peak times and partially/ fully substitutes high-cost generating units and alleviates transmission congestion. Thus, assessing the integration impacts in peak and off-peak times provides more accurate results. FIGURE 3. New Jersey’s county-level aggregated generation mix and capacities in MW in 2018. TABLE 3. Selection criteria and candidate buses for ESS siting. TABLE 3. Selection criteria and candidate buses for ESS siting. Next, the second-stage simulations are rendered to assess the impacts of planned ESS installation scenarios with different locations, configurations, and capacities. Table 5 shows the 7-day average impact-assessment results for twenty-one ESS scenarios in July 2028. The simulation results show that the ESS installa- tion improved the KPIs by reducing the curtailment of low-cost renewable energy generation. In most scenarios, distributed ESS configurations lead to relatively better KPIs than centralized ESSs. Furthermore, unequal capacity installations have better KPIs than the equally-distributed ESSs. Except for the transmission congestion KPI that reflects the transmission network capacity utilization, all the other KPIs are improved with ESS, regardless of location. It is observed that ESS relieves transmission load in some highly congested areas/zones and indirectly increases congestion in others. Since our results are inconclusive and do not show a generalizable improve- ment trends regarding transmission loading and conges- tion, our analysis will be focused on the remaining four KPIs. yearly capacity increments following the NJ targeted ESS installation plans including: 600 MW in 2025; 1,200 MW in 2028; 2,000 MW in 2030; and 2,500 MW in 2035. For each scenario, an incremental analysis that shows the impact of the partial capacity of the State’s target are carried out. This provides insight into the additional value of the individual ESS investments done by private developers. 1) TEST SYSTEM AND INPUT DATA The net-load and RES power generation forecasts are made over a 7-day period for four seasons. For each season, a representative month is selected: January (Winter), April (Spring), July (Summer), and October (Fall). The forecasts are made using our in-house developed load forecasting tool and the GPR (see Section II-B). The KPIs are measured for each period and averaged to find the weekly average values over a planning year. yearly capacity increments following the NJ targeted ESS installation plans including: 600 MW in 2025; 1,200 MW in 2028; 2,000 MW in 2030; and 2,500 MW in 2035. For each scenario, an incremental analysis that shows the impact of the partial capacity of the State’s target are carried out. This provides insight into the additional value of the individual ESS investments done by private developers. The net-load and RES power generation forecasts are made over a 7-day period for four seasons. For each season, a representative month is selected: January (Winter), April (Spring), July (Summer), and October (Fall). The forecasts are made using our in-house developed load forecasting tool and the GPR (see Section II-B). The KPIs are measured for each period and averaged to find the weekly average values over a planning year. To investigate the impacts of different ESS technology, multiple mixed technology scenarios are examined. Fig. 4 shows the measured KPIs for each technology mix, where the ratio refers to the rated capacities of Li-ion (with 4-hr discharge duration), vanadium flow, and hydrogen out of the total aggregate ESS capacity. 2) RESULTS AND DISCUSSIONS As discussed in Section II, the ESS siting selection criteria, including (i) high LMPs, (ii) high-loaded transmission lines, 2) RESULTS AND DISCUSSIONS As discussed in Section II, the ESS siting selection criteria, including (i) high LMPs, (ii) high-loaded transmission lines, 2) RESULTS AND DISCUSSIONS As discussed in Section II, the ESS siting selection criteria, including (i) high LMPs, (ii) high-loaded transmission lines, 134999 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs TABLE 5. The Measured 7-day average KPIs for 21 ESS scenarios in July 2028. TABLE 5. The Measured 7-day average KPIs for 21 ESS scenarios in July 2028. other hand, the flow battery technology is most effective in LMP reduction. A technology mix of the two can alleviate the shortcomings of the individual technologies. 1) TEST SYSTEM AND INPUT DATA Based on our results, the technology mixes of (8:1:1) and (7:1.5:1.5) provide more reduction in the renewable energy curtailment than single-type technologies and still show almost the same effectiveness compared to the best single-type technology scenarios in the other KPIs. FIGURE 4. Measured KPIs for different ESS technology scenarios including mixed technologies. Putting together all the simulation results obtained from the four target planning years, Fig. 5 demonstrates the potential strategic ESS implementation roadmap from 2025 to 2035, given the target ESS aggregate capacities. As can be seen from this figure, different pathways can be selected depending on the most important KPI selected by the decision makers. The red, blue, green, and yellow arrows represent the ‘‘optimal’’ pathways based on the total operation cost, carbon footprint, renewable energy curtailment, and average LMP during peak times, respectively. 1) TEST SYSTEM AND INPUT DATA Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs FIGURE 5. Strategic ESS implementation roadmap from 2025 to 2035 given the target ESS aggregate capacities. FIGURE 5. Strategic ESS implementation roadmap from 2025 to 2035 given the target ESS aggregate capacities. FIGURE 6. NY’s 27-bus representative transmission network with its planned OSWs at A–H expected to be operational from 2023 to 2035. ESS capacities to reflect possible optimistic and pessimistic scenarios alongside the state’s actual planned capacity, which is 3,000 MW. Accordingly, four different aggregate ESS capacities of 500, 1,000, 3,000, and 6,000 MW are examined. Additionally, the ESS siting is assumed to be restricted to either zone J (b10) or K (b11) to comply with the New York State Energy Research and Development Authority’s (NYSERDA) 2022 offshore wind solicitation requirements [18]. The Li-ion battery ESS with a 4-hr discharge duration and 87.6% round-trip efficiency is considered for the primary analysis. The simulation time horizon is the same 7-day representative in two Winter and Summer seasons, where the net-load and RES generation forecasts are obtained following the same methods explained for the NJ study. In order to investigate the potential impacts on the grid from a single developer’s perspective, an additional set of relatively small ESS capacities, e.g., 20 and 40 MW, are also considered. These capacities are on top of the State’s targeted plans. These incremental ESS installations, from a developer’s perspective, provide valuable insights to developers on the marginal impacts their projects would have on the grid. FIGURE 6. NY’s 27-bus representative transmission network with its planned OSWs at A–H expected to be operational from 2023 to 2035. TABLE 6. NY’s Planned OSW Farm Installations With Rated Capacity, POI, and Expected COD. 1) TEST SYSTEM AND INPUT DATA FIGURE 4. Measured KPIs for different ESS technology scenarios including mixed technologies. The state has set targets including 6,000 MW of solar by 2025, 70% RESs by 2030, 9,000 MW of OSW by 2035, 85% reduction in GHG emissions from 1990 levels by 2050, and 3,000 MW of ESSs by 2030. The NY’s eleven control area load zones are considered to model the state’s representative power transmission grid. Fig. 6 depicts the NY’s representative 27-bus power transmission grid with the state’s planned OSW interconnections. Table 6 provides the state’s planned OSW farm characteristics. In Fig. 6, sixteen interconnections are considered between the NY and its neighboring utilities/markets, including PJM (b12–b19), the Independent Electricity System Operator (IESO) which is Ontario’s power system (b20), Hydro-Quebec (b21–b23), and New England Independent System Operator (ISONE) with (b24–b27). The generation fleet data and the state’s From the analysis, a 4-hr Li-ion battery shows the highest reduction in the total operation cost by 5% and the carbon footprint by 4%, while a vanadium flow battery is the most effective technology that decreases the average LMP during the peak time (by 19%). The hydrogen FC shows the slightest reduction in all the KPIs due to its low round-trip efficiency that leads to energy losses despite its large energy capacity. The mixed technology scenarios provide higher effec- tiveness in reducing the curtailment of renewable power generation. For the other KPIs, single and mixed technologies perform similarly. For instance, Li-ion battery provides the best performance in total operation cost and carbon footprint, and low performance in reducing LMP. On the From the analysis, a 4-hr Li-ion battery shows the highest reduction in the total operation cost by 5% and the carbon footprint by 4%, while a vanadium flow battery is the most effective technology that decreases the average LMP during the peak time (by 19%). The hydrogen FC shows the slightest reduction in all the KPIs due to its low round-trip efficiency that leads to energy losses despite its large energy capacity. The mixed technology scenarios provide higher effec- tiveness in reducing the curtailment of renewable power generation. For the other KPIs, single and mixed technologies perform similarly. For instance, Li-ion battery provides the best performance in total operation cost and carbon footprint, and low performance in reducing LMP. On the 135000 135000 VOLUME 11, 2023 VOLUME 11, 2023 F. 2) RESULTS AND DISCUSSIONS Measured 7-day average KPIs for different aggregate ESS capacities located at Zones J or K or both in 2030. locations and configurations, it seems that ESS installations almost always improve the baseline KPIs. There are a few scenarios showing the ESS at either J or K zones may result in higher peak LMP and transmission congestion loading for higher ESS capacities. High congestion triggers the use of more expensive generators, thereby increasing the average peak LMP. Off-peak LMPs may also increase due to the demand generated by ESS charging at off-peak times. by an amount between 0.5%–1.5%, total operation cost by an amount between 1%–10%, renewable energy curtailments by an amount between 1.5%–25%, and carbon footprint by up to 5%. The ESS in Zone K represents more decrease in renewable energy curtailment, whereas the ESS in Zone J shows the tendency to decrease LMP during the peak times. Zone K also reduces the total operation cost more than Zone J when the capacity is 3,000 MW, although other capacities have no significant difference. The distributed ESSs have shown even more decreases in the LMP, total operation cost, and carbon footprint compared to the centralized standalone ESSs. It should be noted that the reported ESS capacities, e.g., 3,000 MW, are the aggregate capacities of multiple ESS projects located in the same zone if not configured as distributed ESSs. In other KPIs, the distribution of the ESS capacities provides an offset of the two zones. Furthermore, the incremental increases in ESS capacity positively impact these five measures. Regardless of by an amount between 0.5%–1.5%, total operation cost by an amount between 1%–10%, renewable energy curtailments by an amount between 1.5%–25%, and carbon footprint by up to 5%. The ESS in Zone K represents more decrease in renewable energy curtailment, whereas the ESS in Zone J shows the tendency to decrease LMP during the peak times. Zone K also reduces the total operation cost more than Zone J when the capacity is 3,000 MW, although other capacities have no significant difference. The distributed ESSs have shown even more decreases in the LMP, total operation cost, and carbon footprint compared to the centralized standalone ESSs. It should be noted that the reported ESS capacities, e.g., 3,000 MW, are the aggregate capacities of multiple ESS projects located in the same zone if not configured as distributed ESSs. 2) RESULTS AND DISCUSSIONS Table 7 shows the 7-day average measured KPIs in the baseline scenarios for the four target aggregate ESS capacities in 2030. Note that the location configuration consists of pre-installed capacities only in Zone J, only in Zone K, and equally-distributed in Zones J and K. Next, the ESS deployment scenarios are simulated and the KPIs are measured and compared to the baseline results. Fig. 7 shows the 7-day average impact-assessment results in 2030 for different Li-ion battery ESS scenarios of 4-hr discharge duration. generation expansion/deactivation plans are adopted from the New York Independent System Operator’s (NYISO) 2022 Gold Book [17]. The hourly nodal net-load and the RES generation forecasts for the target planning year are estimated using similar tools used for the NJ case study. The simulation results reveal that over 90% of scenarios examined produce positive impacts due to ESS installation. Also, the KPIs compared to the baseline reduce the peak LMP by an amount between 0.5%–6% with the highest LMP by an amount between 10%– 60%, peak transmission congestion In contrast to the NJ study, the NY case is carried out for a single target year 2030; but with multiple planned aggregate 135001 135001 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs TABLE 7. Average 7-day KPIs in different locations and target aggregate ESS capacities. FIGURE 7. Measured 7-day average KPIs for different aggregate ESS capacities located at Zones J or K or both in 2030. by an amount between 0.5%–1.5%, total operation cost by FIGURE 8. Average Measured KPIs for different ESS scenarios including mixed technologies. locations and configurations, it seems that ESS installations almost always improve the baseline KPIs. There are a few scenarios showing the ESS at either J or K zones may TABLE 7. Average 7-day KPIs in different locations and target aggregate ESS capacities. TABLE 7. Average 7-day KPIs in different locations and target aggregate ESS capacities. FIGURE 8. Average Measured KPIs for different ESS scenarios including mixed technologies. FIGURE 7. Measured 7-day average KPIs for different aggregate ESS capacities located at Zones J or K or both in 2030. FIGURE 7. Measured 7-day average KPIs for different aggregate ESS capacities located at Zones J or K or both in 2030. FIGURE 8. Average Measured KPIs for different ESS scenarios including mixed technologies. FIGURE 7. 2) RESULTS AND DISCUSSIONS In other KPIs, the distribution of the ESS capacities provides an offset of the two zones. Furthermore, the incremental increases in ESS capacity positively impact these five measures. Regardless of Fig. 8 shows the impacts of ESS technologies, Li-ion battery, hydrogen storage, and their mix technology sce- narios. As can be observed from Fig. 8, Li-ion battery with a 4-hr discharge duration seems to have better impacts than Hydrogen storage in economical-related KPIs; however, Hydrogen storage shows better results in carbon reduction and avoiding renewable curtailments. The results also prove that the technology mix can lead to better KPIs than single-type ESS deployments. Table 8 provides the assessment results of incremental ESS impacts on the KPIs by adding a 20 MW or a 40 MW ESS on 135002 135002 VOLUME 11, 2023 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs TABLE 8. Observed average KPI improvements by additional incremental capacities to the already-installed aggregate 3,000 MW ESS. average KPI improvements by additional incremental capacities to the already-installed aggregate 3,000 MW ESS. top of what is already installed, which is 3,000 MW in this case. than single-type ESS deployments. In terms of the ESS configuration, distributed capacities generated better KPIs than centralized standalone ESSs, and unequally distributed cases outperformed the equally-distributed configurations. Finally, it was observed that regardless of the locations and configurations, ESS installations almost always improve the baseline KPIs in both studies. Overall, the capacity addition decreases the LMPs during peak times, renewable energy curtailment, total operation cost, and carbon footprint. As can be seen from the table, the renewable curtailment from OSWs is zero in the baseline scenarios. This comes from the fact that the generated power from the OSWs is fully utilized to supply the demand and charge the ESSs where the connecting transmission lines have sufficient headroom. Moreover, the results show that adding a small incremental ESS to a zone without an existing ESS further reduces the total operation cost due to the better impact of distributed ESSs on transmission congestion relief that avoids longer power transfers compared to the centralized ESSs. Adding 40 MW in Zone K seems more effective in reducing the LMPs during peak times. Under the distributed ESS scenarios, the location and capacity of the incremental ESS installations become more influential factors. APPENDIX UC-BASED MODEL The objective function of the proposed UC-based model to be minimized is: X t∈T  X g∈G  IgtCg + X j∈J mjgPjgt + CSU gt + CSD gt + µR gRgt  + X e∈E  CePD et + µR e Ret  (5) (5) where Igt is a binary variable represents the commitment status of conventional generating unit g at time t; Cg is the minimum generation cost of unit g, and CSU gt , and CSD gt are the start up and shot down costs of unit g at time t, respectively; ejg and Pjgt are the slope and corresponding power generation in block j of the considered piecewise linear generation cost function of unit g at time t; Ce and PD et are the variable cost and discharged power from energy storage e at time t; and Rgt and Ret are the reserve capacities scheduled for generating unit g and energy storage e at time t, respectively, with the corresponding reserve prices of µR g and µR e . 2) RESULTS AND DISCUSSIONS Adding a 20 MW to Zone K is the most efficient scenario to relieve the LMPs during peak times, while adding a 40 MW to Zone J is more effective in decreasing carbon footprint. Also, adding a 40 MW to Zone K shows the most effective scenario to improve renewable curtailment and total operation cost. [1] V. Stevenson, ‘‘Use of storage and renewable electricity generation to reduce domestic and transport carbon emissions-whole life energy, carbon and cost analysis of single dwelling case study (U.K.),’’ in Proc. MDPI, 2022, pp. 95–130. IV. CONCLUSION Available: http://pvwatts.nrel.gov/pvwatts.php | | g 0 ≤PC et ≤βetPe ∀e, t (18) 0 ≤PD et + Ret ≤(1 −βet)Pe ∀e, t (19) 0 ≤Ret ≤(1 −βet)Re ∀e, t (20) Eet = Ee(t−1) +  ηC e PC et −PD et ηDe  1t ∀e, t (21) Ee ≤Eet ≤Ee ∀e, t (22) X g∈G(b) Pgt + X w∈W(b) Pwt + X s∈S(b) Pst + X e∈E(b) (PD et −PC et) −Dbt = X l∈L(b) Plt ∀b, t (23) −Pl ≤Plt ≤Pl ∀l, t (24) Plt −Bl  θS lt −θR lt  = 0 ∀l, t (25) X g∈G Rgt + X e∈E Ret ≥Rt ∀t (26) Igt, βet ∈{0, 1} ∀g, e, t (27) | | g 0 ≤PC et ≤βetPe ∀e, t (18) 0 ≤PD et + Ret ≤(1 −βet)Pe ∀e, t (19) 0 ≤Ret ≤(1 −βet)Re ∀e, t (20) Eet = Ee(t−1) +  ηC e PC et −PD et ηDe  1t ∀e, t (21) Ee ≤Eet ≤Ee ∀e, t (22) X g∈G(b) Pgt + X w∈W(b) Pwt + X s∈S(b) Pst + X e∈E(b) (PD et −PC et) −Dbt = X l∈L(b) Plt ∀b, t (23) −Pl ≤Plt ≤Pl ∀l, t (24) Plt −Bl  θS lt −θR lt  = 0 ∀l, t (25) X g∈G Rgt + X e∈E Ret ≥Rt ∀t (26) Igt, βet ∈{0, 1} ∀g, e, t (27) [9] Atlantic Shores Buoys, Atlantic Shores OffshoreWind, Atlantic Shores Offshore Wind LLC, Brooklyn, NY, USA, 2022. [Online]. Available: https://www.atlanticshoreswind.com [10] C. Williams and C. Rasmussen, ‘‘Gaussian processes for regression,’’ in Proc. Adv. Neural Inf. Process. Syst., vol. 8, 1995, pp. 1–7. [11] New Jersey Energy Master Plan: Pathway to 2050. (2019). The New Jersey Board of Public Utilities (NJBPU). [Online]. Available: https://nj.gov/emp/docs/pdf/2020_NJBPU_EMP.pdf [12] New York Senate. (2019). Senate Bill S6599–Climate Leadership & Community Protection Act. [Online]. Available: https://www.nysenate.gov/legislation/bills/2019/s6599 [13] IBM Software Group, CPLEX, IBM ILOG, User-Manual CPLEX, vol. 12, 2018. [Online]. Available: http://www.ilog.com/products/cplex [14] (2022). Generation Capacity by Fuel Type, System Operations, PJM Interconnection. [Online]. Available: https://www.pjm.com/markets-and- operations/ops-analysis.aspx [15] U.S. Energy Information Administration (EIA), Electric Power Annual 2021, U.S. Dept. Energy, Washington, DC, USA, 2022. [Online]. Available: https://www.eia.gov/electricity/annual/pdf/epa.pdf [16] New Jersey Energy Storage Analysis (ESA): Responses to the ESA Elements of the Clean Energy Act of 2018, Rutgers, The State University of New Jersey, State New Jersey Board Public Utilities (NJBPU), Trenton, NJ, USA, 2019. [2] P. Denholm, P. Brown, W. Cole, T. Mai, B. Sergi, M. Brown, P. Jadun, J. Ho, J. Mayernik, C. McMillan, and others, ‘‘Examining supply-side options to achieve 100% clean electricity by 2035,’’ Nat. Renew. Energy Lab. (NREL), Golden, CO, USA, Tech. Rep. NREL/TP-6A40-81644, 2022. IV. CONCLUSION [17] Load & Capacity Data (Gold Book), New York Independent System Operator (NYISO), New York, NY, USA, 2022. (27) [18] New York’s 6 GW Energy Storage Roadmap: Policy Options for Continued Growth in Energy Storage, New York State Energy Research and Development Authority (NYSERDA), Albany, NY, USA, 2022. Constraints (6)-(9) and (12)-(17) model the thermal generating units operation including min/max generation and reserve capacities, start-up and shot down costs, ramp up and down and on/off limits. Constraints (10) and (11) restrict captured wind and solar power generations to their maxi- mum available amounts. Constraints (18)-(22) model ESSs charging/discharging events and dynamic energy balance that is bounded by min/max rated power, energy and dedicated reserve capacities. The power balance equation, transmission network, and system reserve requirement constraints are reflected in (23)-(27). FARHAD ANGIZEH (Member, IEEE) received the B.Sc. degree in electrical engineering from the Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran, in 2011, and the M.Sc. degree in energy systems engineering from the Sharif University of Technology, Tehran, Iran, in 2014, and the Ph.D. degree in industrial and systems engineering from Rutgers University, New Brunswick, NJ, USA, in 2023. He is currently a Post-Doctoral Associate with the Institute for Data, Systems, and Society at Massachusetts Institute of Technology, Cambridge, MA, USA. His research interests include demand response and energy management, modeling and integration of distributed and renewable energy resources, and optimization of smart electricity grids. He was a recipient of the IEEE Power and Energy Society (PES) Innovative Smart Grid Technologies (ISGT) North America Conference Best Paper Award, in 2021. IV. CONCLUSION Energy, Wash Available: https://www.eia.gov/elec [16] New Jersey Energy Storage Ana Elements of the Clean Energy Act o of New Jersey, State New Jersey Bo NJ, USA, 2019. IV. CONCLUSION [17] Load & Capacity Data (Gold Bo Operator (NYISO), New York, NY, [18] N Y k’ 6 GW E S R CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) Pgt −Pg(t−1) ≤PRU g Ig(t−1) + SURg(Igt −Ig(t−1)) + Pg(1 −Igt) ∀g, t (14) Pg(t−1) −Pgt ≤PRD g Igt + SDRg(Ig(t−1) −Igt) + Pg(1 −Ig(t−1)) ∀g, t (15) t+T on g −1 X t′=t t=1...|T |−T on g +1 Igt′ ≥T on g (Igt −Ig(t−1)) ∀g (16) t+T off g −1 X t′=t t=1...|T |−T off g +1 (1 −Igt′) ≥T off g (Ig(t−1) −Igt) ∀g (17) 0 ≤PC et ≤βetPe ∀e, t (18) 0 ≤PD et + Ret ≤(1 −βet)Pe ∀e, t (19) 0 ≤Ret ≤(1 −βet)Re ∀e, t (20) Eet = Ee(t−1) +  ηC e PC et −PD et ηDe  1t ∀e, t (21) Ee ≤Eet ≤Ee ∀e, t (22) X g∈G(b) Pgt + X w∈W(b) Pwt + X s∈S(b) Pst + X e∈E(b) (PD et −PC et) −Dbt = X l∈L(b) Plt ∀b, t (23) −Pl ≤Plt ≤Pl ∀l, t (24) Plt −Bl  θS lt −θR lt  = 0 ∀l, t (25) X g∈G Rgt + X e∈E Ret ≥Rt ∀t (26) Igt, βet ∈{0, 1} ∀g, e, t (27) CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) Pgt −Pg(t−1) ≤PRU g Ig(t−1) + SURg(Igt −Ig(t−1)) + Pg(1 −Igt) ∀g, t (14) Pg(t−1) −Pgt ≤PRD g Igt + SDRg(Ig(t−1) −Igt) + Pg(1 −Ig(t−1)) ∀g, t (15) t+T on g −1 X t′=t t=1...|T |−T on g +1 Igt′ ≥T on g (Igt −Ig(t−1)) ∀g (16) t+T off g −1 X t′=t t=1...|T |−T off g +1 (1 −Igt′) ≥T off g (Ig(t−1) −Igt) ∀g (17) 0 ≤PC et ≤βetPe ∀e, t (18) 0 ≤PD et + Ret ≤(1 −βet)Pe ∀e, t (19) 0 ≤Ret ≤(1 −βet)Re ∀e, t (20) Eet = Ee(t−1) +  ηC e PC et −PD et ηDe  1t ∀e, t (21) Ee ≤Eet ≤Ee ∀e, t (22) X g∈G(b) Pgt + X w∈W(b) Pwt + X s∈S(b) Pst + X e∈E(b) (PD et −PC et) −Dbt = X l∈L(b) Plt ∀b, t (23) −Pl ≤Plt ≤Pl ∀l, t (24) Plt −Bl  θS lt −θR lt  = 0 ∀l, t (25) X g∈G Rgt + X e∈E Ret ≥Rt ∀t (26) Igt, βet ∈{0, 1} ∀g, e, t (27) CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) Pgt −Pg(t−1) ≤PRU g Ig(t−1) + SURg(Igt −Ig(t−1)) + Pg(1 −Igt) ∀g, t (14) Pg(t−1) −Pgt ≤PRD g Igt + SDRg(Ig(t−1) −Igt) + Pg(1 −Ig(t−1)) ∀g, t (15) t+T on g −1 X t′=t t=1...|T |−T on g +1 Igt′ ≥T on g (Igt −Ig(t−1)) ∀g (16) t+T off g −1 X t′=t t=1 |T |−T off +1 (1 −Igt′) ≥T off g (Ig(t−1) −Igt) ∀g (17) CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) [3] N. IV. CONCLUSION This paper proposed a two-stage UC-based impact- assessment framework that evaluates the impact of new OSW generation and different ESS technologies, using five KPIs. This framework could be usefully applied in future work to any energy markets, to evaluate the impacts of changing demand, new ESS additions, or major RES deployments, or any combination of those events. New Jersey and New York states, with their ambitious clean power grid targets, were investigated. The simulation results revealed that over 90% of the scenarios examined produce positive impacts due to ESS installations. Furthermore, Li- ion batteries with a 4-hr discharge duration showed better impacts in all KPIs than flow batteries with 8-hr and hydrogen storage with 10-hr, except for average peak LMPs where the flow battery provided better results. Additionally, diversifying ESS technologies can lead to better KPIs g The objective function (5) is subject to prevailing thermal generating units, RESs injections and charging/discharging, and dynamic energy balance of ESSs, transmission grid, and system constraints: Pgt = IgtPg + X j∈J Pjgt ∀g, t (6) 0 ≤Pjgt ≤Pjg ∀j, g, t (7) IgtPg ≤Pgt ≤IgtPg ∀g, t (8) 0 ≤Rgt ≤IgtRg ∀g, t (9) 0 ≤Pwt ≤Pwt ∀w, t (10) 0 ≤Pst ≤Pst ∀s, t (11) Pgt = IgtPg + X j∈J Pjgt ∀g, t (6) 0 ≤Pjgt ≤Pjg ∀j, g, t (7) IgtPg ≤Pgt ≤IgtPg ∀g, t (8) 0 ≤Rgt ≤IgtRg ∀g, t (9) 0 ≤Pwt ≤Pwt ∀w, t (10) 0 ≤Pst ≤Pst ∀s, t (11) (6) (11) VOLUME 11, 2023 F. IV. CONCLUSION Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) Pgt −Pg(t−1) ≤PRU g Ig(t−1) + SURg(Igt −Ig(t−1)) + Pg(1 −Igt) ∀g, t (14) Pg(t−1) −Pgt ≤PRD g Igt + SDRg(Ig(t−1) −Igt) + Pg(1 −Ig(t−1)) ∀g, t (15) t+T on g −1 X t′=t t=1...|T |−T on g +1 Igt′ ≥T on g (Igt −Ig(t−1)) ∀g (16) t+T off g −1 X t′=t t=1...|T |−T off g +1 (1 −Igt′) ≥T off g (Ig(t−1) −Igt) ∀g (17) 0 ≤PC et ≤βetPe ∀e, t (18) 0 ≤PD et + Ret ≤(1 −βet)Pe ∀e, t (19) 0 ≤Ret ≤(1 −βet)Re ∀e, t (20) Eet = Ee(t−1) +  ηC e PC et −PD et ηDe  1t ∀e, t (21) Ee ≤Eet ≤Ee ∀e, t (22) X g∈G(b) Pgt + X w∈W(b) Pwt + X s∈S(b) Pst + X e∈E(b) (PD et −PC et) −Dbt = X l∈L(b) Plt ∀b, t (23) −Pl ≤Plt ≤Pl ∀l, t (24) Plt −Bl  θS lt −θR lt  = 0 ∀l, t (25) X g∈G Rgt + X e∈E Ret ≥Rt ∀t (26) Igt, βet ∈{0, 1} ∀g, e, t (27) [3] N. Kittner, F. Lill, and D. M. Kamm innovation for the clean energy tra pp. 1–6, Jul. 2017. [4] J. Aleluia, P. Tharakan, A. P. Ch ‘‘Accelerating a clean energy tra governments and public policy,’’ R May 2022, Art. no. 112226. [5] I. Khan, A. Zakari, J. Zhang, V trilemma energy balance, clean expansion in the midst of enviro from three trilemma leadership Art. no. 123619. [6] O. Castrejon-Campos, L. Aye, a mixes more robust: An integrativ clean energy transitions,’’ Energy Art. no. 101425. [7] F. Angizeh, A. Ghofrani, and M. demand forecast under data sc top–down and bottom–up approa pp. 2923–2933, Jun. 2022. [8] PVWatts Calculator, Nat. Renew. En 2022. [Online]. Available: http://pv [9] Atlantic Shores Buoys, Atlantic S Offshore Wind LLC, Brooklyn, N https://www.atlanticshoreswind.com [10] C. Williams and C. Rasmussen, ‘‘G Proc. Adv. Neural Inf. Process. Syst [11] New Jersey Energy Master Pla New Jersey Board of Public Uti https://nj.gov/emp/docs/pdf/2020_N [12] New York Senate. (2019) Leadership & Community Pro https://www.nysenate.gov/legislatio [13] IBM Software Group, CPLEX, IBM 2018. [Online]. Available: http://ww [14] (2022). Generation Capacity by Interconnection. [Online]. Availabl operations/ops-analysis.aspx [15] U.S. Energy Information Administ 2021, U.S. Dept. IV. CONCLUSION Kittner, F. Lill, and D. M. Kammen, ‘‘Energy storage deployment and innovation for the clean energy transition,’’ Nature Energy, vol. 2, no. 9, pp. 1–6, Jul. 2017. CSU gt ≥SUCg(Igt −Ig(t−1)) ∀g, t (12) CSD gt ≥SDCg(Ig(t−1) −Igt) ∀g, t (13) [3] N. Kittner, F. Lill, and D. M. Kammen, ‘‘Energy storage deployment and innovation for the clean energy transition,’’ Nature Energy, vol. 2, no. 9, pp. 1–6, Jul. 2017. (13) [4] J. Aleluia, P. Tharakan, A. P. Chikkatur, G. Shrimali, and X. Chen, ‘‘Accelerating a clean energy transition in Southeast Asia: Role of governments and public policy,’’ Renew. Sustain. Energy Rev., vol. 159, May 2022, Art. no. 112226. y [5] I. Khan, A. Zakari, J. Zhang, V. Dagar, and S. Singh, ‘‘A study of trilemma energy balance, clean energy transitions, and economic expansion in the midst of environmental sustainability: New insights from three trilemma leadership,’’ Energy, vol. 248, Jun. 2022, Art. no. 123619. (15) [6] O. Castrejon-Campos, L. Aye, and F. K. P. Hui, ‘‘Making policy mixes more robust: An integrative and interdisciplinary approach for clean energy transitions,’’ Energy Res. Social Sci., vol. 64, Jun. 2020, Art. no. 101425. [7] F. Angizeh, A. Ghofrani, and M. A. Jafari, ‘‘Higher granular sectoral demand forecast under data scarcity: An integrated physics-based top–down and bottom–up approach,’’ IEEE Syst. J., vol. 16, no. 2, pp. 2923–2933, Jun. 2022. [8] PVWatts Calculator, Nat. Renew. Energy Lab. (NREL), Golden, CO, USA, 2022. [Online]. REFERENCES 135004 VOLUME 11, 2023 F. Angizeh et al.: Impact Assessment Framework for Grid Integration of ESSs and RESs ALEXANDER KLEBNIKOV received the B.Sc. and M.Sc. degrees in mechanical engineering from the University of Pennsylvania, in 2016. He worked multiple roles as an Operations, Maintenance and Pressure Equipment Engineer with the Shell Deer Park Refinery, Houston, TX, USA, and Shell Ursa TLP, Gulf of Mexico. He is currently the Project Manager of Atlantic Shores Offshore Wind. His research interests include amorphous plasticity mechanics, wave energy and desalination, hydrogen, and energy policy. ALEXANDER KLEBNIKOV received the B.Sc. and M.Sc. degrees in mechanical engineering from the University of Pennsylvania, in 2016. He worked multiple roles as an Operations, Maintenance and Pressure Equipment Engineer with the Shell Deer Park Refinery, Houston, TX, USA, and Shell Ursa TLP, Gulf of Mexico. He is currently the Project Manager of Atlantic Shores Offshore Wind. His research interests include amorphous plasticity mechanics, wave energy and and energy policy. JINWOO BAE (Student Member, IEEE) received the B.Sc. degree in industrial and management systems engineering from Kyung Hee University, Republic of Korea, in 2016, and the M.Sc. degree in industrial and manufacturing engineering from the University of Wisconsin-Milwaukee, WI, USA, in 2019. He is currently pursuing the Ph.D. degree in industrial and systems engineering with Rutgers University, NJ, USA. His research interests include reliability and uncertainty of estimation from machine learning models, especially in computer vision, robot navigation, and prognostics. MOHSEN A. JAFARI (Member, IEEE) received the Ph.D. degree from Syracuse University, in 1985. He has directed or co-directed a total of over 23 million U.S. dollars in funding from var- ious government agencies, including the National Science Foundation, the Department of Energy, the Office of Naval Research, the Defense Logistics Agency, the NJ Department of Transportation, FHWA, and industry in automation, system opti- mization, data modeling, information systems, and cyber risk analysis. He actively collaborates with universities and research institutes abroad. He has also been a Consultant to several Fortune 500 companies and local and state government agencies. He is currently a Professor and the Chair of Industrial and Systems Engineering with Rutgers University–New Brunswick. His research interests include manufacturing, transportation, healthcare, and energy systems. He is a member of IIE. He received the IEEE Excellence Award in service and research. Shores Offshore Wind. JOYCE CHEN received the master’s degree in international business from Queen’s University, Canada. REFERENCES She has worked across the USA, Canada, The Netherlands, and China, including leading Corporate M&A activities of major renewable company acquisitions, energy market for new country entry, and major divestment program and a Venture Investor in renewable power, hydrogen, storage, and mobility for shell. Currently, she is the Commercial and Finance manager for Atlantic JOYCE CHEN received the master’s degree in international business from Queen’s University, Canada. She has worked across the USA, Canada, The Netherlands, and China, including leading Corporate M&A activities of major renewable company acquisitions, energy market for new country entry, and major divestment program and a Venture Investor in renewable power, hydrogen, storage, and mobility for shell. Currently, she is the Commercial and Finance manager for Atlantic JOYCE CHEN received the master’s degree in international business from Queen’s University, Canada. She has worked across the USA, Canada, The Netherlands, and China, including leading Corporate M&A activities of major renewable company acquisitions, energy market for new country entry, and major divestment program and a Venture Investor in renewable power, hydrogen, storage, and mobility for shell. Currently, she is the Commercial and Finance manager for Atlantic Shores Offshore Wind. 135005 VOLUME 11, 2023
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Dyspraxia and autistic traits in adults with and without autism spectrum conditions
Molecular autism
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cc-by
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Cassidy, S., Hannant, P., Tavassoli, T. ORCID: https://orcid.org/0000-0002-7898-2994, Allison, C., Smith, P. and Baron-Cohen, S. (2016) Dyspraxia and autistic traits in adults with and without autism spectrum conditions. Molecular Autism, 7 (1). 48. ISSN 2040-2392 doi: Cassidy, S., Hannant, P., Tavassoli, T. ORCID: https://orcid.org/0000-0002-7898-2994, Allison, C., Smith, P. and Baron-Cohen, S. (2016) Dyspraxia and autistic traits in adults with and without autism spectrum conditions. Molecular Autism, 7 (1). 48. ISSN 2040-2392 doi: https://doi.org/10.1186/s13229-016-0112-x Available at https://centaur.reading.ac.uk/69936/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1186/s13229-016-0112-x To link to this article DOI: http://dx.doi.org/10.1186/s13229-016-0112-x It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1186/s13229-016-0112-x To link to this article DOI: http://dx.doi.org/10.1186/s13229-016-0112-x Published version at: http://dx.doi.org/10.1186/s132290160112x To link to this article DOI: http://dx.doi.org/10.1186/s13229-016-0112-x Publisher: BioMed Central All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur Article Published Version Creative Commons: Attribution 4.0 (CC-BY) Published Version Creative Commons: Attribution 4.0 (CC-BY) Creative Commons: Attribution 4.0 (CC-BY) © The Author(s). 2016 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. Dyspraxia and autistic traits in adults with and without autism spectrum conditions Sarah Cassidy1,2* , Penelope Hannant1, Teresa Tavassoli2,3, Carrie Allison2, Paula Smith2 and Si * Correspondence: sarah.cassidy@coventry.ac.uk 1Centre for Psychology, Behaviour and Achievement, Coventry University, Priory Street, Coventry CV1 5FB, UK 2Autism Research Centre, Department of Psychiatry, University of Cambridge, Douglas House, 18B Trumpington Road, Cambridge CB2 8AH, UK Full list of author information is available at the end of the article Abstract Background: Autism spectrum conditions (ASC) are frequently associated with motor coordination difficulties. However, no studies have explored the prevalence of dyspraxia in a large sample of individuals with and without ASC or associations between dyspraxia and autistic traits in these individuals. Methods: Two thousand eight hundred seventy-one adults (with ASC) and 10,706 controls (without ASC) self- reported whether they have been diagnosed with dyspraxia. A subsample of participants then completed the Autism Spectrum Quotient (AQ; 1237 ASC and 6765 controls) and the Empathy Quotient (EQ; 1147 ASC and 6129 controls) online through the Autism Research Centre website. The prevalence of dyspraxia was compared between those with and without ASC. AQ and EQ scores were compared across the four groups: (1) adults with ASC with dyspraxia, (2) adults with ASC without dyspraxia, (3) controls with dyspraxia, and (4) controls without dyspraxia. Results: Adults with ASC were significantly more likely to report a diagnosis of dyspraxia (6.9%) than those without ASC (0.8%). In the ASC group, those with co-morbid diagnosis of dyspraxia did not have significantly different AQ or EQ scores than those without co-morbid dyspraxia. However, in the control group (without ASC), those with dyspraxia had significantly higher AQ and lower EQ scores than those without dyspraxia. Conclusions: Dyspraxia is significantly more prevalent in adults with ASC compared to controls, confirming reports that motor coordination difficulties are significantly more common in this group. Interestingly, in the general population, dyspraxia was associated with significantly higher autistic traits and lower empathy. These results suggest that motor coordination skills are important for effective social skills and empathy. Keywords: Autism spectrum conditions, Dyspraxia, Co-morbidity, Autistic traits, Social skills Keywords: Autism spectrum conditions, Dyspraxia, Co-morbidity, Autistic traits, Social skills been recently confirmed in a number of research studies [6, 7]. Children (without ASC), who have dyspraxia, also exhibit social and emotional difficulties [8]. However, lit- tle research in ASC or the general population has ex- plored the association between dyspraxia and social or emotional skills in adulthood [9]. This is the purpose of the current study. Open Access Central Archive at the University of Reading Reading’s research outputs online Cassidy et al. Molecular Autism (2016) 7:48 DOI 10.1186/s13229-016-0112-x Participants Participants completed questionnaires online through one of two websites (www.autismresearchcentre.com or www.cambridgepsychology.com). Controls (without ASC) were only included if they did not report having a child or other family members with autism, to avoid in- cluding those with the ‘broader autism phenotype’ [38]. Individuals with a diagnosis of bipolar disorder, epilepsy, schizophrenia, attention deficit/hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), learning disability (LD), intersex/transsexual condition or psychosis were excluded from the control group. There are however very few studies on dyspraxia in adults with and without ASC—all of the aforementioned studies involved children. However, the limited number of available studies suggests that in those with and without ASC, motor difficulties continue into adulthood [32, 33]. There is also preliminary evidence from a small number of studies that general population young adults (aged 16– 25 years) with dyspraxia may have many of the same diffi- culties as in childhood [34, 35]. There is also high risk of these individuals experiencing mental health problems, low self-esteem and emotional difficulties, exacerbated by low occupational attainment [9]. It is also currently un- clear how many adults with and without ASC have dys- praxia or the impact of this on their social skills. After exclusions, 2871 participants reported having a formal clinical diagnosis of ASC (70% male). A majority (n = 2056) had Asperger syndrome; the remaining partici- pants reported having high-functioning autism (n = 287), autism (n = 302), atypical autism (n=43), pervasive developmental disorder (n = 124) and autism spectrum condition (i.e. participants who did not spe- cify a subtype) (n = 59). The control group (without ASC) was comprised of n = 10,706 individuals (41% male), who reported they had no diagnosis of ASC. Participants were aged between 18 and 75 years old (Table 1). To address this gap in research, the current study aimed to explore the prevalence of dyspraxia in a large population sample of adults with and without ASC and associations between dyspraxia and autistic traits in these individuals. We utilize online self-reported diagno- sis of dyspraxia, alongside validated measures of autistic traits (the Autism Spectrum Quotient (AQ) [36]) and empathy (the Empathy Quotient (EQ) [37]). These mea- sures have been validated for use in those with ASC and the general population, to reliably quantify individual differences in autistic traits and empathy in those with and without ASC. Background ac g ou d Is ability to effectively coordinate, plan and carry out movements associated with successful social function- ing? Dyspraxia is characterized by pronounced difficul- ties in the selection, timing and spatial organization of purposeful movement and coordination [1] and is thought to arise from atypical neural connections in the cerebral cortex [2]. Individuals with autism spectrum conditions (ASC), who have pronounced difficulties with social interaction, also exhibit atypical motor movements [3]. In fact, original clinical reports of ASC reported gen- eral ‘clumsiness’ in these individuals [4, 5], which have Studies of children with ASC have demonstrated sig- nificant motor difficulties in these individuals [10, 11]. Motor skill scores for children with ASC often fall 1.5 standard deviations below the typical mean [12, 13], and approximately 80% have definite pronounced motor dif- ficulties with 10% being borderline [6, 14–18]. Atypical motor skills in ASC are present from early infancy [19–21] and reported by parents as one of their first areas of concern (average age 14.7 months) prior to seeking an ASC diagnosis [22]. Motor difficulties in * Correspondence: sarah.cassidy@coventry.ac.uk 1Centre for Psychology, Behaviour and Achievement, Coventry University, Priory Street, Coventry CV1 5FB, UK 2Autism Research Centre, Department of Psychiatry, University of Cambridge, Douglas House, 18B Trumpington Road, Cambridge CB2 8AH, UK Full list of author information is available at the end of the article Cassidy et al. Molecular Autism (2016) 7:48 Page 2 of 6 children with ASC may be associated with their social and communicative difficulties. For example, children with ASC show significant difficulties in skilled motor gestures, such as imitation [23]. Empathic ability is also reduced in children (without ASC) who exhibit motor difficulties [8], and research has shown correlations between motor coordination and social communication skills in children with ASC [24–26]. Approximately 2–6% of children (without ASC) from the general population have dyspraxia [27]. These children exhibit difficulties in social skills, social phobia, empathy [8, 28], maintaining peer relationships and increased anxiety [29–31]. This suggests that children without ASC, but with dyspraxia, exhibit traits associated with ASC, particularly in social interaction, empathy and social anxiety. individuals (without ASC) who have dyspraxia may also have significantly higher autistic traits than those without dyspraxia or ASC. Background If this were the case, then this would suggest that movement difficulties are associated with aut- istic traits in those with and without ASC and could be a prime target for intervention to improve social skills in these groups. AQ When registering in the CARD, participants provided demographic data, including age, biological birth sex and educational and occupational attainment, and any diagnoses from a trained clinician, including ASC and dyspraxia. Participants then complete questionnaires de- signed to quantify autistic traits. We extracted data from two of these self-report questionnaires. The AQ [36] quantifies individual differences in autistic traits, in adults with average or above average intelligence quo- tient (IQ). The EQ quantifies individual differences in empathizing ability [37]. A between-subjects ANCOVA showed a significant main effect of age (F(1, 7997) = 131, p < 0.001, η2 = 0.02). After controlling for the effect of age, there was a significant main effect of dyspraxia, where participants with a diag- nosis of dyspraxia self-reported significantly higher levels of autistic traits (mean = 33.4) than those without a diagnosis of dyspraxia (mean = 20.7) (F(1, 7997) = 16.58, p < 0.001, η2 = 0.002). Results also showed a significant main effect of ASC diagnosis, where participants with a diagnosis of ASC self-reported significantly higher levels of autistic traits (mean = 38.4) than those without ASC (mean = 17.8) (F(1, 7997) = 624, p < 0.001, η2 = 0.07). Lastly, there was a significant interaction between the presence of dyspraxia and ASC diagnosis (F(1, 7997) = 22, p < 0.001, η2 = 0.003). Simple main effects analysis showed a significant effect of dyspraxia in the control group (F(1, 7997) = 28, p < 0.001, η2 = 0.003); controls with dyspraxia self-reported significantly higher levels of autis- tic traits (mean = 23.4) than controls without dyspraxia (mean = 17.7). There was no significant effect of dyspraxia in the ASC group (F(1, 7997) = 0.3, p = 0.5, η2 = 0.001). Results Table 1 shows the frequency of self-reported dyspraxia in the ASC and control groups. Table 2 shows the means for age, AQ and EQ for (1) adults with ASC with dys- praxia, (2) adults with ASC without dyspraxia, (3) con- trols with dyspraxia, and (4) controls without dyspraxia. Table 2 Mean age, AQ and EQ by diagnostic group Dyspraxia? Age (SD) AQ (SD) EQ (SD) ASC Yes 29 (12.4) 37.6 (7.5) 20 (12) No 35.7 (13.2) 38.5 (6.8) 17.6 (9.9) Control Yes 25.3 (10.1) 23.4 (8.2) 39.8 (14.4) No 31.4 (13.5) 17.7 (7.5) 45 (14.2) Total Yes 27.9 (11.9) 33.4 (10) 25.2 (15.3) No 32.1 (13.5) 20.7 (10) 41 (16.8) Dyspraxia y p Participants with ASC were significantly more likely to report a clinical diagnosis of dyspraxia (6.9%) than con- trols (0.8%) (X2(1) = 400.5, p < 0.001; OR 8.69). Participants with ASC were significantly more likely to report a clinical diagnosis of dyspraxia (6.9%) than con- trols (0.8%) (X2(1) = 400.5, p < 0.001; OR 8.69). EQ A between-subjects ANCOVA showed a significant main effect of age (F(1, 7271) = 18, p < 0.001, η2 = 0.002). After controlling for the effect of age, there was no significant main effect of dyspraxia; participants with a diagnosis of dyspraxia did not self-report significantly different levels of empathy (mean = 25.2) than those without a diagnosis of dyspraxia (mean = 41) (F(1, 7271) = 0.6, p =.4, η2 = 0.001). However, results did show a signifi- cant main effect of ASC diagnosis, where participants with a diagnosis of ASC self-reported significantly lower empathy (mean = 17.8) than those without ASC (mean = 45) (F(1, 7271) = 289, p < 0.001, η2 = 0.04). Lastly, there was a significant interaction between the presence of dyspraxia and ASC diagnosis (F(1, 7271) = 8, p < 0.01, η2 = 0.001). Simple main effects analysis showed a significant effect of dyspraxia in the control group (F(1, 7271) = 4, p = 0.04, η2 = 0.01); controls with dyspraxia self-reported significantly lower empathy (mean = 39.8) than controls without dyspraxia (mean = 45). There was no significant effect of dyspraxia in the ASC group (F(1, 7271) = 3.6, p = 0.06, η2 = 0.001). Participants This allows us to assess whether the presence of dyspraxia is associated with significantly in- creased autistic traits in a large population sample, con- sisting of over 2500 adults with ASC and over 10,000 adults without ASC. Recruitment of high-functioning adults over 18 years old also allows us to explore associ- ations between dyspraxia and autistic traits independent of intellectual disability or age-related effects through development. A majority of the individuals in the ASC group pro- vided information on type of education (mainstream, home, special) (n = 2473, 86%), and of these individuals, a majority reported having attended mainstream school (n = 1949, 78.8%). In total, 1284 (44.7%) of the ASC group also provided information on current occupation, and of these, a majority (n = 846, 65.9%) were employed, n = 233 (18.1%) individuals were in full-time study and n = 202 (15.7%) individuals were unemployed. In the control group, n = 5490 (51.3%) individuals provided in- formation on their education type, and of these, a majority (n = 5358, 97.6%) reported having attended mainstream education. In total, 6011 (56.1%) of the control group provided information on their occupation, and of Table 1 Self-reported dyspraxia in adults with ASC vs. adult controls without ASC Table 1 Self-reported dyspraxia in adults with ASC vs. adult controls without ASC Dyspraxia No dyspraxia ASC (n = 2871) 199 (6.9%) 2672 (93.1%) Control (n = 10,706) 91 (0.8%) 10,615 (99.2%) Total (n = 13,577) 290 (2.1%) 13,287 (97.9%) If movement difficulties are significantly associated with social, communication skills and empathy, then individ- uals with ASC and co-morbid dyspraxia may have a sig- nificantly higher number of autistic traits than individuals with ASC without co-morbid dyspraxia. Additionally, Page 3 of 6 Cassidy et al. Molecular Autism (2016) 7:48 Page 3 of 6 these, n = 4931 (82%) were currently employed, n = 967 (16.1%) were in full-time study and n = 113 (1.9%) were unemployed. Statistical analysis Chi-square analyses were used to compare the preva- lence of dyspraxia in the ASC and control groups, with odds ratios used as a measure of effect size. Large sam- ples increase the robustness of ANOVA to violation of normality and homogeneity of variance. Separate two- way ANCOVAs, including age as a covariate, were con- ducted on AQ and EQ data, with two between-subjects factors of ‘diagnosis’ (ASC vs. control) and ‘dyspraxia’ (dyspraxia vs. no dyspraxia). The presence of significant diagnosis-by-dyspraxia interaction effects indicates that the effect of dyspraxia on autistic traits is dependent on ASC diagnosis. Significant interaction effects were followed up by simple main effects analysis, to establish whether the effect of dyspraxia on autistic traits was present in each diagnostic group. Effect sizes were calcu- lated using partial eta squared (η2) for main effects, in- teractions and simple main effects. For partial eta squared (η2), 0.01 represents a small, 0.06 a medium and 0.14 a large effect. Discussion This study aimed to explore for the first time whether dyspraxia was significantly more prevalent in adults with Cassidy et al. Molecular Autism (2016) 7:48 Page 4 of 6 Page 4 of 6 ASC compared to controls without ASC and associa- tions between dyspraxia and autistic traits in adults with and without ASC. Results showed that adults with ASC self-reported a significantly higher rate of dyspraxia (6.9%) than adults without ASC (0.8%); the relative odds of having a diagnosis of dyspraxia were 8 times higher in adults with ASC compared to controls without ASC. These results show for the first time that the prevalence of dyspraxia is significantly higher in adults with ASC compared to controls without ASC. These findings reflect previous research, showing that motor coordination diffi- culties are highly prevalent in ASC [6, 7]. Furthermore, these findings add to the small body of currently available evidence showing that the difficulties associated with dys- praxia in childhood persist into adulthood [33, 35]. ASC compared to controls without ASC and associa- tions between dyspraxia and autistic traits in adults with and without ASC. Results showed that adults with ASC self-reported a significantly higher rate of dyspraxia (6.9%) than adults without ASC (0.8%); the relative odds of having a diagnosis of dyspraxia were 8 times higher in adults with ASC compared to controls without ASC. These results show for the first time that the prevalence of dyspraxia is significantly higher in adults with ASC compared to controls without ASC. These findings reflect previous research, showing that motor coordination diffi- culties are highly prevalent in ASC [6, 7]. Furthermore, these findings add to the small body of currently available evidence showing that the difficulties associated with dys- praxia in childhood persist into adulthood [33, 35]. with ASC, recognition and diagnosis of these difficulties is key to access appropriate support and treatment. A small body of evidence suggests high risk of adults with dyspraxia, without co-morbid ASC, experiencing mental health problems, low self-esteem and emotional difficul- ties, exacerbated by low occupational attainment [9]. Re- sults from the current study add to this literature, suggesting that these individuals also experience difficul- ties in social skills and empathy, characteristic of ASC. Future research will need to explore whether improving motor coordination early in childhood, or in adulthood, improves these poor outcomes. p p The current study has a number of strengths as well as limitations. Discussion It contributes to an under-explored area of research—dyspraxia in adulthood and the prevalence and impact of dyspraxia on autistic traits in adults with and without ASC. It also utilized measures of self- reported autistic traits (AQ) and empathy (EQ), which both have undergone substantial reliability tests and have excellent psychometric properties [40, 41]. The current study also analysed data from a very large popu- lation sample—over 2800 adults with ASC and 10,000 control adults without ASC. These large numbers were necessary in order to explore differences in autistic traits between the groups, considering that only 0.8% of con- trol adults had a diagnosis of dyspraxia. One limitation is that in order to achieve this large sample, self-report measures of dyspraxia and ASC diagnoses were neces- sary. However, previous research has shown significantly high concordance rates between self-reported and clinic- ally confirmed diagnoses [42, 43]. Additionally, partici- pants provide details on when and where they received their ASC diagnosis when they register in the research database, to ensure that these self-reported diagnoses are valid. Hence, it is unlikely that the self-report methods utilized in the study significantly invalidated the results. Results also showed that the association between dys- praxia and levels of autistic traits and empathy differed ac- cording to the presence of co-morbid ASC. Specifically, diagnosis of dyspraxia was only significantly associated with self-reported autistic traits and empathy if partici- pants did not have co-morbid ASC. Controls without ASC, with a diagnosis of dyspraxia, self-reported a signifi- cantly higher number of autistic traits and significantly lower levels of empathy than controls without ASC or dyspraxia, whereas those with ASC and co-morbid dys- praxia did not self-report significantly different levels of autistic traits or empathy compared to those with ASC without co-morbid dyspraxia. These results suggest that motor coordination difficul- ties are significantly associated with social skills and em- pathy in adults without ASC, whereas co-morbid dyspraxia in adults with ASC is not significantly associ- ated with increased difficulties in social communication skills and empathy. One possible explanation for this finding is that dyspraxia and ASC symptoms may over- lap, particularly as both conditions are seemingly associ- ated with atypical development of neurons within the cerebral cortex [2, 7, 39]. For example, previous research has shown that the difficulties individuals with dyspraxia experience are somewhat similar to the difficulties people with ASC experience. References 1 G i J The role of sensorimotor difficulties in the development of autism spectrum conditions. Frontiers Neurology: Movement Disorders. 2016; 7. 8. Cummins A, Piek JP, Dyck MJ. Motor coordination, empathy, and social behaviour in school-aged children. Dev Med Child Neurol. 2005;47:437–42. 9. Kirby A, Williams N, Thomas M, Hill EL. Self-reported mood, general health, wellbeing and employment status in adults with suspected DCD. Res Dev Disabil. 2013;34:1357–64. 10. Berkeley SL, Zittel LL, Pitney LV, Nichols SE. Locomotor and object control skills of children diagnosed with autism. Adapt Phys Act Q. 2001;18:405–16. 11. Hughes C. Brief report: planning problems in autism at the level of motor control. J Autism Dev Disord. 1996;26:99–107. 12. Miyahara M, Tsujii M, Hori M, Nakanishi K, Kageyama H, Sugiyama T. Brief report: motor incoordination in children with Asperger syndrome and learning disabilities. J Autism Dev Disord. 1997;27:595–603. 13. Fournier KA, Hass CJ, Naik SK, Lodha N, Cauraugh JH. Motor coordination in autism spectrum disorders: a synthesis and meta-analysis. J Autism Dev Disord. 2010;40:1227–40. Availability of data and materials 9. Kirby A, Williams N, Thomas M, Hill EL. Self-reported mood, general health, wellbeing and employment status in adults with suspected DCD. Res Dev Disabil. 2013;34:1357–64. The datasets analysed during the current study are not publicly available due to the terms and conditions participants agree to when they register in CARD, but are available from the corresponding author on reasonable request. 10. Berkeley SL, Zittel LL, Pitney LV, Nichols SE. Locomotor and object control skills of children diagnosed with autism. Adapt Phys Act Q. 2001;18:405–16. Funding SAC SAC was supported by the ESRC Future Research Leaders programme; SAC and PH were supported by the Centre for Research in Psychology, Behaviour and Achievement, Coventry University; and SB-C was supported by the MRC, the Autism Research Trust and the Wellcome Trust during the period of this work. This project was conducted in association with the NIHR CLAHRC for Cambridgeshire and Peterborough NHS Foundation Trust. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. No funding body had any role in the design of the study, data collection, analysis, interpretation of data or manuscript writing. 4. Kanner L. Autistic disturbances of affective contact. Publisher not identified; 1943. p. 217-250. 5. Asperger H. Die “Autistischen Psychopathen” im Kindesalter. Eur Arch Psychiatry Clin Neurosci. 1944;117:76–136. 6. Gowen E, Hamilton A. Motor abilities in autism: a review using a computational context. J Autism Dev Disord. 2013;43:323–44. 7. Hannant P, Cassidy SA, Tavissoli T. The role of sensorimotor difficulties in the development of autism spectrum conditions. Frontiers Neurology: Movement Disorders. 2016; 7. 8. Cummins A, Piek JP, Dyck MJ. Motor coordination, empathy, and social behaviour in school-aged children. Dev Med Child Neurol. 2005;47:437–42. Competing interests Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate A favourable ethical opinion was obtained from the Psychology Research Ethics Committee, Coventry University, UK, to conduct secondary analysis of anonymized data from the Cambridge Autism Research Database (CARD), from the Autism Research Centre, University of Cambridge, UK. Participants register to join the CARD and provide their consent online, where they also have opportunity to read the Terms and Conditions, which includes details of how their data will be stored and used in a variety of research projects in an anonymized form. Participants with and without ASC then provide questionnaire and performance data and indicate their willingness to be re-contacted with invitations to participate in new research studies. This consent procedure for participants to register in the CARD was approved by the Psychological Research Ethics Committee (PREC), University of Cambridge, UK. Received: 15 July 2016 Accepted: 17 November 2016 Received: 15 July 2016 Accepted: 17 November 2016 Discussion All authors read and approved the final manuscript. Authors’ information Not applicable. Authors’ information Not applicable. Authors’ information Not applicable. Competing interests The authors declare that they have no competing interests. Acknowledgements Not applicable. 2. Portwood M. Developmental dyspraxia: identification and intervention: a manual for parents and professionals. London: Routledge; 2013. p p g 3. American Psychiatric Association. Diagnostic and statistical manual of mental disorders (DSM-5®). American Psychiatric Pub; 2013. Consent for publication Not applicable. In conclusion, the current study reports the first evi- dence that dyspraxia is significantly more prevalent in adults with ASC compared to controls without ASC, confirming previous reports that motor coordination dif- ficulties are highly prevalent in these individuals. Inter- estingly, the presence of dyspraxia was significantly associated with difficulties in social skills and empathy, particularly in those without co-morbid ASC. These re- sults suggest that adults with dyspraxia demonstrate a significantly increased number of autistic traits com- pared to the general population (without ASC or dys- praxia) and thus experience similar difficulties to adults with ASC. This is the first evidence of the significant as- sociation between motor coordination difficulties with social skills and empathy in adults in the general popula- tion and adds to the limited available literature showing a host of poor outcomes in adults with dyspraxia (with- out ASC). Clinicians must be aware of the impact and importance of motor coordination skills for wider social functioning and empathy and offer appropriate support and treatment for these individuals. Abbreviations AQ: Autism Spectrum Quotient; ASC: Autism spectrum condition; EQ: Empathy Quotient Discussion Empathy, for example, is significantly reduced in children (without ASC) who ex- hibit motor difficulties [8]. Hence, the association be- tween dyspraxia with social skills and empathy in those without diagnosis of ASC would be greater than in those with co-morbid ASC. Another potential limitation is the use of the term ‘dyspraxia’ in the current study. This could have led to an under-reporting of this diagnosis in the control group, due to lack of familiarity with this term. It may also be the case that the likelihood of receiving a dys- praxia diagnosis maybe unevenly distributed across sub- sets of those with ASC. As discussed above, it is possible that dyspraxia is under-diagnosed in those with ASC, and this may differ by subtype. This could have meant that the rate of dyspraxia in both the control and ASC groups could have been under-estimated in the current study. However, if anything, this means that the rate of dyspraxia diagnosis is a conservative estimate in the current study. If an alternative label, or in person mea- sures, were used, the rates could potentially have been higher in both groups. Future research studies will need to explore whether these rates of dyspraxia are replicable Another possibility is that dyspraxia may be under- diagnosed in those with ASC. Motor difficulties are highly prevalent in people with ASC [6, 7], and these dif- ficulties may be viewed as part of their ASC, as opposed to requiring another co-morbid diagnosis. However, given the small but growing body of evidence showing the importance of motor coordination in social skills in both the general population (without ASC) and those Cassidy et al. Molecular Autism (2016) 7:48 Page 5 of 6 Page 5 of 6 Page 5 of 6 Page 5 of 6 in a large representative sample of those with and with- out ASC using in person assessments, across the autism spectrum. However, taken together, this is the only and the largest study to date that has explored the preva- lence of dyspraxia and associations between dyspraxia and autistic traits in those with and without ASC. critical revision and final approval of the manuscript. SBC helped design the study, critical revision and final approval of the manuscript. All authors read and approved the final manuscript. critical revision and final approval of the manuscript. SBC helped design the study, critical revision and final approval of the manuscript. References 1 G i J 1. Grieve J, Gnanasekaran L. Neuropsychology for occupational therapists: cognition in occupational performance. Oxford: Blackwell Publishing; 2008. 2. Portwood M. Developmental dyspraxia: identification and intervention: a manual for parents and professionals. London: Routledge; 2013. 3. American Psychiatric Association. Diagnostic and statistical manual of mental disorders (DSM-5®). American Psychiatric Pub; 2013. 4. Kanner L. Autistic disturbances of affective contact. Publisher not identified; 1943. p. 217-250. 5. Asperger H. Die “Autistischen Psychopathen” im Kindesalter. Eur Arch Psychiatry Clin Neurosci. 1944;117:76–136. 6. Gowen E, Hamilton A. Motor abilities in autism: a review using a computational context. J Autism Dev Disord. 2013;43:323–44. 7. Hannant P, Cassidy SA, Tavissoli T. The role of sensorimotor difficulties in the development of autism spectrum conditions. Frontiers Neurology: Movement Disorders. 2016; 7. 8. Cummins A, Piek JP, Dyck MJ. Motor coordination, empathy, and social behaviour in school-aged children. Dev Med Child Neurol. 2005;47:437–42. 9. Kirby A, Williams N, Thomas M, Hill EL. Self-reported mood, general health, wellbeing and employment status in adults with suspected DCD. Res Dev Disabil. 2013;34:1357–64. 10. Berkeley SL, Zittel LL, Pitney LV, Nichols SE. Locomotor and object control skills of children diagnosed with autism. Adapt Phys Act Q. 2001;18:405–16. 11. Hughes C. Brief report: planning problems in autism at the level of motor control. J Autism Dev Disord. 1996;26:99–107. 12. Miyahara M, Tsujii M, Hori M, Nakanishi K, Kageyama H, Sugiyama T. Brief report: motor incoordination in children with Asperger syndrome and learning disabilities. J Autism Dev Disord. 1997;27:595–603. 13. Fournier KA, Hass CJ, Naik SK, Lodha N, Cauraugh JH. Motor coordination in autism spectrum disorders: a synthesis and meta-analysis. J Autism Dev Disord. 2010;40:1227–40. 1. Grieve J, Gnanasekaran L. Neuropsychology for occupational therapists: cognition in occupational performance. Oxford: Blackwell Publishing; 2008. 1. Grieve J, Gnanasekaran L. Neuropsychology for occupational therapists: cognition in occupational performance. Oxford: Blackwell Publishing; 2008. 2. Portwood M. Developmental dyspraxia: identification and intervention: a manual for parents and professionals. London: Routledge; 2013. 3. American Psychiatric Association. Diagnostic and statistical manual of mental disorders (DSM-5®). American Psychiatric Pub; 2013. 4. Kanner L. Autistic disturbances of affective contact. Publisher not identified; 1943. p. 217-250. 5. Asperger H. Die “Autistischen Psychopathen” im Kindesalter. Eur Arch Psychiatry Clin Neurosci. 1944;117:76–136. 6. Gowen E, Hamilton A. Motor abilities in autism: a review using a computational context. J Autism Dev Disord. 2013;43:323–44. 7. Hannant P, Cassidy SA, Tavissoli T. Author details 1 1Centre for Psychology, Behaviour and Achievement, Coventry University, Priory Street, Coventry CV1 5FB, UK. 2Autism Research Centre, Department of Psychiatry, University of Cambridge, Douglas House, 18B Trumpington Road, Cambridge CB2 8AH, UK. 3The Centre for Research in Autism and Education, UCL Institute of Education, University College London, 55-59 Gordon Square, London WC1H 0NU, UK. 4CLASS Clinic, Cambridgeshire and Peterborough NHS Foundation Trust, Cambridge, UK. Received: 15 July 2016 Accepted: 17 November 2016 Authors’ contributions Personal Individ Differ. 2011;51:829–35. 19. Teitelbaum P, Teitelbaum O, Nye J, Fryman J, Maurer RG. Movement analysis in infancy may be useful for early diagnosis of autism. Proc Natl Acad Sci. 1998;95:13982–7. 42. Auyeung B, Allison C, Wheelwright S, Baron-Cohen S. Brief report: development of the adolescent empathy and systemizing quotients. J Autism Dev Disord. 2012;42:2225–35. 20. Matson JL, Mahan S, Fodstad JC, Hess JA, Neal D. Motor skill abilities in toddlers with autistic disorder, pervasive developmental disorder—not otherwise specified, and atypical development. Res Autism Spectr Disord. 2010;4:444–9. 43. Daniels AM, Rosenberg RE, Anderson C, Law JK, Marvin AR, Law PA. Verification of parent-report of child autism spectrum disorder diagnosis to a web-based autism registry. J Autism Dev Disord. 2012;42:257–65. 21. Bhat AN, Galloway JC, Landa RJ. Relation between early motor delay and later communication delay in infants at risk for autism. Infant Behav Dev. 2012;35:838–46. 22. Chawarska K, Paul R, Klin A, Hannigen S, Dichtel LE, Volkmar F. Parental recognition of developmental problems in toddlers with autism spectrum disorders. J Autism Dev Disord. 2007;37:62–72. 23. Mostofsky SH, Dubey P, Jerath VK, Jansiewicz EM, Goldberg MC, Denckla MB. Developmental dyspraxia is not limited to imitation in children with autism spectrum disorders. J Int Neuropsychol Soc. 2006;12:314–26. 24. MacDonald M, Lord C, Ulrich DA. Motor skills and calibrated autism severity in young children with autism spectrum disorder. Adapt Phys Act Q. 2014;31:2. 25. Dziuk MA, Larson JC, Apostu A, Mahone EM, Denckla MB, Mostofsky SH. Dyspraxia in autism: association with motor, social, and communicative deficits. Dev Med Child Neurol. 2007;49:734–9. 26. Hilton C, Wente L, LaVesser P, Ito M, Reed C, Herzberg G. Relationship between motor skill impairment and severity in children with Asperger syndrome. Res Autism Spectr Disord. 2007;1:339–49. 27. Lingam R, Hunt L, Golding J, Jongmans M, Emond A. Prevalence of developmental coordination disorder using the DSM-IV at 7 years of age: a UK population-based study. Pediatrics. 2009;123:e693–700. 28. Pratt ML, Hill EL. Anxiety profiles in children with and without developmental coordination disorder. Res Dev Disabil. 2011;32:1253–9. 28. Pratt ML, Hill EL. Anxiety profiles in children with and without developmental coordination disorder. Res Dev Disabil. 2011;32:1253–9. 29. Pearsall-Jones JG, Piek JP, Rigoli D, Martin NC, Levy F. Motor disorder and anxious and depressive symptomatology: a monozygotic co-twin control approach. Res Dev Disabil. 2011;32:1245–52. 30. Dewey D, Kaplan BJ, Crawford SG, Wilson BN. Authors’ contributions 12. Miyahara M, Tsujii M, Hori M, Nakanishi K, Kageyama H, Sugiyama T. Brief report: motor incoordination in children with Asperger syndrome and learning disabilities. J Autism Dev Disord. 1997;27:595–603. SAC study conception and design, data analysis, manuscript writing and final approval of the manuscript. PH contributed to the conception and design, critical revision and final approval of the manuscript. TT contributed to the design, critical revision and final approval of the manuscript. CA helped in the data extraction and preparation, critical revision and final approval of the manuscript. PS was responsible for the data extraction and preparation, 13. Fournier KA, Hass CJ, Naik SK, Lodha N, Cauraugh JH. Motor coordination in autism spectrum disorders: a synthesis and meta-analysis. J Autism Dev Disord. 2010;40:1227–40. 13. Fournier KA, Hass CJ, Naik SK, Lodha N, Cauraugh JH. Motor coordination in autism spectrum disorders: a synthesis and meta-analysis. J Autism Dev Disord. 2010;40:1227–40. Page 6 of 6 Cassidy et al. Molecular Autism (2016) 7:48 Page 6 of 6 14. Whyatt CP, Craig CM. Motor skills in children aged 7–10 years, diagnosed with autism spectrum disorder. J Autism Dev Disord. 2012;42:1799–809. 38. Piven J, Palmer P, Jacobi D, Childress D, Arndt S. Broader autism phenotype: evidence from a family history study of multiple-incidence autism families. Am J Psychiatr. 1997;154:185–90. 15. Whyatt C, Craig C. Sensory-motor problems in Autism. Front Integr Neurosci. 2013;7:1–12. 39. Fatemi SH, Aldinger KA, Ashwood P, Bauman ML, Blaha CD, Blatt GJ, Chauhan A, Chauhan V, Dager SR, Dickson PE, Estes AM. Consensus paper: pathological role of the cerebellum in autism. Cerebellum. 2012;11(3):777–807. 16. Green D, Charman T, Pickles A, Chandler S, Loucas T, Simonoff E, Baird G. Impairment in movement skills of children with autistic spectrum disorders. Dev Med Child Neurol. 2009;51:311–6. 40. Ruzich E, Allison C, Smith P, Watson P, Auyeung B, Ring H, Baron-Cohen S. Measuring autistic traits in the general population: a systematic review of the Autism-Spectrum Quotient (AQ) in a nonclinical population sample of 6,900 typical adult males and females. Mol Autism. 2015;6:1. 17. Ming X, Brimacombe M, Wagner GC. Prevalence of motor impairment in autism spectrum disorders. Brain Dev. 2007;29:565–70. 18. Page J, Boucher J. Motor impairments in children with autistic disorder. Child Lang Teach Ther. 1998;14:233–59. 6,900 typical adult males and females. Mol Autism. 2015;6:1. 41. Allison C, Baron-Cohen S, Wheelwright SJ, Stone MH, Muncer SJ. Psychometric analysis of the Empathy Quotient (EQ). Authors’ contributions Developmental coordination disorder: associated problems in attention, learning, and psychosocial adjustment. Hum Mov Sci. 2002;21:905–18. 31. Smyth MM, Anderson HI. Coping with clumsiness in the school playground: social and physical play in children with coordination impairments. Br J Dev Psychol. 2000;18:389–413. 31. Smyth MM, Anderson HI. Coping with clumsiness in the school playground: social and physical play in children with coordination impairments. Br J Dev Psychol. 2000;18:389–413. 32. Cantell MH, Smyth MM, Ahonen TP. Two distinct pathways for developmental coordination disorder: persistence and resolution. Hum Mov Sci. 2003;22:413–31. 32. Cantell MH, Smyth MM, Ahonen TP. Two distinct pathways for developmental coordination disorder: persistence and resolution. Hum Mov Sci. 2003;22:413–31. 33. Losse A, Henderson SE, Elliman D, Hall D, Knight E, Jongmans M. Clumsiness in children—do they grow out of it? A 10‐year follow‐up study. Dev Med Child Neurol. 1991;33:55–68. 33. Losse A, Henderson SE, Elliman D, Hall D, Knight E, Jongmans M. Clumsiness in children—do they grow out of it? A 10‐year follow‐up study. Dev Med Child Neurol. 1991;33:55–68. 34. Hill EL, Brown D, Sorgardt KS. A preliminary investigation of quality of life satisfaction reports in emerging adults with and without developmental coordination disorder. J Adult Dev. 2001;18:130–4. 34. Hill EL, Brown D, Sorgardt KS. A preliminary investigation of quality of life satisfaction reports in emerging adults with and without developmental coordination disorder. J Adult Dev. 2001;18:130–4. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 35. Kirby A, Edwards L, Sugden D. Emerging adulthood in developmental co- ordination disorder: parent and young adult perspectives. Res Dev Disabil. 2011;32:1351–60. 35. Kirby A, Edwards L, Sugden D. Emerging adulthood in developmental co- ordination disorder: parent and young adult perspectives. Res Dev Disabil. 2011;32:1351–60. 36. Baron-Cohen S, Wheelwright S, Skinner R, Martin J, Clubley E. The autism- spectrum quotient (AQ): evidence from Asperger syndrome/high- functioning autism, males and females, scientists and mathematicians. J Autism Dev Disord. 2001;31:5–17. 37. Baron-Cohen S, Wheelwright S. The empathy quotient: an investigation of adults with Asperger syndrome or high functioning autism, and normal sex differences. J Autism Dev Disord. 2004;34:163–75. 37. Baron-Cohen S, Wheelwright S. The empathy quotient: an investigation of adults with Asperger syndrome or high functioning autism, and normal sex differences. J Autism Dev Disord. 2004;34:163–75.
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Spinal tumors: literature review
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Д. Е. Закондырин1, А. А. Гринь1, 2 1ФГБОУ ВО «Московский государственный медико-стоматологический университет им. А. И. Евдокимова» Минздрава России; Россия, 127473 Москва, ул. Делегатская, 20, стр. 1; 2ГБУЗ «Научно-исследовательский институт скорой помощи им. Н. В. Склифосовского Департамента здравоохранения г. Москвы»; Россия, 129090 Москва, Большая Сухаревская пл., 3 2ГБУЗ «Научно-исследовательский институт скорой помощи им. Н. В. Склифосовского Департамента здравоохранения г. Москвы»; Россия, 129090 Москва, Большая Сухаревская пл., 3 К о н т а к т ы : Рассмотрены вопросы эпидемиологии, классификации, диагностики и методов лечения опухолей позвоночника. Приведены данные о частоте встречаемости и особенностях различных гистологических форм доброкачественных и злокачественных новообразований позвонков. Обсуждаются особенности диагностики опухолей позвоночника, классические рентгенографические признаки и современные неинвазивные визуализационные и инвазивные (биопсия) методы. Проанализированы литературные данные о методах и возможностях хирургического лечения. Исследованы современные тенденции в выборе степени радикальности хирургического вмешательства в зависи‑ мости от вида опухоли позвоночника с использованием классификаций стадийности новообразований по Ennеking, Weinstein–Boriani–Biagini и Tomita. Уделено внимание современным показаниям к проведению радикальной ре‑ зекции en bloc на позвоночнике, выполняемой в ограниченном числе случаев. Обсуждаются технологии MISS-хи‑ рургии: от вертебропластики, радиочастотной абляции и интервенционного удаления метастатической опухоли до сепарационной хирургии при эпидуральной компрессии. Помимо хирургического лечения, рассматриваются методы неоадъювантной и адъювантной лучевой терапии новообразований позвонков: конвенциальная, конформ‑ ная, в том числе стереотаксическая, ЛТ и радиохирургия. Представлены данные о современных тенденциях в вы‑ боре тактики лечения в зависимости от гистологической природы опухоли, ее радиочувствительности и возмож‑ ности постлучевой злокачественной трансформации. Медикаментозное лечение, в частности химиотерапия, служит незаменимым методом в лечении вторичных и некоторых первичных злокачественных опухолей позвоночника. Приводятся данные о химиочувствительности различных новообразований и тактике комбинированного и ком‑ плексного лечения. Очевидно, что современная тенденция – уменьшение степени хирургической инвазии и выбор в сторону малоинвазивных методов хирургического лечения. При этом сделан вывод, что с учетом биологической природы гемопоэтических опухолей и некоторых сарком позвоночника на современном этапе хирургическое ле‑ чение не служит основным методом лечения данных злокачественных новообразований. Ключевые слова: опухоль позвоночника, хирургическое лечение, лучевая терапия, химиотерапия, нейрохирургия Для цитирования: Закондырин Д. Е., Гринь А. А. Опухоли позвоночника: обзор литературы. Нейрохирургия 2022; 24(2):94–104. DOI: 10.17650/1683‑3295‑2022‑24‑2‑94‑104. НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы 4.0 DOI: 10.17650/1683‑3295‑2022‑24‑2‑94‑104 Spinal tumors: literature review D. E. Zakondyrin1, A. A. Grin1, 2 1A.I. Evdokimov Moscow State University of Medicine and Dentistry, Ministry of Health of Russia; Bld. 1, 20 Delegatskaya St., Moscow 127473, Russia; 2N.V. Sklifosovsky Research Institute for Emergency Medicine, Moscow Healthcare Department; 3 Bolshaya Sukharevskaya Sq., Moscow 129090, Russia 1A.I. Evdokimov Moscow State University of Medicine and Dentistry, Ministry of Health of Russia; Bld. 1, 20 Delegatskaya St., Moscow 127473, Russia; 2N.V. Sklifosovsky Research Institute for Emergency Medicine, Moscow Healthcare Department; 3 Bolshaya Sukharevskaya Sq., Moscow 129090, Russia 2’ 2022 2’ 2022 2’ 2022 НЕЙРОХИРУРГИЯ Том 24 Volume 24 ВВЕДЕНИЕ качественных опухолей (ДНО) наиболее часто встре- чаются гемангиомы (93,8 %), которые, по  данным литературы, обнаруживаются у 10–14 % пациентов [3]. В 12 % случаев наблюдается агрессивный рост геман- гиомы с мягкотканевым компонентом, прорастающим в позвоночный канал и сдавливающим невральные структуры. Новообразования (НО) позвоночного столба встре­ чаются значительно реже по сравнению с дегенератив- но-дистрофическими заболеваниями и травмами по- звоночника – с частотой 2,5–8,5 случаев на 100 тыс. населения в течение 1 года [1]. Большинство специа- листов (неврологи, травматологи-ортопеды, нейрохи- рурги) не склонны рассматривать их как возможную причину болей в спине, считая крайне редкой патоло- гией, что приводит к отсроченной постановке пра- вильного диагноза. Таким образом, проблема диагнос­ тики и выбора тактики лечения опухолей позвоночника продолжает оставаться актуальной проблемой клиниче- ской медицины в целом и вертебрологии в частности. структуры. Кроме гемангиом, наиболее распространенными среди ДНО позвоночника считаются следующие: остеоид-остеома, остеобластома, остеохондрома, ги- гантоклеточная опухоль, аневризматическая киста, эозинофильная гранулема, или гистиоцитоз Х, и ней- рофиброма. Остеобластомы – наиболее распростра- ненные (до 10 %) доброкачественные костные опухо- ли позвоночника [4]. Преимущественно поражают задние элементы позвонка – ножку дуги и дужку. Гис­ тологически они неотличимы от  остеоид-остеом, но отличаются по клиническому течению. Остеоид- остеомы обычно не превышают 1,5 см, а остеобласто- мы, как правило, больше 2 см в диаметре и склонны к прорастанию в позвоночный канал. Гигантоклеточ- ная опухоль, аневризматическая киста также склонны к агрессивному росту. Гигантоклеточная опухоль пора- жает преимущественно крестец; аневризматическая костная киста – задние элементы позвонков [2] и мо- жет быть первичным НО или возникать на фоне других доброкачественных костных опухолей (остеобласто- мы, хондробластомы, гигантоклеточной опухоли) [5]. В группе первично злокачественных НО позвоноч- ника преобладают гемопоэтические опухоли и хордо- ма. В структуре заболеваемости пациентов с пораже- нием позвоночника опухолями гемопоэтической и лимфоидной тканей множественная миелома состав- ляет 40–45 % от всех первичных ЗНО этого костного отдела, в то время как другие нозологии составляют лишь незначительную ее часть. Вторая по  частоте группа опухолей кроветворной и лимфоидной тканей, при которых наблюдается поражение позвоночника, – первичные костные лимфомы: поражение ими позво- ночника встречается у 1,7 % больных. Вторичное во- влечение костной ткани в  опухолевый процесс при лимфоме Ходжкина происходит у 5–15 %, при не- ходжкинских лимфомах – у 30–53 % пациентов [6]. ру ур Кроме гемангиом, наиболее распространенными среди ДНО позвоночника считаются следующие: остеоид-остеома, остеобластома, остеохондрома, ги- гантоклеточная опухоль, аневризматическая киста, эозинофильная гранулема, или гистиоцитоз Х, и ней- рофиброма. Остеобластомы – наиболее распростра- ненные (до 10 %) доброкачественные костные опухо- ли позвоночника [4]. ВВЕДЕНИЕ Преимущественно поражают задние элементы позвонка – ножку дуги и дужку. Гис­ тологически они неотличимы от  остеоид-остеом, но отличаются по клиническому течению. Остеоид- остеомы обычно не превышают 1,5 см, а остеобласто- мы, как правило, больше 2 см в диаметре и склонны к прорастанию в позвоночный канал. Гигантоклеточ- ная опухоль, аневризматическая киста также склонны к агрессивному росту. Гигантоклеточная опухоль пора- жает преимущественно крестец; аневризматическая костная киста – задние элементы позвонков [2] и мо- жет быть первичным НО или возникать на фоне других доброкачественных костных опухолей (остеобласто- мы, хондробластомы, гигантоклеточной опухоли) [5]. Russian Journal of Neurosurgery Обзор литературы surgical treatment, neoadjuvant and adjuvant radiotherapy of vertebral tumors are analyzed: conventional, conformal, including stereotaxic, beam therapy and radiosurgery. Data on current trends in treatment selection depending on histological nature of the tumor, its radiosensitivity and probability of post-radiation malignant transformation are presented. Drug treatment, in particular chemotherapy, is an indispensable in treatment of secondary and some prima‑ ry spinal tumors. Data on chemosensitivity of various tumors and tactics of combination and complex treatment are presented. Evidently, the current trend is to decrease the level of surgical invasiveness and selection of minimally invasive methods of surgical treatment. Moreover, it is concluded that considering the biological nature of hemopoietic tumors and some sarcomas, currently surgical treatment is not the main method of treatment of these malignant tumors. surgical treatment, neoadjuvant and adjuvant radiotherapy of vertebral tumors are analyzed: conventional, conformal, including stereotaxic, beam therapy and radiosurgery. Data on current trends in treatment selection depending on histological nature of the tumor, its radiosensitivity and probability of post-radiation malignant transformation are presented. Drug treatment, in particular chemotherapy, is an indispensable in treatment of secondary and some prima‑ ry spinal tumors. Data on chemosensitivity of various tumors and tactics of combination and complex treatment are presented. Evidently, the current trend is to decrease the level of surgical invasiveness and selection of minimally invasive methods of surgical treatment. Moreover, it is concluded that considering the biological nature of hemopoietic tumors and some sarcomas, currently surgical treatment is not the main method of treatment of these malignant tumors. Key words: spinal tumor, surgical treatment, radiation therapy, chemotherapy, neurosurgery For citation: Zakondyrin D. E., Grin A. A. Spinal tumors: literature review. Neyrokhirurgiya = Russian Journal of Neuro‑ surgery 2022;24(2):94–104. (In Russ.). DOI: 10.17650/1683‑3295‑2022‑24‑2‑94‑104. C o n t a c t s : Dmitriy Evgenevich Zakondyrin russiandoctor@mail.ru C o n t a c t s : Dmitriy Evgenevich Zakondyrin russiandoctor@mail.ru Problems epidemiology, classification, diagnosis, and treatment of spinal tumors are considered. Data on morbidity and characteristics of different histological forms of benign and malignant vertebral tumors are presented. Features of spinal tumor diagnosis, classic radiological signs, and current noninvasive visualization and invasive (biopsy) techniques are discussed. Literature data on techniques and capabilities of surgical treatment are analyzed. Current trends in selection of surgical intervention radicality level depending on the type of spinal tumor using staging classifications by Ennеking, Weinstein–Boriani–Biagini and Tomita are described. Current indications for radical en bloc resection performed in a li­ mited number of cases is considered. Minimally invasive spine surgery is discussed: from vertebroplasty, radiofrequen‑ cy ablation and intervention removal of metastatic tumor to separation surgery for epidural compression. Apart from 94 94 2’ 2022 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Russian Journal of Neurosurgery НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurger НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы «шапочка» (экзостоза), напоминающая по структуре цветную капусту. Аневризматическая костная киста на рентгенограммах характеризуется вздутием и истон- чением коркового слоя и специфическими «пузыря- ми» (множественные камеры кисты). При метастати- ческом поражении и гемопоэтических опухолях на рентгенограммах преимущественно диагностируются литические формы (чаще это метастазы рака легкого, щитовидной железы и почки), выглядящие как зоны де- струкции кости, дефекты кортикального слоя ножек дуг и замыкательных пластин тел позвонка, а также приво- дящие к патологическим переломам тел позвонков. Хордома – наиболее часто (около 20 %) встречаю- щееся ЗНО позвоночника, причем в отличие от других опухолей локализуюшееся только в  позвоночнике. Хордома встречается в крестце (50–55 %), основании черепа (35 %) и реже – в грудопоясничном отделе по- звоночника (10–15 %) [7]. Саркомы в  позвоночнике развиваются редко. В их структуре преобладают остеосаркома, саркома Юинга и хондросаркома. Хондросаркома составляет от 7 до 12 % всех ЗНО позвоночника, остеосаркома – от 3 до 14 %, саркома Юинга – от 5 до 8 % [8]. Инвазивная диагностика Предполагается выполнение биопсии НО. Выде- ляют 4 вида биопсии: Предполагается выполнение биопсии НО. Выде- ляют 4 вида биопсии: 1) тонкоигольная аспирационная; Классическая рентгенография позвоночного стол- ба служит 1‑й линией диагностики и широко приме- няется в амбулаторном звене. Информативность вы- явления крупных ДНО позвонков составляет 80 %, метастатических опухолей – 40 % [2]. В большинстве случаев ДНО дают достаточно специфическую карти- ну изменения позвонков. Остеоид-остеома, как и осте- областома, имеет участки разреженной костной ткани и зону ее склероза. Гемангиома рентгенологически определяется по вертикальным трабекулам в виде пче- линых сот, наиболее часто – в теле позвонка. Хондро- бластома выглядит на снимках как обширная область остеолизиса, окруженная зоной остеосклероза, а ги- гантоклеточная опухоль – как четко отграниченная зона лизиса костной ткани без остеосклерозного обо- дка. Остеохондрома обладает достаточно специфичной рентгенологической картиной: на кости присутствует 2) трепанобиопсия (толстой иглой с забором столби- ка ткани); 3) открытая инцизионная (забор фрагмента НО); 4) эксцизионная (тотальное удаление НО с биопсией). Открытая инцизионная биопсия – стандарт во всех случаях отсутствия уверенности в гистологическом характере опухоли [9, 10]. Открытая инцизионная биопсия – стандарт во всех случаях отсутствия уверенности в гистологическом характере опухоли [9, 10]. КЛИНИЧЕСКИЕ ПРОЯВЛЕНИЯ ОПУХОЛЕЙ ПОЗВОНОЧНИКА По сравнению с классической рентгенографией: По сравнению с классической рентгенографией: • КТ обладает большей информативностью вследст- вие большей разрешающей способности и возмож- ности построить изображение в различных проек- циях; Наиболее частый симптом при опухолях позвоноч- ника – боль (у 84 % больных). Боль возникает на фоне незначительной травмы или видимого благополучия и чаще всего локальная; обычно не регрессирует на фоне консервативного лечения и усиливается в тече- ние нескольких недель. Патогномоничный симптом для опухолей позвоночника – усиление болей в гори- зонтальном положении (в ночное время) [9], что свя- зано с расширением эпидуральных вен над  очагом поражения и застоем в них крови или раздражением периостальных нервных окончаний при прорастании опухоли в позвоночник [2]. • МРТ не позволяет в полной мере оценивать сте- пень костных деструкций, но  дает возможность более точно определять размеры мягкотканевого компонента опухоли, его взаимоотношения с не- вральными и сосудистыми структурами, а также кровоизлияния в строму НО; позволяет дифферен- цировать патологические переломы остеопорозной и  опухолевой природы в  отсроченном периоде после перелома. Так, при МРТ-исследовании через 3–6 нед после перелома позвонка, при опухолевой природе компрес- сии отмечается низкая интенсивность изображения зоны перелома в режиме Т1 и высокая в режиме Т2 (при остеопорозной природе отмечается низкая ин- тенсивность сигнала в обоих режимах). В остром пе- риоде дифференцировка характера патологического перелома возможна только с использованием остеос- цинтиграфии. Резкое усиление болей возникает при патологи­ ческом переломе тела позвонка вследствие остеодест­ рукции на фоне опухолевого процесса. Неинвазивные методики К  ним относят рентгенологические исследова- ния – рентгенографию, компьютерную томографию (КТ), магнитно-резонансную томографию (МРТ), а также радиологический метод – сцинтиграфию. Инвазивная диагностика ДИАГНОСТИКА Диагностика НО позвоночного столба включает в себя неинвазивные и инвазивные методы. ЭПИДЕМИОЛОГИЯ ОПУХОЛЕЙ ПОЗВОНОЧНИКА Новообразования позвоночника могут возникать из местных источников, т. е. быть первичными опухо- лями костной, жировой, фиброзной, нервной ткани, нервных оболочек, смежных паравертебральных мяг- ких тканей и лимфатических сосудов. Но могут быть и  вторичными, или метастатическими, опухолями, когда клетки НО попадают в позвоночник гематоген- ным или лимфатическим путем из отдаленных злока- чественных очагов. Метастатические образования значительно прео- бладают над первичными, составляя до 97 % от всех НО [2]. Поскольку позвоночник хорошо васкуляризо- ван и имеет тесную связь с регионарными лимфатиче- скими и венозными дренажными сосудами, он, как правило, подвержен метастазированию. Исследования показывают, что почти у 70 % пациентов с наиболее распространенными злокачественными новообразо- ваниями (ЗНО) – рак молочной железы, легких, пред- стательной железы – выявляются метастазы в костях, в том числе в позвоночнике. Метастатическое пораже- ние костей скелета встречается у 70–80 % больных раком молочной железы или раком простаты и у 40 % пациентов с распространенным раком легкого [3]. В группе первично злокачественных НО позвоноч- ника преобладают гемопоэтические опухоли и хордо- ма. В структуре заболеваемости пациентов с пораже- нием позвоночника опухолями гемопоэтической и лимфоидной тканей множественная миелома состав- ляет 40–45 % от всех первичных ЗНО этого костного отдела, в то время как другие нозологии составляют лишь незначительную ее часть. Вторая по  частоте группа опухолей кроветворной и лимфоидной тканей, при которых наблюдается поражение позвоночника, – первичные костные лимфомы: поражение ими позво- ночника встречается у 1,7 % больных. Вторичное во- влечение костной ткани в  опухолевый процесс при лимфоме Ходжкина происходит у 5–15 %, при не- ходжкинских лимфомах – у 30–53 % пациентов [6]. Первичные опухоли позвоночника встречаются редко, часто асимптомны, поэтому реальная частота их встречаемости неизвестна. Среди первичных добро- 95 2’ 2022 2’ 2022 Классификация опухолей Классификация опухолей При злокачественных образованиях позвоночника хирургическая тактика определяется как степенью рас- пространенности опухоли, так и прогнозом заболева- ния и состоянием пациента. W. F. Enneking и соавт. (1980) предложили классификацию ЗНО по степени их распространения в кости и предпочтительным ме- тодам ХЛ: 1) расположение на уровне Th3–Th9‑позвонков; 2) тотальное поражение тела позвонка; 3) распространение опухолевого процесса на ножку дуги позвонка; • IА стадия  – низкая степень злокачественности, НО не выходит за пределы пораженной кости; 4) костная экспансия с выпячиванием кортикально- го слоя с нечеткими краями; • IB стадия – низкая степень злокачественности, НО выходит за пределы кости; 5) неравномерная трабекулярная структура геман­ гиомы; • II А стадия – высокая степень злокачественности, НО не выходит за пределы пораженной кости без метастазов; 6) наличие эпидурального или паравертебрального компонента опухоли; • IIВ стадия – высокая степень злокачественности, НО выходит за пределы пораженной кости без ме­ тастазов; 7) низкий сигнал на Т1- и высокий – на Т2‑взвешен- ном изображении на МРТ, накопление контраст- ного препарата при проведении КТ. • III cтадия – НО кости с метастазами. Метод выбора – пункционная вертебропластика. Выделяют 2 клинические формы агрессивных геман- гиом: осложненные (с экстравертебральным компо- нентом и клиническими признаками сдавления спин- ного мозга и  корешков спинномозговых нервов) и неосложненные. Осложненные агрессивные геман- гиомы встречаются крайне редко (1 % случаев) и тре- буют применения декомпрессивно-стабилизирующих вмешательств [15, 16]. Доброкачественные опухоли в дальнейшем добави- ли в доработанную классификацию Enneking. Стадий- ность ДНО в данной доработанной классификации принято обозначать арабскими цифрами: 1‑я стадия – латентная, 2‑я – активная, 3‑я – агрессивная [11]. Свод­ ная классификация представлена в таблице. K. Tomita и соавт. (2001) предлагали выполнять ра­ дикальную резекцию en bloc при всех видах ЗНО [22]. Однако S. Boriani (2018) показал, что радикальная ре- зекция en bloc должна применяться при ДНО 3‑й сте- пени (с агрессивным ростом, например, остеобластомы и  гигантоклеточные опухоли), злокачественных  – при низкой степени злокачественности (IA и IB) (на- пример, хондрома и  хондросаркома) [23]. Однако при ЗНО IIА и В (например, остеосаркома и саркома Юинга) радикальная резекция en bloc не имеет преи- муществ перед ЛТ и химиотерапией (ХТ) [23]. При ДНО, склонных к локальному инвазивному росту, общепринятой считается агрессивная хирурги- ческая тактика. При остеобластомах рекомендуют ра- дикальное удаление опухоли, которое дает лучшие результаты, чем внутриопухолевый кюретаж с после- операционной лучевой терапией (ЛТ), кроме того, ре- комендуют предоперационную эмболизацию сосудов опухоли, учитывая ее обильную васкуляризацию [4]. 3) широкая резекция (в пределах здоровых тканей); 4) радикальная резекция en bloc [11]. 3) широкая резекция (в пределах здоровых тканей); 4) радикальная резекция en bloc [11]. При  ДНО возможности ХЛ наиболее высоки. При остеоид-остеомах вследствие их небольших раз- меров активно применяют методы перкутанной абля- ции (радиочастотной, лазерной) [12]. При гемангио- мах ХЛ показано при агрессивном типе течения [13]. J. D. Laredo и соавт. (1986) определили понятие «агрес- сивность гемангиом» [14] и предложили рассматривать гемангиому как агрессивную при наличии 3 и более из 7 рентгенологических признаков: 1. Хирургический метод Cовременное хирургическое лечение (ХЛ) обеспе- чивает локальный контроль за распространением опу- холи, декомпрессию невральных структур и ликви­ дацию нестабильности позвоночного столба. Спектр 96 2’ 2022 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы ческому удалению аневризматической костной кисты P. Tsagozis и соавт. (2015) рекомендуют склеротерапию или предоперационную эмболизацию сосудов опухоли [18]. H. Aiba и соавт. (2018), напротив, сообщают о до- пустимости использования методики внутриопухоле- вого эндоскопического кюретажа при отсутствии риска патологического перелома [19]. При гигантоклеточной опухоли радикальная резекция также служит методом выбора, в отличие от кюретажа опухоли как единст- венного метода лечения (18 % риска рецидива vs. 71 %) [20]. При невозможности выполнения радикаль- ной операции методом выбора, по мнению C. Martin и E. F. McCarthy (2010), служит предоперационная эм- болизация и последующий внутриопухолевый кюре- таж [21]. оперативных вмешательств при опухолях позвоночно- го столба велик: от перкутанной радиочастотной абля- ции до резекции en bloс. Принципиальные факторы при выборе хирургиче- ского метода – предполагаемая природа опухоли и сте- пень ее распространенности. W. F. Enneking и соавт. (1980) сформулированы 4 метода ХЛ опухолей костей: 1) внутриопухолевое удаление (кюретаж и кускова- ние); 2) краевая резекция; Russian Journal of Neurosurgery Обзор литературы Классификация опухолей позвоночника по W. F. Enneking Classification of spinal tumors per W. F. Enneking Тип опу- холи Tumor type Ста- дия Stage Рекомендуемый вид лечения Recommended treatment Доброкаче- ственная Benign 1 Нехирургическое (за исключением случаев, когда требуется деком- прессия и / или стабилизация) Nonsurgical (except for cases requiring decompression and / or stabilization) 2 Внутриопухолевое удаление Intralesional excision 3 Краевая резекция Marginal excision Злокачест- венная Malignant IА IB Широкая резекция Wide en bloc excision IIA IIB Широкая резекция с адъювантной терапией Wide en bloc excision with adjuvant therapy III Паллиативная хирургия и адъювантная терапия Palliative surgery with adjuvant therapy наличия парализации, статуса по шкале Karnofsky, чи- сла метастазов – экстраспинальных костных, в по- звонки и внутренние органы [25]. Авторы предлагали выполнять удаление опухоли при хорошем прогнозе (13–15 баллов), паллиативное ХЛ  – при  промежу­ точном прогнозе (9–11 баллов), консервативное ле­ чение – у пациентов, набравших 8 и менее баллов. Z. Pennington и соавт. (2018) на основе анализа доступ- ных публикаций делают вывод и  подчеркивают, что большинство авторов считают ХЛ целесообразным при ожидаемой продолжительности жизни не менее 3–6 мес [26]. наличия парализации, статуса по шкале Karnofsky, чи- сла метастазов – экстраспинальных костных, в по- звонки и внутренние органы [25]. Авторы предлагали выполнять удаление опухоли при хорошем прогнозе (13–15 баллов), паллиативное ХЛ  – при  промежу­ точном прогнозе (9–11 баллов), консервативное ле­ чение – у пациентов, набравших 8 и менее баллов. Z. Pennington и соавт. (2018) на основе анализа доступ- ных публикаций делают вывод и  подчеркивают, что большинство авторов считают ХЛ целесообразным при ожидаемой продолжительности жизни не менее 3–6 мес [26]. Объем ХЛ при метастатическом поражении позво- ночника – тема, вызывающая дискуссии. Термин en bloc spondylectomy введен K. Tomita и соавт. (1994) [27]. Согласно предложенной ими шкале, больным с хоро- шим прогнозом (2–3 балла) рекомендована широкая резекция опухоли, при 4–5 баллах – краевая или вну- триопухолевая резекция, при 6–7 баллах – паллиатив- ные вмешательства, при плохом прогнозе (8–10) бал- лов  – отказ от  ХЛ. По  мнению S. Boriani (2018), радикальная резекция en bloc при метастатическом поражении должна проводиться только в ограничен- ном числе случаев [23]. Одним из показаний к исполь- зованию данной методики может быть единичный метастаз резистентной к ЛТ опухоли (рак почки, гепа- тоцеллюлярный рак, рак толстой кишки, щитовидной железы, немелкоклеточный рак легкого и меланома) или гормонпродуцирующей опухоли (феохромоцито- ма, карциноид, параганглиома, хориокарцинома) [28]. M. Ohashi и  соавт. Классификация опухолей При аневризматической костной кисте методом выбо- ра служит радикальная резекция или удаление en bloc, так как частичное удаление и внутриопухолевый кю- ретаж дают большой рецидив НО (до 25–35,7 %) [17]. В качестве дополнения к нерадикальному хирурги­ Вследствие неполного соответствия Enneking-клас- сификации задачам ХЛ опухолей позвонков в 1997 г. была разработана другая шкала S. Boriani и соавт. [24]. Данная шкала Weinstein–Boriani–Biagini имеет 97 2’ 2022 2’ 2022 Russian Journal of Neurosurgery (2019) указывают на  тот факт, что блок-резекция эффективна в отношении локаль- ного контроля метастазов, но только при условии вы- полнения ее в пределах здоровых тканей, и неспособ- на предотвратить прогрессирование метастатического поражения в целом. Проблемы применения спонди- лэктомии en bloc – техническая сложность и высокая частота интра- и  послеоперационных осложнений (от 40 до 76,5 %) [29]. 12 секторов (на циферблате часов) и проецируется на горизонтальный срез позвонка в зоне поражения опухолью: в зависимости от расположения опухоли по  отношению к  позвоночному каналу возможно определение предпочтительного доступа. На попереч- ном срезе выделены следующие уровни: • А – экстраоссальное распространение; • B – поверхностный периферический отдел ко • C – глубокая внутрикостная (центральная) лока- лизация, прилежащая к позвоночному каналу; • D – экстраоссальное эпидуральное распространение; • D – экстраоссальное эпидуральное распространение; • E – интрадуральное распространение опухоли; • E – интрадуральное распространение опухоли; • F (только для шейного отдела) – поражение опу- холью канала позвоночной артерии. Предпочтительный доступ для удаления: опухоли, расположенные в секторах 3–10 – задний доступ; не- большие опухоли, расположенные в телах грудных и поясничных позвонков – изолированный передний доступ; в случаях других локаций опухолей – необхо- димы комбинации доступов [23]. Малоинвазивные методики Учитывая положительный эффект декомпрессии невральных структур и известные ограничения для проведения ЛТ, авторами сделан вывод, что у больных с метастатическими опухолями позвоночника реко- мендовано проводить минимальное (циторедуктив- ное) хирургическое удаление опухоли лишь с целью отделения ее края от спинного мозга на 2–3 мм для дальнейшего использования стереотаксической ЛТ. Так появился термин separation surgery [33]. Авторы описали стандартную методику – ламинэктомию на 3 уровнях (на уровне опухоли, а также на уровень вы- ше и ниже ее) и стабилизацию (по необходимости). Основное проявление множественной миеломы – формирование множественных очагов остеодеструк- ции в плоских костях скелета и, в частности, в позвонках. Основной метод ХЛ – пункционная вертебро- и ки- фопластика [6, 39], которая служит паллиативным вмешательством, выполняемым на фоне проведения адъювантной терапии. При выраженных разрушениях позвонков с развитием нестабильности показаны де- компрессивно-стабилизирующие вмешательства [40]. Солитарная плазмоцитома  – первичная локальная опухоль, поэтому основным методом ХЛ служит ре- зекция новообразования. Рекомендуется радикальная или краевая резекция опухоли, а при внутриопухоле- вом удалении необходима адъювантная ЛТ [39]. Роль ХЛ при лимфоме до конца не определена, вследствие высокой химиолучевой чувствительности опухоли ряд авторов даже отрицают целесообразность хирургиче- ской декомпрессии при сдавлении спинного мозга [41]. E. Harris и соавт. (2014) считают необходимым начинать лечение с комбинированной химиолучевой терапии, а операцию выполнять лишь при клиниче- ском ухудшении [42]. Малоинвазивные технологии в хирургии спинальных метастазов – второй современный тренд. Травматич- ность классических доступов к грудным и поясничным позвонкам может быть уменьшена применением тора- коскопической хирургии, эндоскопической ассистенции и малоинвазивных открытых доступов [31, 34]. Вне- дрение опыта MISS-хирургии – минимально инвазив- ной спинальной хирургии (minimally invasive spine surgery, MISS) – в практику лечения метастазов в по- звоночник предполагает использование транскутан- ных методик стабилизации, мини-доступов с исполь- зованием тубулярных ретракторов [35]. Z. С. Pennington и соавт. (2018), проанализировав результаты лечения больных с метастазами в позвоночник, оперированных с использованием стандартных и MISS-методик, при- шли к выводу, что малоинвазивная хирургия служит методом выбора для той категории пациентов, кото- рые в силу тяжести не подлежат открытому вмешатель- ству с большими продолжительностью и интраопера- ционной кровопотерей [26]. Хордома отличается практически полной нечувст- вительностью к ЛТ и ХТ, поэтому основной метод ле- чения в ее случае – хирургический. И для первичной, и для рецидивной хордомы методом выбора служит радикальная резекция en bloc [42]. Особенность опу- холи  – склонность к  упорному рецидивированию, ­частота которого даже после радикальной операции составляет в среднем около 40 % [43]. Малоинвазивные методики Малоинвазивные методики, направленные на деком- прессию невральных структур в комбинации с адъю- вантными методами, в последние годы становятся по- пулярной эффективной альтернативой радикальным вмешательствам [30, 31]. S. Spinazze и соавт. (2005) ввели термин «эпидуральная компрессия спинного мозга» (epidural spinal cord compression, ESCC) для на- иболее частых осложнений с неврологическими про- явлениями при метастазах в позвоночник [32]. M. Bil- sky и  M. Smith (2006), члены Spine Oncology Study Group (SOSG), предложили шкалу степени эпидураль- ной компрессии, в которой 5 градаций-стадий: K. Tomita и соавт. (2001) предложили свою класси- фикацию опухолей позвоночника, включающую 7 ти- пов распространения опухоли в  зависимости от  ее расположения относительно границ пораженного по- звонка [22]. В той же работе авторами предложена бал- льная прогностическая шкала для  больных с мета­ статическим поражением позвоночника, основанная на гистологическом типе первичной опухоли, наличии метастазов в кости и внутренние органы (легкие, пе- чень, почки и головной мозг). Y. Tokuhashi и соавт. (2005) предложена прогностическая балльная система с учетом клинических факторов: первичной опухоли, K. Tomita и соавт. (2001) предложили свою класси- фикацию опухолей позвоночника, включающую 7 ти- пов распространения опухоли в  зависимости от  ее расположения относительно границ пораженного по- звонка [22]. В той же работе авторами предложена бал- льная прогностическая шкала для  больных с мета­ статическим поражением позвоночника, основанная на гистологическом типе первичной опухоли, наличии метастазов в кости и внутренние органы (легкие, пе- чень, почки и головной мозг). Y. Tokuhashi и соавт. (2005) предложена прогностическая балльная система с учетом клинических факторов: первичной опухоли, • 0 – поражены только кости; • 1а – распространение в эпидуральное пространст- во без деформации дурального мешка; 98 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы • 1b – деформация дурального мешка без признаков воздействия на спинной мозг; ких миллилитров костного цемента (вертебропластика). Авторы методики оценили ее эффективность и без- опасность по сравнению с обычной вертебропласти- кой в ходе проспективного исследования на 124 паци- ентах, выявив значительный регресс болевого синдрома в послеоперационном периоде и увеличение мобиль- ности пациентов [38]. • 1c  – деформация и  компрессия спинного мозга с сохранением резервных ликворных пространств; р р р р р р • 3  – компрессия спинного мозга без  сохранения ликворных пространств [33]. 2. Лучевой метод В ряде случаев ЛТ может не только дополнять ХЛ (адъювантная терапия), но и предшествовать ему (нео­ адъювантная терапия) или служить основным методом лечения. Традиционно стандартом лечения метастатической эпидуральной компрессии спинного мозга считалось применение кортикостероидов и конвенциальной ра- диотерапии, что у 50 % больных позволяло получить клинически значимое восстановление моторных функ- ций [51]. Существует 2 схемы назначения ЛТ: корот- кий и длинный курс. D. Rades и соавт. (2016) изучили ­эффективность коротких курсов радиотерапии (8 Гр за 1 фракцию; 20 Гр за 5 фракций) и длинных (30 Гр за 10 фракций; 37,5 Гр за 15 фракций) и пришли к вы- воду о сопоставимости функциональных исходов. Вы- бор между длинным и коротким курсом ЛТ должен основываться на прогнозе выживаемости больного: длинный курс показан больным с прогнозом выжива- емости более 6 мес [50]. В настоящее время ЛТ принципиально представ- лена 3 методиками: дистанционная, контактная (брахитерапия) и системная (радионуклидная). В ле- чении опухолей позвоночника актуальна дистанци- онная радиотерапия (external beam radiotherapy). Условно все методы дистанционной радиотерапии можно разделить на 2 группы: конвенциальное об- лучение (conventional irradiation); конформное (conformal irradiation) [47]. Основное отличие кон- формной ЛТ от конвенциальной – создание поля облучения заданной формы с минимальным воздей- ствием на окружающие ткани. Конформная ЛТ – современный метод, имеющий несколько разновид- ностей: В настоящее время конформные методы ЛТ в ле- чении метастазов в  кости вытесняют конвенцио- нальную ЛТ, так как возможность концентрации дозы облучения без  ее увеличения при  конформной ЛТ в области опухоли служит, в том числе, и способом преодоления радиорезистентности опухоли. Однако J. H. Lee, S. H. Lee (2019) подчеркивают преимущество современной конвенциальной ЛТ (по  сравнению с конформными методами), обусловленное более ко- ротким периодом подготовки больного, необходимым для планирования облучения [52]. Альтернативой изо- лированного применения стереотаксической ЛТ стала технология сепарационной хирургии (операции отде- ления) – separation surgery, описанная выше. • с модуляцией интенсивности мощности дозы в ди- намике (RapidArc); намике (RapidArc); • с контролем по изображению (IGRT); • стереотаксическая ЛТ (SRT); • стереотаксическая радиохирургия (SRS); • корпускулярная ЛТ (particle RT) [48]. Большинство радиологических отделений России продолжают применять конвенциональную ЛТ [48]. В лечении ДНО позвоночника послеоперационную ЛТ рекомендуют применять после внутриопухолевого удаления в случаях агрессивного роста новообразова- ния (Enneking – 3) и невозможноcти радикальной ре- зекции en bloc только при  остеобластоме [4, 23]. При гигантоклеточной опухоли и аневризматической костной кисте облучение показано только при реци- диве новообразований, что обусловлено их радиочувст­ вительностью и возможностью саркоматозной транс- формации [5, 18]. НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы en bloc и для остеосаркомы [45], и для саркомы Юин- га [46], обеспечивающее достоверно лучший локаль- ный контроль по сравнению с внутриопухолевым уда- лением. По  мнению S. Boriani и  cоавт. (2018), внутриопухолевое удаление саркомы Юинга в комби- нации с адъювантной ХТ или ЛТ даже менее эффек- тивно, чем ЛТ или ХТ как единственный метод лече- ния [23]. 2) умеренно радиочувствительные (рак молочной же- лезы, простаты, яичника и  нейроэндокринные опухоли, мелкоклеточный рак легкого); 3) радиорезистентные (рак почки, щитовидной же- лезы, гепатоцеллюлярный рак, немелкоклеточные опухоли легких и меланома). При множественной миеломе ЛТ считается палли- ативным методом, оказывает исключительно симпто- матический эффект, не влияя на выживаемость таких больных [49]. При лимфоме позвонков ЛТ – основной метод лечения [50]. Малоинвазивные методики Технически ра- дикальное удаление опухоли вызывает значительные трудности, поэтому в литературе встречаются сообще- ния о внутриопухолевой и частичной резекции НО, а также о влиянии радикальности удаления только на частоту локального рецидивирования, но не продол- жительность жизни [44]. Паллиативные малоинвазивные хирургические вме- шательства при болевом синдроме на фоне метастати- ческого поражения позвонков – вертеброплатика, ки- фопластика и радиочастотная абляция – третий важный тренд [30]. Пункционная вертебропластика признана эффективным вмешательством при остеолитических метастазах в тела позвонков, особенно при множест- венном поражении и патологических переломах по- звонков, в том числе в составе комбинированного ле- чения [6]. Радиочастотная абляция метастазов в позвоночник предложена M. P. Goetz и соавт. (2004) [36]. Методика наиболее эффективна в комбинации с  вертебропластикой и  не  должна применяться как изолированный метод лечения. Cреди сарком, поражающих позвонки, хондросар- кома обладает гистологическим полиморфизмом, имея несколько форм, отличающихся по степени злокаче- ственности (от GI до GIII). Хондросаркома малочув- ствительна к ЛТ и ХТ, поэтому основной метод лече- ния в ее случае – хирургический, как и при хордоме. На  практике вследствие технических трудностей при удалении данного новообразования часто ограни- чиваются частичным удалением. H. M. Song и соавт. (2014) предложили новый метод паллиативного ХЛ метастазов в позвоночник – интер- венционное удаление опухоли [37]. В зону вмешатель- ства в конце операции проводится инъекция несколь- Остеосаркома и саркома Юинга относятся к опу- холям с высокой степенью злокачественности (GII). Общепринятый стандарт ХЛ, принятый на основании мультицентровых исследований, – радикальное удаление 99 2’ 2022 ОБСУЖДЕНИЕ – – тестикулярная карцинома; Анализ многочисленных публикаций позволяет оценить состояние проблемы лечения опухолей позво- ночника на сегодняшний день. A. C. Disch и соавт. (2020) [10] провели опрос членов крупнейшего в Ев- ропе Немецкого общества спинальных хирургов (Ger- man Spine Society), представляющих 84 центра, где оказывают хирургическую помощь пациентам с опу- холями позвоночника. Авторы выяснили, что подав- ляющее большинство хирургических вмешательств (85 ± 18,7 %) носило паллиативный характер и выпол- нялось при  диссеминированных ЗНО, 2 / 3 вмеша- тельств были открытыми. В большинстве учреждений проводилась нео- или  адъювантная химиотерапия, а в 62 из 84 стационаров стандартно применяли после- операционную конвенциональную ЛТ при метастати- ческом поражении позвоночника радиочувствитель- ными ЗНО. – – острая промиелолитическая лейкемия; – – нефробластома; – – саркома Юинга; – – рабдомиосаркома; 2) условно чувствительные к ХТ: 2) условно чувствительные к ХТ: – – аденокарцинома молочной железы; – – аденокарцинома молочной – – неходжкинская лимфома; – – остеосаркома; – – карцинома предстательной железы; – – колоректальный рак; – – рак шейки матки, рак эндометрия и рак яични- ков; 3) минимально чувствительные к ХТ опухоли: – – низкодифференцированный эндокринный рак; – – злокачественная меланома; – – злокачественная меланома; – – гепатоцеллюлярная карцинома – – рак почки; – – рак почки; Учитывая паллиативный характер большинства операций при опухолях позвоночника, внедрение ма- лоинвазивных технологий – один из трендов развития спинальной онкологии. Спектр малоинвазивных вме- шательств достаточно широк, и  выбор конкретной методики определяется характером патологии и целя- ми вмешательства (лечение боли, нестабильности или устранение эпидуральной компрессии спинного мозга). Нестабильность позвоночника, возникшая на фоне разрушения опухолью костных структур по- звоночного столба, служит показанием для паллиатив- ного ХЛ при  радио- и  химиочувствительных ЗНО и распространенных ДНО. Перспективно внедрение перкутанных методик транспедикулярной фиксации, дополняемых при необходимости вертебропластикой. – – рак поджелудочной железы [56]. Гемопоэтические опухоли традиционно считают чувствительными к радиотерапии и ХТ. Обязательные компоненты ХТ-лечения множественной миеломы с поражением позвонков – глюкокортикоидные гор- моны (дексаметазон или преднизолон) и цитостатики, а также бифосфонаты – блокаторы резорбции кости (золедроновая кислота и т. д.), блокаторы протеасом (бортезомиб), иммуномодулирующие препараты и пере­ садка костного мозга [49]. Стандартной для B-кле­ точной неходжкинской лимфомы считается иммуно- химиотерапевтическая схема R-CHOP, включающая несколько химиопрепаратов (доксорубицин, винкристин, полицикламид), гормональный препарат (преднизо- лон) и синтетический аналог моноклональных анти- тел, обладающих специфичностью к CD20‑антигену В-лимфоцитов (ритуксимаб) [57]. Удаление опухоли путем радикальной резекции en bloc продолжает оставаться «золотым стандартом» в ХЛ инвазивно растущей опухоли позвонка, но пра- ктика показывает невозможность такого выполнения в большинстве случаев. 3. Медикаментозный метод Среди современных разновидностей медикамен- тозной терапии ЗНО позвоночника выделяют ХТ, им- мунотерапию и таргетную терапию. В лечении метастатического поражения позвоноч- ника в качестве дополнения к другим методам комби- нированного лечения применяется ХТ в составе ком- плекса медикаментозной терапии, включающего также применение стероидных гормонов и бифосфонатов. Наиболее эффективна ХТ при раке груди, предстатель- ной железы и мелкоклеточном раке легкого [59]. В лечении метастатического поражения позвоноч- ника в качестве дополнения к другим методам комби- нированного лечения применяется ХТ в составе ком- плекса медикаментозной терапии, включающего также применение стероидных гормонов и бифосфонатов. Наиболее эффективна ХТ при раке груди, предстатель- ной железы и мелкоклеточном раке легкого [59]. Химиотерапия – наиболее значимый метод и, так же как и ЛТ, может дополнять ХЛ (неоадъювантная или адъювантная терапия) или быть единственным методом лечения. I. H. Krakoff (1977) выделил 3 группы опухолей по степени чувствительности: 1) высокочувствительные опухоли: 1) высокочувствительные опухоли: – – лимфома; – – лимфома; – – хориокарцинома; 2. Лучевой метод Показания к применению ЛТ при агрессивных гемангиомах в послеоперационном пе- риоде: заполнение полиметилметакрилатом менее 80 % от общего объема гемангиомы, а также наличие остаточного паравертебрального компонента новоо- бразования [15]. Хордома и хондросаркома – радиорезистентные опухоли: для достижения значимого клинического эф- фекта при адъювантной ЛТ в случаях неполного или внутриопухолевого удаления новообразования необ- ходима доза не менее 60–65 Гр [53]. Анализ результатов лечения 863 больных с хондросаркомой и 715 – с хонд­ ромой, получавших протонную или конвенциальную ЛТ в дозе от 20 до 80 Гр с периодом наблюдения 15 мес, показал, что увеличение дозы облучения и протонная терапия коррелируют с улучшением выживаемости пациентов [54]. В лечении остеосаркомы роль ЛТ окончательно не определена, так как остеосаркома не считается опу- холью с высокой радиочувствительностью [45]. Напро- тив, саркома Юинга – радиочувствительна, и некото- рые авторы отмечают хорошие показатели локального Радиочувствительность во многом определяет так- тику лечения ЗНО позвонков. По своей чувствитель- ности к конвенциальной ЛТ злокачественные опухоли подразделяются на 3 группы: 1) высокочувствительные (миелома и лимфома); 100 2’ 2022 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Russian Journal of Neurosurgery Обзор литературы контроля (у 84 % пациентов) при использовании ра- диохирургии как  единственного метода ее лечения [55]. контроля (у 84 % пациентов) при использовании ра- диохирургии как  единственного метода ее лечения [55]. Наиболее распространены схемы из комбинации ан- трациклинового антибиотика (доксорубицин, адриами- цин) и цитостатика (цисплатин), а также комбинации указанных препаратов и антиметаболита (метотрексат) [58]. Для саркомы Юинга ХТ также служит основным методом лечения и включает набор препаратов из док- сорубицина, винкристина и циклофосфамида, комби- нируемых с этопозидом и ифосфамидом [58]. Л И Т Е Р А Т У Р А / r e f e r e n c e s Surgery 2015;12(3):72–82. (In Russ.)]. DOI: DOI: 10.14531/ss2015.3.72-82. 1. Dreghorn C.R., Newman R.J., Hardy G.J., Dickson R.A. Primary tumours of the axial skeleton. Experience of the Leeds Regional Bone Tumour Registry. Spine (Phila Pa 1976) 1990;15(2):137–40. DOI: 10.1097/ 00007632-­199002000-00018. 6. Валиев А.К., Соколовский А.В., ­Неред А.С., Мусаев Э.Р. Малоинвазив- ные хирургические технологии при по- ражениях позвоночника в онкогемато- логии. Клиническая онкогематология 2013;6(2):177–94. [Valiyev A.K., Sokolovskiy A.V., Nered A.S., Musaev E.R. Minimally invasive surgical techniques in hematological malignancies with spinal involvement. Klinicheskaya onkogematologiya = Clinical onco­ hematology 2013;6(2):177–94. (In Russ.)]. 10. Disch A.C., Kleber C., Redemann D. et al. Current surgical strategies for treating spinal tumors: results of a questionnaire survey among members of the German Spine Society (DWG). Eur J Surg Oncol 2020;46(1):89–94. DOI: 10.1016/j.ejso.2019.08.019. 2. Ciftdemir M., Kaya M., Selcuk E., Yalniz E. Tumors of the spine World J Orthop 2016;7(2):109–16. DOI: 10.5312/wjo.v7.i2.109. 11. Enneking W.E., Spanier S.S., Goodman M.A. A System for the surgical staging of musculoskeletal sarcoma. Clin Orthop Relat Res 1980;(153):106–20. / j 3. Заборовский Н.С., Пташников Д.А., Топузов Э.Э и др. Эпидемиология опу- холей позвоночника у пациентов, по- лучивших специализированную орто- педическую помощь. Травматология и ортопедия России 2019;25(1):104–12. [Zaborovsky N.S., Ptashnikov D.A., Topuzov E.E. et al. Spine tumor epidemiology in patients who underwent orthopaedic surgery. Travmatologiya i ortopediya Rossii = Traumatology and orthopedics of Russia 2019;25(1):104–12. (In Russ.)]. DOI: 10.21823/2311-2905- 2019-25-1-104-112. 7. Мусаев Э.Р. Первичные опухоли по- звоночника: обзор литературы. Пра- ктическая онкология 2010;11(1):19–24. [Musayev E.R. Primary spine tumors: a comprehensive review. Prakticheskaya onkologiya = Practical oncology 2010;11(1):19–24 (In Russ.)]. 12. Tomasian A., Wallace A.N., Jennings J.W. Benign spine lesions: advancesin tech­ niques for minimally invasive percuta­ neous treatment. Am J Neuroradiol 2017;38(5):852–61. DOI: 10 3174/ajnr A5084 ( ) DOI: 10.3174/ajnr.A5084 / j 13. Wang B., Meng N., Zhuang Н. et al. The role of radiotherapy and surgery in the management of aggressive vertebral hemangioma: a retrospective study of 20 patients. Med Sci Monit 2018;24:6840–50. DOI: 10.12659/ MSM.910439. 8. Cho W., Chang U.-K. Survival and recurrence rate after treatment for primary spinal sarcomas. J Korean Neurosurg Soc 2013;53(4):228–34. DOI: 10.3340/jkns.2013.53.4.228. 4. Galgano M.A., Goulart C.R., Iwenofu H. et al. Osteoblastomas of the spine: a comprehensive review. Neurosurg Focus 2016;41(2):E4. DOI: 10.3171/2016.5.FOCUS16122. 9. Гуща А.О., Коновалов Н.А., Арес­ тов С.О и др. Тактика и результаты хи- рургического лечения пациентов с пер­ вичными опухолями позвоночника. Хирургия позвоночника 2015;12(3):72– 82 [Gushcha A.O., Konovalov N.A., Arestov S.O. et al. ЗАКЛЮЧЕНИЕ Таким образом, важным вектором развития спиналь- ной онкологии стала минимализация операционной травмы за счет применения как современных хирургиче- ских технологий, так и адъювантных методов с учетом радио- и химиочувствительности различных новообра- зований. Научно-технический прогресс значительно расширил возможности лучевой терапии, как пример: внедряются конформные методы облучения с возмож- ностью концентрации дозы облучения в ограниченной зоне, позволяющей преодолеть проблемы радиорези- стентности некоторых новообразований. Комбинация возможностей радиохирургии и малоинвазивной деком- прессии позвоночного канала привела к созданию ново- го направления в лечении метастатической эпидураль- ной компрессии – сепарационной хирургии. Большое количество публикаций посвящено воз- можностям стереотаксической ЛТ, и особенно радио- хирургии, в комплексе комбинированного лечения ЗНО, в том числе и радиорезистентных. Техническая возможность фокусирования большей части дозы об- лучения на  опухоли с  минимальным воздействием на такие радиочувствительные структуры, как спинной мозг, привела к появлению методики сепарационной хирургии. При эпидуральной компрессии опухолью в процессе операции достаточно создать минимальный зазор между патологической тканью и твердой мозго- НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Russian Journal of Neurosurgery Обзор литературы и не дает 100 % гарантии, что опухоль не будет реци- дивировать. Рациональное уменьшение радикальности удаления новообразования за счет комбинации ХЛ и ЛТ (в том числе и современных конформных мето- дик), а в ряде случаев и ХТ, также становится трендом спинальной онкологии. Принятие решения о внутри- опухолевом удалении новообразования значительно расширяет возможности минимализации операцион- ной травмы за счет использования тубулярных систем (уменьшение операционного доступа), эндоскопи­ ческой техники (подход к передним отделам тел по- звонков), интервенционного удаления и радиочастот- ной абляции опухоли. вой оболочкой (около 2–3 мм), которого достаточно для предотвращения лучевого поражения невральных структур при адъювантном облучении. Проблемы: не- широкая доступность радиохирургии для большинст- ва пациентов, даже за рубежом, вследствие дороговиз- ны метода и сложностей, связанных с необходимостью планирования сеанса облучения. ОБСУЖДЕНИЕ Вмешательство дает большое число послеоперационных осложнений, сопровожда- ется большой интраоперационной кровопотерей Остеосаркома и  саркома Юинга чувствительны к ХТ, обязательному компоненту комбинированного лечения [58]. Неоадъювантная ХТ, дополняемая хирур- гическим вмешательством, признана стандартом ле- чения остеосаркомы с поражением позвонков [7, 45]. 101 2’ 2022 НЕЙРОХИРУРГИЯ Том 24 Volume 24 Varga P.P., Szoverfi Z., Fisher C.G. et al. Surgical treatment of sacral chordoma: prognostic variables for local recurrence and overall survival. Eur Spine J 2015;24(5):1092–101. DOI: 10.1007/s00586-014-3728-6. 20. Li G., Fu D., Chen K. et al. Surgical strategy for the management of sacral giant cell tumors: a 32-case series. Spine J 2012;12(6):484–91. DOI: 10.1016/j.spinee.2012.06.014. 33. Bilsky M., Smith M. Surgical approach to epidural spinal cord compression. Hematol Oncol Clin North Am 2006;20(6):1307–17. DOI: 10.1016/j.hoc.2006.09.009. 45. Dekutoski M.B., Clarke M.J., Rose P. et al. Osteosarcoma of the spine: prognostic variables for local recurrence and overall survival, a multicenter ambispective study. J Neurosurg Spine 2016;25:59–68. DOI: 10.3171/2015.11.SPINE15870. 21. Martin С., McCarthy E.F. 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(In Russ.)]. 50. Rades D., Сonde-Moreno A.J., Cacicedo J. et al. Radiation therapy alone provides excellent outcomes for spinal cord compression from vertebral lymphoma. Anticancer Res 2016;36:3081–3. 54. Palm R.F., Oliver D.E., Yang G.Q. et al. The role of dose escalation and proton therapy in perioperative or definitive treatment of chondrosarcoma and chordoma: an analysis of the National Cancer Data Base. Cancer 2019;125(4):642–51. DOI: 10.1002/cncr.31958. 51. Patchell R.A., Tibbs P.A., Regine W.F et al. Direct decompressive surgical resection in the treatment of spinal cord compression caused by metastatic cancer: a randomised trial. Lancet 2005;366(9486):643–8. DOI: 10.1016/S0140-6736(05)66954-1. 55. Chang U.K., Lee D.H., Kim M.S. Stereotactic radiosurgery for primary malignant spinal tumors. Neurol Res 2014;36(6):597–606. DOI: 10.1179/1743132814Y.000000038 59. Makatsoris T., Kalofonos H.P. The role of chemotherapy in the treatment of bone metastases. In: Bone Metastases. Cancer Metastasis – Biology and Treatment. Eds.: D. Kardamakis, V. Vassiliou, E. Chow. Dordrecht: Springer Netherlands, 2009. Pp. 287–98. DOI: https://doi. org/10.1007/978-1-4020-9819-2_14. 52. Lee J.-H., Lee S.H. Selecting the appropriate radiation therapy technique for malignant spinal cord compression syndrome. Front Oncol 2019;9:65. DOI 10 3389/f 2019 00065 Вклад авторов Вклад авторов Д.Е. Закондырин: литературный обзор, написание статьи; А.А. Гринь: разработка дизайна исследования. Authors’ contributions D.E. Zakondyrin: literature review, article writing; A.A. Grin: research design of the study. ORCID авторов / ORCID of authors Д.Е. Закондырин / D.E. Zakondyrin: https://orcid.org/0000‑0002‑0925‑415X А.А. Гринь / A.A. Grin: https://orcid.org/0000-0003-3515-8329 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Conflict of interest. The authors declare no conflict of interest. Финансирование. Исследование проведено без спонсорской поддержки. Financing. The study was performed without external funding. Вклад авторов Д.Е. Закондырин: литературный обзор, написание статьи; А.А. Гринь: разработка дизайна исследования. Authors’ contributions D.E. Zakondyrin: literature review, article writing; A.A. Grin: research design of the study. D.E. Zakondyrin: literature review, article writing; D.E. Zakondyrin: literature review, article writing; A.A. Grin: research design of the study. ORCID авторов / ORCID of authors Д.Е. Закондырин / D.E. Zakondyrin: https://orcid.org/0000‑0002‑0925‑415X А.А. Гринь / A.A. Grin: https://orcid.org/0000-0003-3515-8329 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Conflict of interest. The authors declare no conflict of interest. Финансирование. Исследование проведено без спонсорской поддержки. h d f d h l f d Финансирование. Исследование проведено без спонсорской поддержки. Финансирование. Исследование проведено без спонсорской поддержки. Financing. The study was performed without external funding. Financing. The study was performed without external funding. Статья поступила: 08.11.2021. Принята к публикации: 01.02.2022. Article submitted: 08.11.2021. Accepted for publication: 01.02.2022. 104
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https://zenodo.org/records/5576547/files/State_Capture_Deconstructed_WEB.pdf
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State Capture Deconstructed: Risk Measurement in Vulnerable Economic Sectors in Europe
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Risk Measurement in Vulnerable Economic Sectors in Europe State Capture Deconstructed Risk Measurement in Vulnerable Economic Sectors in Europe State Capture Deconstructed STATE CAPTURE DECONSTRUCTED Risk Measurement in Vulnerable Economic Sectors in Europe The current policy environment is particularly favourably disposed towards strengthening the European Union’s response to state capture vulnerabilities. In 2020 the EU introduced a range of new initiatives, including the Eu­ ropean Rule of Law Mechanism, the European Democracy Action Plan, and a ew Security Strategy with a strong focus on anti-corruption. The report complements previous CSD efforts to understand and monitor state capture vulnerabilities and provides a useful practical framework for risk assessment, which could guide EU policy and law enforcement efforts. It presents the findings from the pilot implementation of an innovative methodology for assessing state capture and corruption at the economic sector level based on red-flag indicators and big data. The analysis covers three economic sectors (construction, wholesale of fuels and wholesale of pharmaceuticals) in four EU countries (Bulgaria, Italy, Romania and Spain). Dr. Todor Galev, Director of Research, Center for the Study of Democracy Dr. Alexander Gerganov, Senior Analyst, Center for the Study of Democracy; Assistant Professor, Institute of Philosophy and Sociology, Bulgarian Academy of Science Boyko Todorov, Senior Fellow, Center for the Study of Democracy Authors: Authors: Dr. Todor Galev, Director of Research, Center for the Study of Democracy Dr. Alexander Gerganov, Senior Analyst, Center for the Study of Democracy; Assistant Professor, Institute of Philosophy and Sociology, Bulgarian Academy of Science Boyko Todorov, Senior Fellow, Center for the Study of Democracy Dr. Todor Galev, Director of Research, Center for the Study of Democracy Dr. Alexander Gerganov, Senior Analyst, Center for the Study of Democracy; Assistant Professor, Institute of Philosophy and Sociology, Bulgarian Academy of Science Boyko Todorov, Senior Fellow, Center for the Study of Democracy Dr. Todor Galev, Director of Research, Center for the Study of Democracy Dr. Alexander Gerganov, Senior Analyst, Center for the Study of Democracy; Assistant Professor, Institute of Philosophy and Sociology, Bulgarian Academy of Science Boyko Todorov, Senior Fellow, Center for the Study of Democracy Editorial Board: Dr. Ognian Shentov Ruslan Stefanov Dr. Alexander Stoyanov Editorial Board: Dr. Ognian Shentov Ruslan Stefanov Dr. Alexander Stoyanov State Capture Estimation and Monitoring of Anti-Corruption Policies at the Sectoral Level /SceMaps/ This publication was funded by the European Union’s Internal Security Fund – Police. The content of this publication represents the views of the authors only and is their sole responsibility. The European Commis­ sion does not accept any responsibility for use that may be made of the information it contains. CONTENTS EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 From accidental to systemic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Sharpening the tool: zooming in on sectoral captures . . . . . . . . . . . . . . . . 10 STATE CAPTURE AS A GOVERNANCE THREAT . . . . . . . . . . . . . . . . . . . . . 13 From accidental to systemic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Lacking policy integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 FACTORS FACILITATING BUSINESS CAPTURE IN BULGARIA, ITALY, ROMANIA AND SPAIN . . . . . . . . . . . . . . . . . . . . . . . . . 27 State Capture and Corruption Risks in Public Procurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Institutional Enablers of State Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Anti-corruption policy implementation on institutional level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACKNOWLEDGEMENTS: The extensive research informing this report is the product of the collective effort of an international team of experts: Alexander Stoyanov, Senior Fellow at the Center for the Study of Democracy, Bulgaria, David Cabo, Co-Direc­ tor and Miguel Gavilanes, Journalist at CIVIO, Spain, Sorin Ionita, President, Laura Stefan, Executive Director and Rule of Law and Anti-Corruption Co­ ordinator, and Septimius Parvu, Expert, Good Governance at Expert Forum, Romania, and Daniela Andreata, then Research Fellow and member of the e-Crimes Research Group at University of Trento, Italy. CSD would particularly like to thank the management and employees of the nine public institutions involved in the assessment of their respective an­ ti-corruption policies, which is at the core of the current analysis. Our thanks extend to: the National Revenue Agency and the Directorate for National Construction Control (Bulgaria), the Chamber of Commerce of Trento and the Emilia-Romagna Regional Government (Italy), the Romanian Competi­ tion Council, the National Integrity Agency and Sinaia Municipality (Roma­ nia), the Valencian Anti-Fraud Agency and the Government of the Region of Murcia (Spain). The policy analysis in the report has greatly benefitted from discussions with Ramona Strugariu, Member of the European Parliament, Renew Europe Group, Committee on Civil Liberties, Justice and Home Affairs (LIBE) and Anti-Corruption Intergroup, as well as Dr. Anitta M. Hipper, Team Leader for Anti-Corruption, Directorate-General Migration and Home Affairs for Euro­ pean Commission, who took part in the international conference on state cap­ ture and corruption assessment at the sectoral level held in April 2021. CSD has worked closely with Martin Tsanov, CEO of BizPortal, to streamline and test the SceMaps interactive web platform. The publication would not have been possible without the technical editing of CSD’s associate researcher Matthew Dumigan and the meticulous proof­ reading of CSD’s publishing coordinator Galina Sapundzhieva. LIST OF FIGURES Figure 1. The building blocks of state capture . . . . . . . . . . . . . . . . . . . . . . . . . 14 Figure 2. SceMaps interactive web platform: Red flags . . . . . . . . . . . . . . . . . 15 Figure 3. State capture assessment on sectoral level – concept and research instruments . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Figure 4. Funnel-like approach to state capture diagnostics . . . . . . . . . . . . . 18 Figure 5. Measured concepts and indicators . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Figure 6. All sectors captured at over 50% . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Figure 7. General monopolisation pressure in Bulgaria is highest . . . . . . . . 30 Figure 8. State capture through anti-monopoly laws is highest in Spain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Figure 9. Construction is the most vulnerable sector to illegitimate activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Figure 10. Total value of public procurement in selected sectors 2010 – 2019 (EUR billion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Figure 11. Buyer concentration index (2011 – 2019) . . . . . . . . . . . . . . . . . . . . . . 34 Figure 12. CONTENTS . . . . 38 WHAT’S NEXT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 EXCUTIVE SUMMARY (IN BULGARIAN LANGUAGE) . . . . . . . . . . . . . . . 43 EXCUTIVE SUMMARY (IN ITALIAN LANGUAGE) . . . . . . . . . . . . . . . . . . . 47 EXCUTIVE SUMMARY (IN ROMANIAN LANGUAGE) . . . . . . . . . . . . . . . . 51 EXCUTIVE SUMMARY (IN SPANISH LANGUAGE) . . . . . . . . . . . . . . . . . . . 55 ¹ Stoyanov, A., Gerganov, A., and Yalamov, T., State Capture Assessment Diagnostics, Sofia: Center for the Study of Democracy, 2019. EXECUTIVE SUMMARY The notion of state capture has long been used to explicate the practice of pri­ vate business subverting government policy and decision-making in its fa­ vour. It has typically referred to a series of individual corrupt transactions at the senior government level. Developments across many European coun­ tries and further afield, however, indicate that the practice has gone beyond a simple deviation in the functioning of a given public institution and increas­ ingly reflects a stable pattern of institutional behaviour, which is resistant to the application of standard, generalised anticorruption policies. This report presents the results from the application of an innovative analytical tool, the State Capture Assessment Diagnostics (SCAD) on sectoral level, which provides policy relevant findings about state capture, characterising it as a systemic failure of public governance. LIST OF FIGURES Procurement exposure per employee ratio (2011 – 2019) . . . . . . . . 35 Figure 13. Procurement exposure to revenue ratio (2011 – 2019) . . . . . . . . . . 36 Figure 14. Institutional environment in Bulgaria is most vulnerable to state capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 From accidental to systemic The new reality of state capture as a de facto, wholesale privatisation of gov­ ernment decisions and the monopolisation of entire economic sectors requires the development of new tools of analysis that will inform a new generation of good governance policies. SCAD reveals the exploitation of the power of government for private benefit in a systematic and permanent manner, involving various forms of corruption and illegitimate activities. 1 The build­ ing blocks of state capture include a variety of tools, such as power over the enforcement of regulations, privileged access to public resources, asymmetric control over the media and the financial sector, and influence over domes­ tic and foreign policy. SCAD exposes the mechanisms through which the drafting, adoption and enforcement of government rules and regulations is warped in favour of a small number of captors (actors with privileged status enjoying undue advantage in economic and/or political terms). SCAD reveals how state capture is enabled by weak governance mechanisms by highlighting four dimensions of capture (business, institutional, political and black market) and two types of enablers, which refer to the institutional and environmental characteristics that affect the system of governance, thus allowing or facilitating state capture (see the figure below). 10 State Capture Deconstructed State capture model Source: Stoyanov, A., Gerganov, A., and Yalamov, T., State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. Institutional enablers Environmental enablers Corruption and AC inefectiveness Captured media State capture dimensions Business capture Institutional capture Black market capture Political capture Monopolisation Privileged access to procurement Lobbyist laws Privileged status (control and sanctions are applied selectively) Concentration of direct subsidies Inefectiveness of anti-monopoly laws Lack of transparency Lack of impartiality Private interest bias Administrative corruption Judiciary corruption State capture model Monopolisation Lack of impartiality Lack of transparency Private interest bias Institutional capture Concentration of direct subsidies Judiciary corruption Inefectiveness of anti-monopoly laws Source: Stoyanov, A., Gerganov, A., and Yalamov, T., State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. ² https://analytics.scemaps.eu. ³ Initially developed and implemented as a separate tool, it was integrated in the methodo­ logical framework for assessing state capture on sectoral level. See: Stoyanov A. et al., Moni­ toring Anti-Corruption in Europe. Bridging Policy Evaluation and Corruption Measurement, Sofia: Center for the Study of Democracy, 2015. Sharpening the tool: zooming in on sectoral captures Built upon a decade-long analysis of corruption and state capture across sev­ eral European countries, SCAD measures the results and effects of busi­ ness capture, as well as the institutional and environmental enablers at the national level. In addition, the tool allows for a closer examination to be made of how individual public institutions, economic sectors and business organisations are affected, thus increasing the efficacy of the respective in­ stitutional and sectoral policies. This report presents the findings of such an examination by assessing on a sectoral level the key dimension of business state capture and the institutional characteristics that enable it in several economic sectors (construction and the wholesale of fuels and pharmaceu­ ticals) within four European countries (Bulgaria, Italy, Romania and Spain). The new sectoral level methodology adds two important elements. First, tak­ ing into account the importance of privileged access to procurement as part of the business capture dimension, the report provides an analysis of state cap­ ture risks and corruption-related behaviour in public procurement based on integrated big-data. Second, the measurement of institutional enablers through index-based expert assessments was complemented by a methodol­ ogy for monitoring the implementation of anti-corruption policies in key regulatory and control institutions, identified through expert assessments. Although differing in nature, when collated, the findings of these two optics in relation to the same phenomenon allow the identification of risks and vul­ 11 Executive Summary Executive Summary nerabilities that might not be visible to a single analytical tool. Moreover, the results offer the possibility for conducting robust monitoring and analysis, as well as for advising management decisions within a particular public organ­ isation or company. The findings were produced as a result of the application of three mutual­ ly-complementary methodologies and their respective research instruments (see the figure below): • State Capture Assessment Diagnostics on Economic Sector Level (SCAD-ESL) assesses state capture risks and vulnerabilities at the sec­ toral level using index-based expert assessments. It also focuses the atten­ tion on the ineffectiveness of anticorruption policies, the lack of integrity and impartiality, and private interest bias. Sharpening the tool: zooming in on sectoral captures • Analysis of the risks of state capture and corruption-related behaviour in public procurement through “red flag” indicators based on integrat­ ed data and implemented through a specially designed and developed interactive web platform.2 The platform pioneers a three-dimensional ap­ proach for analysing state capture risks and vulnerabilities on both the side of buyers (contracting authorities) and suppliers (companies), combin­ ing public procurement data, company financial and ownership informa­ tion, and a media alert system, which identifies alleged cases of miscon­ duct related to procurement. The assessment is carried out on the basis of a combination of red flags, each indicating a risk situation that might be the result of corruption or state capture.i • Monitoring Anticorruption Policy Implementation (MACPI)3 identifies vulnerabilities and potential gaps between high corruption risk practices in individual public institutions (identified as key for the regulation of the sectors through SCAD-ESL) and the availability of anticorruption policies addressing these risks. It then evaluates the ease of implementation, actual implementation, and the subsequent enforcement of these policies. While state capture assessment at the national level through the application of SCAD provides valuable and insightful knowledge on vulnerable areas in the entire economy, its sector-specific tools are of greater practical relevance at the level of economic sectors due to their specific characteristics. Moreover, many of the information sources (including the knowledge and know-how of experts used in the MACPI tool), as well as the vulnerabilities and policy gaps, differ across sectors and thus, the sectoral assessment produces more robust and reliable results. The State Capture Assessment Diagnostics demonstrates that state capture vul­ nerabilities are sizable at the national level in Europe and are particularly problematic in certain Eastern European countries. SCAD further uncovers that several sectors, such as pharmaceuticals, fuels and construction, deserve special policy attention as sources of state capture vulnerabilities. The results of the piloting of the SCAD-ESL (sectoral level) and the red-flagging in public procurement (achieved by the analysis of big data) presented in the current 12 State Capture Deconstructed State capture assessment on sectoral level – concept and research instruments Source: CSD, 2021. ⁴ Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019, p. 27. STATE CAPTURE AS A GOVERNANCE THREAT Upon its introduction in the late 1990s, the notion of state capture was sup­ posed to mark an evolution in the understanding of corruption, reflecting the practice of private business subverting government policies and decision making in its favour. Nevertheless, the concept has been viewed as a series of individual corrupt transactions rather than as the systemic change in the na­ ture of a governance regime resulting from sustained pressure from captors (companies or persons with privileged access to government decision-mak­ ing). Today, in many European countries and elsewhere, the practice has evolved beyond a simple deviation in the functioning of a given public insti­ tution and has morphed into a stable pattern of institutional behaviour that is resistant to the application of standard, generalised anticorruption policies. Sharpening the tool: zooming in on sectoral captures Institutional enablers Corruption and AC inefectiveness State capture dimensions Business capture Monopolisation Privileged access to procurement Lobbyist laws Privileged status (control and sanctions are applied selectively) Concentration of direct subsidies Inefectiveness of anti-monopoly laws Lack of transparency Lack of impartiality Private interest bias MACPI SCAD ESL expert assessment Red-fags risk analysis State capture assessment on sectoral level – concept and research instruments Monopolisation Lack of impartiality Inefectiveness of anti-monopoly laws SCAD ESL expert assessment Source: CSD, 2021. study confirm that state capture risks in these three sectors are higher than those at the national level in all four of the countries in question (Bulgaria, Italy, Romania and Spain). Nevertheless, Bulgaria and, partially, Romania, have been revealed as the countries with a higher degree of state capture vul­ nerability on both sectoral and public procurement level. It appears likely that the disruption of competitive market forces and the undoing of democratic checks and balances across European economies during the Covid-19 pan­ demic has further exacerbated state capture vulnerabilities within member states and sectors. The issue of state capture is still not adequately captured in European pol­ icy debates, which appear much more centred on different forms of corrup­ tion, thus neglecting a systematic evaluation of the linkages between them. Supplementing the SCAD model with instruments capable of deciphering media and judiciary capture is required as the next step forward when it comes to responding to the need of the EU’s Rule of Law mechanism for scala­ ble tools to perform an integrated analysis of state capture. In many European countries, oligarchic groups insist on having complete discretion in domestic affairs while claiming the benefits of good governance at the European level. In order to expose this discrepancy – and thus challenge it – the EU needs to ensure its policies are grounded in verifiable evidence concerning the specific mechanisms through which state power is being hijacked for private inter­ ests. This is exactly what the State Capture Assessment Diagnostics provides. From accidental to systemic The new reality of state capture as a de facto wholesale privatisation of govern­ ment decisions and the monopolisation of entire economic sectors requires the development of new tools of analysis which would inform a new gen­ eration of good governance policies. Introduced by the Center for the Study of Democracy (CSD), the State Capture Assessment Diagnostics (SCAD) reveals the exploitation of the power of government for private benefit in a sys­ tematic and permanent manner, involving various forms of corruption and illegitimate activities.4 It exposes the mechanism through which the drafting, adoption and enforcement of government rules and regulations is warped in favour of a small number of captors at the expense of society and business at large. These could be economic actors (e.g., business enterprises or persons who control them), but also institutional actors (e.g., public officials, political parties or groups inside them), or even illegal actors (e.g., black market play­ ers). In reality the lines between these distinctions are often blurred, and a captor could represent a complex network of intertwined actors who mutual­ ly reinforce each other through the privatisation of different state functions or institutions. In this way, they ensure systematic and permanent privileges to the whole network. This underlines the functional and process-wise na­ ture of state capture, which allows captors to gain privileged status in a given economic sector or public institution (e.g., judicial or media capture). The building blocks of state capture include a variety of tools, such as power over the enforcement of regulations, privileged access to public resources, asymmetric control over the media and the financial sector, influence over domestic and foreign policy to name just a few. As a next generation analytical tool, the model used by SCAD reveals the path-dependant nature of state capture as enabled by weak governance mechanisms. This is achieved by highlighting four possible dimensions of capture (business, institutional, political and black market) and two types of 14 State Capture Deconstructed State Capture Deconstructed enablers, which refer to the institutional and environmental characteristics that affect the system of governance, thus allowing or facilitating state cap­ ture (Figure 1). Figure 1. The building blocks of state capture Figure 1. The building blocks of state capture Figure 1. The building blocks of state capture Source: CSD, 2021, based on: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, 2019. ⁵ Stefanov, R., Karaboev, S., and Yalamov, T., Evaluating Governance and Corruption Risk in Bul­ garia, Sofia: Center for the Study of Democracy, 2017; Center for the Study of Democracy, Shadow Power: Assessment of Corruption and Hidden Economy in Southeast Europe, Sofia: Center for the Study of Democracy, 2016; Stoyanov, A., Gegranov, A. and Stefanov, R., State Capture Diagnostics Roadmap, Working Paper, Sofia: Center for the Study of Democracy, 2016; Center for the Study of Democracy, State Capture Unplugged: Countering Administrative and Political Corruption in Bulgaria, Sofia: Center for the Study of Democracy, 2016. ⁶ Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. From accidental to systemic Institutional enablers Environmental enablers Corruption and AC inefectiveness Captured media State capture dimensions Business capture Institutional capture Black market capture Political capture Monopolisation Privileged access to procurement Lobbyist laws Privileged status (control and sanctions are applied selectively) Concentration of direct subsidies Inefectiveness of anti-monopoly laws Lack of transparency Lack of impartiality Private interest bias Administrative corruption Judiciary corruption Lack of impartiality Business capture Institutional capture Judiciary corruption Inefectiveness of anti-monopoly laws Source: CSD, 2021, based on: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, 2019. While state capture is a hidden phenomenon (most of the evidence for its presence is anecdotal) it nonetheless leaves behind tangible public traces. Furthermore, since it needs to affect public policy, it is bound to leave visi­ ble effects that can be subsequently discovered by suitable pattern-finding methods. It is these public traces from the existence of state capture activity that SCAD identifies, which can then be used to inform policy adjustments in relation to the improvement of the integrity of government. Built upon a decade-long analysis of corruption and state capture across several Europe­ an countries5, SCAD measures the results and effects of business capture and institutional and environmental enablers at national level.6 Thus, it is an instrument that focuses the attention of policymakers, researchers and practitioners on certain vulnerabilities existent within national level public institutions, which enable captors to successfully pursue their objectives. 15 State Capture as a Governance Threat State Capture as a Governance Threat State Capture as a Governance Threat State Capture as a Governance Threat • replicable and scalable across other economic sectors and countries; ⁷ For detailed description see Gerganov A., Mineva D., and Galev T., State Capture Assessment on Sectoral Level: Methodological Toolkit. Sofia: Center for the Study of Democracy, 2021. ⁸ https://analytics.scemaps.eu. g 10 Initially developed and implemented as a separate tool, it was integrated in the method­ ological framework for assessing state capture on sectoral level. For information on it see: Stoyanov et al, Monitoring Anti-Corruption in Europe. Bridging Policy Evaluation and Corruption Measurement, Center for the Study of Democracy, 2015. Sharpening the tool: zooming in on sectoral captures The application of SCAD at the national level provides evidence for the de­ sign of strategic policies designed to counteract state capture. Additionally, the tool allows for a closer examination to be made of how individual pub­ lic institutions, economic sectors or business organisations are affected, thus supporting the improvement of the respective institutional and sectoral poli­ cies. This report presents the findings of such an examination by assessing the key dimension of “business state capture” and the institutional char­ acteristics that enable it on sectoral level (i.e., the institutional enablers). Furthermore, the new sectoral-level methodology adds two important ele­ ments. First, taking into account the importance of privileged access to pro­ curement as part of the business capture dimension, an original methodol­ ogy was developed for the analysis of state capture risks and corruption behaviour in public procurement based on integrated big-data. Second, the measurement of institutional enablers through index-based expert assess­ ments was complemented by a methodology for monitoring the implemen­ tation of anti-corruption policies in key regulatory and control institutions, identified through the expert assessment. Although differing in nature, when collated the findings of these two optics in relation to the same phenomenon enable the identification of risks and vulnerabilities that may not be visible to a single analytical tool. Moreover, the results offer the possibility for con­ ducting robust monitoring and analysis, as well as for advising management decisions within a particular public organisation or company. Figure 2. SceMaps interactive web platform: Red flags The new methodology is a practical instrument, which empowers policymak­ ers and officials, think-tanks, watchdog organisations, investigative journal­ ists, and researchers to monitor state capture pressure at the sectoral level. The entire methodology and each of its elements is designed to be: • replicable and scalable across other economic sectors and countries; g p pl g Source: https://analytics.scemaps.eu Source: https://analytics.scemaps.eu The new methodology is a practical instrument, which empowers policymak­ ers and officials, think-tanks, watchdog organisations, investigative journal­ ists, and researchers to monitor state capture pressure at the sectoral level. t p a a y i e ap eu ⁹ The methodology of the risk analysis in public procurement, including the computation of filters, rankings and red flags, is derived theoretically from the list of indicators, indicating state capture and corruption pressure, and used as basis of the SCAD-ESL assessment, e.g. tenders are won by very few ultimate owners or tenders are often won by very new, un­ known companies, etc. For a detailed description, see: State Capture Assessment on Sectoral Level: Methodological Toolkit ⁷ For detailed description see Gerganov A., Mineva D., and Galev T., State Capture Assessment on Sectoral Level: Methodological Toolkit. Sofia: Center for the Study of Democracy, 2021. ⁸ https://analytics.scemaps.eu. ⁹ The methodology of the risk analysis in public procurement, including the computation of filters, rankings and red flags, is derived theoretically from the list of indicators, indicating state capture and corruption pressure, and used as basis of the SCAD-ESL assessment, e.g. tenders are won by very few ultimate owners or tenders are often won by very new, un­ known companies, etc. For a detailed description, see: State Capture Assessment on Sectoral Level: Methodological Toolkit. 10 Initially developed and implemented as a separate tool, it was integrated in the method­ ological framework for assessing state capture on sectoral level. For information on it see: Stoyanov et al, Monitoring Anti-Corruption in Europe. Bridging Policy Evaluation and Corruption Measurement, Center for the Study of Democracy, 2015. 11 Kaufmann, D., and Vicente, P., Legal corruption, Economics & Politics, 23(2), 2011, pp. 195-219. Sharpening the tool: zooming in on sectoral captures 17 State Capture as a Governance Threat State Capture as a Governance Threat While state capture assessment at the national level through the application of SCAD provides valuable knowledge (unobtainable through other means) on vulnerable areas across the entire economy and could focus and priori­ tise the further assessment of state capture, quantitative analysis is of great­ er practical relevance at the level of economic sectors due to their specific characteristics. Moreover, because many of the information sources (incl. the knowledge and know-how of experts) and the vulnerabilities and policy gaps differ across sectors, the sectoral assessments produce more robust and reli­ able results. Figure 3. State capture assessment on sectoral level – concept and research instruments Source: CSD, 2021. Institutional enablers Corruption and AC inefectiveness State capture dimensions Business capture Monopolisation Privileged access to procurement Lobbyist laws Privileged status (control and sanctions are applied selectively) Concentration of direct subsidies Inefectiveness of anti-monopoly laws Lack of transparency Lack of impartiality Private interest bias MACPI SCAD ESL expert assessment Red-fags risk analysis Figure 3. State capture assessment on sectoral level – concept and research instruments Source: CSD, 2021. Institutional enablers Corruption and AC inefectiveness State capture dimensions Business capture Monopolisation Privileged access to procurement Lobbyist laws Privileged status (control and sanctions are applied selectively) Concentration of direct subsidies Inefectiveness of anti-monopoly laws Lack of transparency Lack of impartiality Private interest bias MACPI SCAD ESL expert assessment Red-fags risk analysis Figure 3. State capture assessment on sectoral level – concept and research instruments Lack of impartiality Inefectiveness of anti-monopoly laws SCAD ESL expert assessment Source: CSD, 2021. While state capture assessment at the national level through the application of SCAD provides valuable knowledge (unobtainable through other means) on vulnerable areas across the entire economy and could focus and priori­ tise the further assessment of state capture, quantitative analysis is of great­ er practical relevance at the level of economic sectors due to their specific characteristics. Moreover, because many of the information sources (incl. the knowledge and know-how of experts) and the vulnerabilities and policy gaps differ across sectors, the sectoral assessments produce more robust and reli­ able results. Sharpening the tool: zooming in on sectoral captures The entire methodology and each of its elements is designed to be: • replicable and scalable across other economic sectors and countries; 16 State Capture Deconstructed • applicable to both designing preventive measures and driving investiga­ tions of particular malpractices; • valuable and usable for a broad group of stakeholders, including policy­ makers, civil society, watchdog organisations, investigative journalists, law enforcement officials, and researchers. This monitoring framework combines three mutually-complementary meth­ odologies and their respective research instruments7: • State Capture Assessment Diagnostics on Economic Sector Level (SCAD-ESL) assesses state capture risks and vulnerabilities at the sectoral level using index-based expert assessments. It also focuses the attention on the regulatory and control institutions that demonstrate risky profiles according to the levels of the institutional enablers (i.e. ineffectiveness of anticorruption policies, lack of integrity, lack of impartiality and private interest bias). • Analysis of the risks of state capture and corruption behaviour in public procurement through “red flag” indicators. These are based on integrated data and implemented through a specially designed interactive web plat­ form.8 The platform pioneers a three-dimensional approach for analysing state capture risks and vulnerabilities on both the side of buyers (contract­ ing authorities) and suppliers (companies), combining public procurement data, company financial and ownership information, and a media alert system, which identifies alleged cases of misconduct related to procure­ ment. The assessment is carried out on the basis of a combination of red flags, each indicating a risk situation that may be the result of corruption or state capture. A single red flag is not necessarily a sign of suspicious be­ haviour, however, the accumulation of red flags for a particular company, contracting authority, sector or country raises serious concern regarding existing problems in the procurement processes on the micro (single com­ pany or contracting entity), meso (sector), or macro (country) level.9 • Monitoring Anticorruption Policy Implementation (MACPI)10 detects vulnerabilities and potential gaps between high corruption risk practices in individual public institutions (identified as key for the regulation of the sectors through SCAD-ESL) and the availability of anticorruption policies addressing these risks. It then evaluates the implementability, implemen­ tation, and enforcement of these policies. Figure 4. Funnel-like approach to state capture diagnostics Figure 4. Funnel-like approach to state capture diagnostics Figure 4. Funnel-like approach to state capture diagnostics Source: CSD, 2021. The approach offers two possibilities for addressing state capture vulnera­ bilities and risks identified on the sectoral level: (i) improving the resilience against state capture pressure of relevant public institutions through internal or sectoral policies on corruption, monopoly, lobbying, conflicts of interests, etc. and (ii) investigations of specific cases by responsible authorities or inde­ pendent experts (incl. journalists) exposing unlawful and illegitimate activi­ ties of particular captor networks.12 12 For more details see: Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021, p. 11. 13 Center for the Study of Democracy, Examining the links between organised crime and corruption, Sofia: Center for the Study of Democracy, 2010. y y p 13 Center for the Study of Democracy, Examining the links between organised crime and corruption, Sofia: Center for the Study of Democracy, 2010. 12 For more details see: Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021, p. 11. Sharpening the tool: zooming in on sectoral captures While state capture assessment at the national level through the application of SCAD provides valuable knowledge (unobtainable through other means) on vulnerable areas across the entire economy and could focus and priori­ tise the further assessment of state capture, quantitative analysis is of great­ er practical relevance at the level of economic sectors due to their specific characteristics. Moreover, because many of the information sources (incl. the knowledge and know-how of experts) and the vulnerabilities and policy gaps differ across sectors, the sectoral assessments produce more robust and reli­ able results. The funnel-like approach, implemented in the current analysis, enables the quantitative assessment of key components of state capture on the sectoral level, offering a possibility for combining them with national-level findings about the environmental characteristics of the studied domain (i.e. environ­ mental enablers). This approach aids the design of preventive policies and measures on the national and sectoral levels all the way down to the level of a single public organisation or company. It also helps law enforcement au­ thorities by allowing them to trace the systematic problems seen on a macro level to a small number of practical cases of misconduct on a sectoral or insti­ tutional level that could be further investigated and sanctioned. The findings of this method could also prompt researchers and investigative journalists to further unravel illegitimate schemes, such as cases of legal corruption or illegitimate but legal practises, which hamper competition and act contrary to the public good11. 18 State Capture Deconstructed Figure 4. Funnel-like approach to state capture diagnostics Estimates at the national level ●Capture of judiciary, media, key public organisations ●Monopolisation of economic sectors Vulnerabilities at the sectoral level ●Business state capture indicators ●Big data based red-flags ●Assessment of particular institution Ad-hoc investigations ●Prevention ●Sanctions Design and implementation of general policies: ● Rule of law ● Media freedom ● Justice system Design and implementation of specific policies: ● Sectoral or domain policies (e.g. on conflicts of interests) ● Institutional internal policies and measures Investigations by: ● law enforcement authorities ● regulatory and control institutions ibid. 17 Promoted through the European Network on the Administrative Approach, this is a method whereby local authorities, in collaboration with law enforcement authorities and civil socie­ ty, use administrative tools such as procedures for obtaining permits, tenders and subsidies to prevent organised crime infiltration of legal businesses and administrative infrastruc­ ture. The approach could add an important local dimension to the activities against state capture, as it often develops and manifests itself in local institutions (e.g. privileged access to public procurement or local subsidies). 14 European Commission, Fight against organised crime: New 5-year strategy for boosting coopera­ tion across the EU and for better use of digital tools for investigations. Press release, Brussels, April 14, 2021. 15 European Commission, Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions on the EU Strategy to Tackle Organised Crime 2021-2025. COM(2021) 170 final, European Commission, Brussels, 2021, p. 20. Policy intersection #1: State capture and organised crime Moreover, as noted above, state capture is used to maximize the profit of criminal groups from both legal and il­ legal activities across different economic sectors. Ultimately, the strategy calls for specific measures and instruments that could strengthen the fight against state capture, provided it is recognised and addressed properly. Among them include support for more effective investigations to disrupt organised crime structures and a renewed focus on high and specific priority crimes (e.g., revising the EU rules against environmental crime, establishing an EU toolbox against counter­ feiting, applying stricter anti-money loundering regulations, and reinforcing law enforcement and the judiciary for international investigations, etc.). Above and beyond this, the wider application of the “Administrative approach” to serious and organised crime as complementary to traditional law enforcement activities is included.17 Environmental crimes, money laundering, counterfeiting of goods, misuse of public funds (incl. through public procurement) are examples of such crimes. The EU’s new 5-year strategy against organised crime outlines that over 60% of criminal networks active within the EU are engaged in corruption and more than 80% of them use legitimate businesses as a front for their activities.14 In partic­ ular, the strategy highlighted that the existing instruments and rules, includ­ ing criminalising both active and passive corruption of public officials, do not cover certain offences such as trading in influence, abuse of power, illic­ it enrichment, misappropriation or other diversion of property by a public of­ ficial.15 Each of these are often elements of state capture, even if not named as such in the strategy. In addition, Member States are required to introduce new legislation protecting whistle-blowers, as well as the creation of safe channels for reporting corrupt practices,16 which could contribute significantly to the fight against state capture while its alleged cases are reported very often initially namely by watch-dogs organisations and investigative journalists. Tackling state capture in its complexity will also strengthen the fight against the infiltration of criminal groups in the economy and society, based on investment of part of their considerable earnings in legal businesses. Moreover, as noted above, state capture is used to maximize the profit of criminal groups from both legal and il­ legal activities across different economic sectors. Ultimately, the strategy calls for specific measures and instruments that could strengthen the fight against state capture, provided it is recognised and addressed properly. Policy intersection #1: State capture and organised crime Policy intersection #1: State capture and organised crime As based on the various forms of corruption that constitute part of the system­ atic and long-term relations between the captor and the captured institutions, state capture processes could also be used by organised crime, as it is corrup­ tion.13 On the one hand, organised crime groups use state capture mechanisms to influence the government institutions in favour of their private gains, irre­ spective of whether they are part of their legal or illegal business. On the oth­ er hand, state capture could also produce specific types of relationships and dependence between public officials and the captors even if the former do not directly support the illegal aspects of the business interests of the latter. Cer­ tain types of organised crime could be considered more prone to engage in state capture as they involve or make use of a complex network of regulatory, control and financial institutions. Moreover, their capture gives enhanced guarantees for expected long-term favourable reaction rather than single corruption act. State Capture as a Governance Threat State Capture as a Governance Threat 19 Environmental crimes, money laundering, counterfeiting of goods, misuse of public funds (incl. through public procurement) are examples of such crimes. The EU’s new 5-year strategy against organised crime outlines that over 60% of criminal networks active within the EU are engaged in corruption and more than 80% of them use legitimate businesses as a front for their activities.14 In partic­ ular, the strategy highlighted that the existing instruments and rules, includ­ ing criminalising both active and passive corruption of public officials, do not cover certain offences such as trading in influence, abuse of power, illic­ it enrichment, misappropriation or other diversion of property by a public of­ ficial.15 Each of these are often elements of state capture, even if not named as such in the strategy. In addition, Member States are required to introduce new legislation protecting whistle-blowers, as well as the creation of safe channels for reporting corrupt practices,16 which could contribute significantly to the fight against state capture while its alleged cases are reported very often initially namely by watch-dogs organisations and investigative journalists. Tackling state capture in its complexity will also strengthen the fight against the infiltration of criminal groups in the economy and society, based on investment of part of their considerable earnings in legal businesses. 16 ibid. 18 European Commission, Communication from the Commission to the European Parliament, the European Council, the Council, the European Economic and Social Committee and the Committee of the Regions on the EU Security Union Strategy, COM/2020/605 final, European Commission, Brussels, 2020.il ibid. 22 Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019, p. 30. 20 For a description of the use of state capture mechanisms by foreign adversary states in Europe, see also: Conley, H. et al, The Kremlin Playbook 2: The Enablers, Center for Strategic and International Studies, Washington D.C., 2019; Stefanov, R. et al., The Kremlin Playbook in Southeast Europe: Economic Influence and Sharp Power, Sofia: Center for the Study of Democra­ cy, 2020. , , 19 The term refers to the use of capital as a foreign policy tool, when opaque financial flows from authoritarian states aim to undermine the rule of law and democratic governance in other countries, irrespective if the capital if formally private or state-owned. Using state cap­ ture tactics, the adversary state power exploits the governance deficits in key markets and institutions of the targeted country. See: Stefanov, R., and Vladimirov, M., Deals in the Dark: Russian Corrosive Capital in Latin America, National Endowment for Democracy, Washington D.C., 2021. Policy intersection #1: State capture and organised crime Among them include support for more effective investigations to disrupt organised crime structures and a renewed focus on high and specific priority crimes (e.g., revising the EU rules against environmental crime, establishing an EU toolbox against counter­ feiting, applying stricter anti-money loundering regulations, and reinforcing law enforcement and the judiciary for international investigations, etc.). Above and beyond this, the wider application of the “Administrative approach” to serious and organised crime as complementary to traditional law enforcement activities is included.17 20 State Capture Deconstructed State Capture Deconstructed Policy intersection #2: State capture and security Policy intersection #2: State capture and security In the latest EU Security Union Strategy, security is seen as a cross-cutting issue that impacts every sphere of life and affects a multitude of policy areas.18 The strategy outlines four pillars: building a future-proof security environment, tack­ ling evolving threats, protecting Europeans from terrorism and organised crime and building a strong security ecosystem. As an abuse of good governance rules in the process of drafting, adoption and enforcement of the rules in favour of pri­ vate interests, state capture could seriously and systematically weaken each of the pillars through distorting the activities of key institutions, including law enforcement and the judiciary. In particular, serving the private gains of specific business, criminal or (foreign or domestic) political interests, it could worsen na­ tional security through bad governance of public policies and spending in areas such as the defence industry, energy security, penetration of authoritarian cor­ rosive capital19 in financial and political system of the country20, prevention and detection of hybrid threats, and increasing the resilience of critical infrastructure. Policy intersection #3: State capture and foreign influence In particular, the complexity of the issues and mechanisms that facilitate these cases, and that are described separately by the existing analysis, fits into the overall analytical framework of state capture and could be tackled more effectively if addressed properly as an integrated phenomenon. In previous years, professional ethics and integrity of public officials have been outlined by both the European Anti-Fraud Office (OLAF) 23 and the Commission Anti-Fraud Strategy24 as a strategic objective and one of the guiding principles and standards for the common fight against fraud across the EU. As an integral part of the institutional characteristics that affect the system of governance al­ lowing or facilitating state capture, professional integrity is a basic component, the importance of which goes above and beyond the issue of fighting fraud.25 In the past few years, various areas of disbursement and redistribution of EU funds (e.g. the Common Agricultural Policy) have raised attention of European and na­ tional policy makers as “fuelling fraud and corruption and the rise of rich busi­ nessmen” 26 across the Member States, and particularly in Central and Eastern Europe. 27 Cases of financial frauds, including with European funds, are reported to be based on exploiting political ties with ruling parties and governments, con­ flicts of interests, lack of transparency and accountability and proper scrutiny both during and after the distribution process.28 Both the EU Anti-Fraud Strategy and the Joint Anti-Fraud Strategy for the European Structural and Investment Funds29 seek to improve fraud detection by introducing big data technologies and new methods for tackling complex offenses and fraud at the national and in­ ternational level. In particular, the complexity of the issues and mechanisms that facilitate these cases, and that are described separately by the existing analysis, fits into the overall analytical framework of state capture and could be tackled more effectively if addressed properly as an integrated phenomenon. 29 European Commission, Joint anti-fraud strategy for shared & indirect management 2020-2025, DG REGIO, DG EMPL, DG MARE, Brussels, 2019. 23 European Anti-Fraud Office, Management Plan 2021, European Commission, Brussels, 2021. 24 European Commission, Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee, the Committee of the Regions and the Court of Auditors on Commission Anti-Fraud Strategy: enhanced action to protect the EU budget. COM(2019) 196 final, European Commission, Brussels, 2019. 25 ibid., p. 18. 26 Dordevic, N., “Fraud, corruption, and misuse of EU agricultural funds a major problem in CEE, say MEPs,” Emerging Europe, February 26, 2021. 27 Sabev, D. et al. Where does the EU money go? An analysis of the implementation of CAP funds in Bulgaria, the Czech Republic, Hungary, Slovakia and Romania, A Report commissioned by the Greens/EFA group in the European Parliament, Brussels, 2021. 28 Organisation for Economic Cooperation and Development, Fraud and corruption in European Structural and Investment Funds. A spotlight on common schemes and preventive actions, OECD, 2019. 29 European Commission, Joint anti-fraud strategy for shared & indirect management 2020-2025, DG REGIO, DG EMPL, DG MARE, Brussels, 2019. 23 European Anti-Fraud Office, Management Plan 2021, European Commission, Brussels, 2021. 24 European Commission, Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee, the Committee of the Regions and the Court of Auditors on Commission Anti-Fraud Strategy: enhanced action to protect the EU budget. COM(2019) 196 final, European Commission, Brussels, 2019. 25 ibid p 18 Policy intersection #3: State capture and foreign influence Policy intersection #3: State capture and foreign influence Policy intersection #3: State capture and foreign influence State capture is considered as a local or national phenomenon depending on the activities of domestic institutions, while foreign countries are considered in most cases to be enablers of specific mechanisms that facilitate the process (e.g., use of offshore or tax heavens for hiding the beneficial owner or the illicit financial flows). However, in the last few years, studies of malign foreign influence and its political, economic and hybrid instruments on the democratic institutions in the European countries have suggested that state capture could be deployed as a foreign policy tool, or at least could facilitate its aims, as mentioned above.21 Local captors could become enablers of foreign malign influence and allow the foreign state to achieve its end and avoid some of the consequences of its be­ haviour.22 State Capture as a Governance Threat State Capture as a Governance Threat 21 Policy intersection #4: State capture and financial frauds In previous years, professional ethics and integrity of public officials have been outlined by both the European Anti-Fraud Office (OLAF) 23 and the Commission Anti-Fraud Strategy24 as a strategic objective and one of the guiding principles and standards for the common fight against fraud across the EU. As an integral part of the institutional characteristics that affect the system of governance al­ lowing or facilitating state capture, professional integrity is a basic component, the importance of which goes above and beyond the issue of fighting fraud.25 In the past few years, various areas of disbursement and redistribution of EU funds (e.g. the Common Agricultural Policy) have raised attention of European and na­ tional policy makers as “fuelling fraud and corruption and the rise of rich busi­ nessmen” 26 across the Member States, and particularly in Central and Eastern Europe. 27 Cases of financial frauds, including with European funds, are reported to be based on exploiting political ties with ruling parties and governments, con­ flicts of interests, lack of transparency and accountability and proper scrutiny both during and after the distribution process.28 Both the EU Anti-Fraud Strategy and the Joint Anti-Fraud Strategy for the European Structural and Investment Funds29 seek to improve fraud detection by introducing big data technologies and new methods for tackling complex offenses and fraud at the national and in­ ternational level. g g p y 27 Sabev, D. et al. Where does the EU money go? An analysis of the implementation of CAP funds in Bulgaria, the Czech Republic, Hungary, Slovakia and Romania, A Report commissioned by the Greens/EFA group in the European Parliament, Brussels, 2021. 26 Dordevic, N., “Fraud, corruption, and misuse of EU agricultural funds a major problem in CEE, say MEPs,” Emerging Europe, February 26, 2021. g p p 28 Organisation for Economic Cooperation and Development, Fraud and corruption in European Structural and Investment Funds. A spotlight on common schemes and preventive actions, OECD, 2019. Lacking policy integration The notion of state capture is used widely by the media, politicians, and ex­ perts in Europe, but it does not exist as a term in the national or the EU legis­ lation, unlike the terms of corruption, conflicts of interest or abuse of power, which are each well defined. Thus, there are no specific regulations focused on tackling state capture in its intricacy and complexity. However, in both European and national contexts there are specialised policies, institutional and legal frameworks focused on different issues central to the state capture concept (e.g., corruption, anti-monopoly, conflicts of interests and integrity of public officials). One of the main reasons for the absence of an integrated approach could be down to the lack of comprehensive analytical framework and respective policy tools for assessing the current state and for monitoring the development of state capture processes, unlike the issue of corruption, 22 State Capture Deconstructed for example, which has been widely studied and subsumed into the political discourse. The current report fills this gap, at least partially, by piloting a new ap­ proach for sectoral assessment of state capture, focusing on four European countries (Bulgaria, Italy, Romania and Spain) and three economic sectors found by previous national-level assessments to be of high risk with strong vulnerabilities to state capture and corruption30: • Wholesale of solid, liquid and gaseous fuels31 – a heavily regulated sector, dominated by large multinational and domestic companies; • Wholesale of pharmaceutical goods – a multinational market, character­ ised by large corporations, and prone to strong influence and (illegal) lob­ bying; • Construction – a sector, vulnerable to multiple state-capture threats, most prominently: procurement concentration. The state capture assessment on the sectoral level should also take into con­ sideration the national characteristics that facilitate state capture pressure and the level of resilience of public institutions.32 The pilot countries, like others in the EU, do not apply the state capture concept in their legislation and pol­ icy, even if it is used by media, politicians and experts, as mentioned above. While they have specialised institutional and regulatory frameworks focused on several of the issues central to the concept (e.g., corruption, anti-monopoly, or integrity of public officials,) they do not have a systematic approach to the phenomenon. While the existence of several risks and vulnerabilities that could be attributed to state capture (incl. 30 Stoyanov, Gerganov and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. 31 According to the Statistical classification of economic activities in the European Community, NACE rev.2. EUROSTAT 2008. 32 The chapter, presented here with limited revisions, was originally prepared for: Gerganov, A., and Galev, T., Assessing state capture vulnerabilities and pressure at the sectoral level, Sofia: Center for the Study of Democracy, 2021 (forthcoming). 32 The chapter, presented here with limited revisions, was originally prepared for: Gerganov, A., and Galev, T., Assessing state capture vulnerabilities and pressure at the sectoral level, Sofia: Center for the Study of Democracy, 2021 (forthcoming). y 31 According to the Statistical classification of economic activities in the European Community, NACE rev.2. EUROSTAT 2008. 30 Stoyanov, Gerganov and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019.i Lacking policy integration corruption) have been highlight­ ed as a serious problem in each of the four countries over the last decade, they have been considered only as separate issues. These include specific cor­ ruption risks and governance deficits in public procurement, the lack of or inefficient implementation of regulations regarding conflicts of interests and lobbying, as well as the regulatory and administrative burdens on free com­ petition, which create favourable conditions for high market concentration in specific sectors. State Capture as a Governance Threat State Capture as a Governance Threat 23 National context: Bulgaria Bulgaria is the country most vulnerable to corruption and regulatory and pol­ icy instability due to the lack of transparency and predictability of the legisla­ tive process, coupled with low efficiency of the judiciary and the specialised an­ ti-corruption bodies.33 A warning sign of state capture is political interference in the work of the public administration, which leads to frequent legislative changes.34 Despite comprehensive reform of the country’s legal and institutional anti-corruption frameworks in 2017 and 2018, the results have remained under­ whelming,35 while some of the reforms were assessed by independent experts as facilitating stronger state and judiciary capture. In particular, the country still lacks “solid track record of concrete results in the investigation and prosecu­ tion of high-level corruption”.36 The interference or even control of the judiciary by powerful political and economic lobbies has been a notable obstacle for both business environment and public sector reforms. 33 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Bulgaria, 2020, p. 12.; European Commission, European Semester Country Report Bulgaria 2020, p. 7. p p y p g p 35 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Bulgaria, 2020, p. 11. p 34 European Commission, European Semester Country Report Bulgaria 2019, p. 56. g p 36 European Commission, European Semester Country Report Bulgaria 2020, p. 58 p p y p g p 37 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Bulgaria, 2020, pp. 4-5. g pp 38 European Council, Council Recommendation on the 2016 National Reform Programme of Bulgaria and delivering a Council opinion on the 2016 Convergence Programme of Bulgaria, 2016, p. 3. 39 E C i i E S t C t R t B l i 2020 58 g pp 38 European Council, Council Recommendation on the 2016 National Reform Programme of Bulgaria 39 European Commission, European Semester Country Report Bulgaria 2020, p. 58. 40 ibid 58 38 European Council, Council Recommendation on the 2016 National Reform Programme and delivering a Council opinion on the 2016 Convergence Programme of Bulgaria, 2016, p 39 European Commission, European Semester Country Report Bulgaria 2020, p. 58. 40 ibid 58 and delivering a Council opinion on the 2016 Convergence Programme of Bulgaria, 2016, 39 European Commission, European Semester Country Report Bulgaria 2020, p. 58. 40 ibid p 58 41 European Commission, European Semester Country Report Italy 2016, p. 68. 51 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Romania, 2020, p. 10. p p y p y p 43 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Italy, 2020, p. 1. y p 44 European Commission, Recommendation for a Council Recommendation on the 2019 National Reform Programme of Italy and delivering a Council opinion on the 2019 Stability Programme of Italy, 2019, p. 12. Lacking policy integration Ultimately, however, the most serious issue remains the lack of accountability of the Prosecutor General and the position’s exceptional power over the work of the entire prosecution service, as well as influence on the governing body of the judiciary, namely, the Supreme Judicial Council.37 Public procurement in Bulgaria has remained a focal point of corruption risks and governance deficits and has “suffered from structural weaknesses, includ­ ing systematic irregularities in procurement procedures, lack of administrative capacity and deficient control mechanisms”.38 The changes in the Public Procure­ ment Act from 2018 aimed at increasing transparency and limiting corruption risks, and the introduction and the mandatory use of the e-procurement sys­ tem since the early 2020, have not yet given rise to tangible outcomes.39 At the same time, the limited results in the fight against corruption – and particular­ ly against top-level political corruption – are reflected in public perceptions, which rank Bulgaria as one of the most corrupt countries in Europe.40 Thus, the regulatory and control institutions and law enforcement organisations have also been suspected of being captured by private (political or economic) interests rath­ er than being instrumental for tackling state capture. 24 State Capture Deconstructed State Capture Deconstructed National context: Italy Italy has made continuous progress in its anti-corruption policies with positive results, but some challenges remain. In 2016, corruption had been highlighted as a critical issue in the country, with references to organised crime, public procure­ ment and affecting the private sector and large public works41. In 2019 and 2020, a new anti-corruption law, combined with stronger prevention measures ensured by the National Anti-corruption Authority, has considerably strengthened the country’s anti-corruption framework.42 Particularly, “the capacity to detect, in­ vestigate and prosecute corruption is very effective and benefits from the exper­ tise of the law enforcement authorities in the fight against organised crime”.43 At the same time, the country still fails to address critical vulnerabilities to state capture, such as over-regulation and restrictions of competition in important sectors including retail, business services, local public services, concessions and transport44, the fragmented regime of addressing conflicts of interest, and lobby­ ing and “revolving doors”45, which create favourable conditions for monopolisa­ tion, inefficiency of public spending and deteriorating governance. 48 European Commission, European Semester Country Report Romania 2019, p. 56. 49 Ibid. y p 46 European Commission, European Semester Country Report Romania 2017, p. 11. p y p y n Commission, European Semester Country Report Italy 2019, National context: Romania National context: Romania Romania highlighted as an example of a country that went through a phase of widespread political corruption in the period after joining the EU. However, in 2017, the country made “substantial progress on much of the reform of the judi­ cial system and the investigation of high-level corruption”.46 Nevertheless, since 2018 “the progress in the fight against corruption has suffered significant set­ backs”47 due to the government’s pressure on key anticorruption institutions (e.g. the National Anti-Corruption Directorate) trying to influence their work and to limit their independence.48 In addition, numerous amendments to anti-corrup­ tion and other laws have undermined the independence of judges and prose­ cutors, as well as the overall public confidence in the judiciary.49 Thus, the latest assessments have highlighted that “corruption continues to be a major problem for the business environment in Romania”.50 While the government currently supports the fight against corruption, Romania is still facing important challeng­ es to restore the progress since the period before 2017 due to the damage done through legislative amendments and continued pressure on judicial institu­ tions, which deteriorates its capacity to investigate high-level corruption.51 In this situation, state capture remains a serious threat to the country, even despite the renewed commitment of the current government to make progress on the preventative side through the comprehensive National Anti-Corruption Strategy. y p 44 European Commission, Recommendation for a Council Recommendation on the 2019 National Reform Programme of Italy and delivering a Council opinion on the 2019 Stability Programme of Italy, 2019, p. 12. p 5 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Italy, 2020, p. 11. p 45 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Italy, 2020, p. 11. y p 46 European Commission, European Semester Country Report Romania 2017, p. 11. p European Commission, European Semester Country Report 48 European Commission, European Semester Country Report Romania 2019, p. 56. 49 Ibid 51 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Romania, 2020, p. 10. 52 European Commission, European Semester Country Report Spain 2019, p. 73. p p y p p p 53 European Commission, European Semester Country Report Spain 2019, p. 73. p p y p p p 54 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Spain, 2020, p. 7. p p 55 Law 19/2013, of December 9, on transparency, access to public information and good governance. Official State Gazette, no. 295, of December 10, 2013. p f p y p f Spain, 2020, p. 7. 55 Law 19/2013, of December 9, on transparency, access to public information and good governance. Official State Gazette, no. 295, of December 10, 2013. 54 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Spain, 2020, p. 7. 55 Law 19/2013, of December 9, on transparency, access to public information and good governance. Official State Gazette no 295 of December 10 2013 52 European Commission, European Semester Country Report Spain 2019, p. 73. 53 European Commission, European Semester Country Report Spain 2019, p. 73. 54 European Commission, 2020 Rule of Law Report: Country Chapter on the rule of law situation in Spain, 2020, p. 7. 55 Law 19/2013, of December 9, on transparency, access to public information and good governance. Official State Gazette, no. 295, of December 10, 2013. National context: Romania State Capture as a Governance Threat State Capture as a Governance Threat 25 National context: Spain Spain has strengthened its anti-corruption institutional and regulatory frame­ work in recent years, including through the establishment of respective agencies in some autonomous regions and separate municipalities.52 However, the coun­ try failed to develop a national anti-corruption strategy, as well as an inte­ grated and systematic policy approach towards various risks and vulnerabili­ ties.53 While in recent years there has been a surge in corruption investigations involving cases at the local and regional levels, the government has made slower progress towards improving the regulatory and institutional framework at the central level and has failed to ensure harmonisation across government levels, which has created wide regional variations in the quality of governance. Simi­ larly, issues presenting serious risks for state capture have been strengthened and improved, but without consistency across various levels of government and categories of officials, remaining divided between several law enforcement authorities. This refers primarily to the improved legal framework for integrity in the public sector, which was put in place to strengthen the integrity mechanisms in parliament, as well as to reinforce the regimes of asset disclosure, conflict of interest and incompatibilities of high-ranking officials in the central state admin­ istration.54 Meanwhile, there is no national level legislation to regulate lobbying. The Transparency Act55 regulates what information state authorities are required to make publicly available, but does not refer to lobbying and the accountability of public officials is left to the discretion of the respective authority or person. 56 Longer version of this chapter was originally prepared for: Gerganov and Galev, Assessing state capture vulnerabilities and pressure at the sectoral level. Center for the Study of Democracy, 2021 (forthcoming) 57 The assessment of business capture is based on a large sample expert survey, which pro­ vides assessment scores for each of the empirical indicators. The indicators are constructed to measure not only the existence of specific institutional or regulatory frameworks but also their real implementation. Following the three-levels of indicators operationlisation and the respective indicators grouping, index and sub-indexes’ scores are computed, based on the predefined methodology. For detailed explanation, incl. indicators and computations of scores, see: Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021. 56 Longer version of this chapter was originally prepared for: Gerganov and Galev, Assessing state capture vulnerabilities and pressure at the sectoral level. Center for the Study of Democracy, 2021 (forthcoming) FACTORS FACILITATING BUSINESS CAPTURE IN BULGARIA, ITALY, ROMANIA AND SPAIN Despite widespread recognition concerning the existence of different forms and trends of risks and vulnerabilities that could be attributed to state cap­ ture in the four countries, they do not have integrated policies against it, and the institutional and regulatory frameworks suffer from varying degrees of fragmentation and inefficiency.56 Accordingly, the identified risks and vul­ nerabilities are not addressed systematically and in future-proof manner. The current analysis assesses and quantifies the results and effects of several of these risks and vulnerabilities, focusing on two key elements central to the state capture concept: business capture and institutional enablers. Employing the concept described above, the assessment of business capture57 covers two groups of factors: • assessment of the public organisations regulating and/or controlling the sectoral market, evaluated in terms of integrity, impartiality, inclination to private bias, and the effectiveness of their anti-corruption policies; • assessment of the economic sector itself in regard to the overall level of monopolisation and ineffectiveness of anti-monopoly laws, as well as the existence of four categories of non-market mechanisms, which provide il­ legitimate competitive advantage and, when occurring systemically, are a strong symptom of state capture in a sector: (1) privileged access to pro­ curement, (2) laws providing illegitimate competitive advantage to cer­ tain businesses, (3) selective application of control and/or sanctions, and (4) concentration of public grants and subsidies to selected companies in the sector. The index of businesses state capture pressure (BSCP) indicates the ex­ istence of systematic problems of well-established and long-term forms of state capture in the three sectors of all studied countries. Spain ranks first with highest score of the BSCP index in two sectors (construction and wholesale of pharmaceuticals), followed by the marginally lower scores of Romania and Bulgaria within a single sector each (respectively wholesale of pharmaceuticals and wholesale of fuels). Meanwhile, Italy remains last with the lowest index value. The index values for the four countries are very high (the lowest is 57%), which underlines the need for specific preventive policies and measures in each of them. 28 State Capture Deconstructed Figure 5. Measured concepts and indicators Figure 5. Measured concepts and indicators g p Source: CSD, 2021, based on Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021, p. 19. FACTORS FACILITATING BUSINESS CAPTURE IN BULGARIA, ITALY, ROMANIA AND SPAIN Business state capture pressure ●Assessed overall level of monopolisation in the sector Institutional enablers General monopolisation pressure Ineffectiveness of antimonopoly laws Specific monopolisation pressure Lack of integrity Lack of impartiality Private interest bias Ineffectiveness of anti-corruption policies ●Laws regulating the sector help/hinder/not related to the formation of monopolistic, oligopolistic or cartel structure ●A specific company or a small number of companies win disproportionately high number of public tenders ●Laws provide illegitimate competitive advantage ●Selective application of control and/or sanctions ●Concentration of public funds in the sector (euro funds, direct subsidies, etc.) ●Activities are not transparent ●Not accountable for actions ●No checks and balances ●Often serves private interests ●Would never sanction certain people/firms ●Rules of operation are violated often ●Private interest bias ●Estimated external corruption pressure ●Estimated pressure from above ●Estimated involvement in corruption General monopolisation pressure ●Assessed overall level of monopolisation in the sector Business state capture pressure Specific monopolisation pressure Lack of impartiality Institutional enablers Private interest bias Ineffectiveness of anti-corruption policies Source: CSD, 2021, based on Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021, p. 19. Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain 29 Figure 6. All sectors captured at over 50% * scores, 0-100% Source: SCAD ESL 2020. 73% 57% 65% 63% 66% 59% 74% 75% 69% 60% 59% 75% Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceutical Constructuon Figure 6. All sectors captured at over 50% Source: SCAD ESL 2020. The differences among the countries are more evident when the sub-compo­ nents of the BSCP58 and the factors that affect them are considered. Bulgaria ranks first according to the general monopolisation pressure in all sectors:59 • Civil engineering (as sub-sector of construction) is the sector regarded as the most vulnerable to monopolisation in Bulgaria (index value 93%), as well as among the other countries. In Bulgaria, this is likely to be as a re­ sult of the existence of multi-billion publicly funded programmes for large infrastructure projects implemented since the country’s entry into the EU in 2007, which have been regularly accompanied by numerous journalis­ tic and expert investigations of alleged cases of high-level corruption and state capture, but nevertheless remain unprosecuted by the law enforce­ ment authorities. g yp ( ) p 60 Investor.bg, „КЗК: Няма картел на пазара на горива, а обмен на търговска информация“ [Commission for Protection of Competition – there is no cartel in the fuels market but only exchange of commercial information], March 31, 2017; Mediapool.bg, „КЗК отново не видя картел и монопол при горивата“ [Commission for Protection of Competition again does not see a cartel and monopoly in the fuels market], March 12, 2019. selective public support measures). 59 The Construction sector is assessed with its three sub-sectors: (i) civil engineering, (ii) con­ struction of buildings of all types, and (iii) specialized construction activities. 58 As explained above, the BSCP index is composed of three sub-components that measure different elements of business capture - General monopolisation pressure, which reflects the existence of different forms of market concentration (monopoly, oligopoly or cartel), Inef­ fectiveness of antimonopoly laws, which is a stand-alone indicator, and Specific monopolisation pressure, which refers to a set of illegitimate and illegal practices resulting in undue advan­ tages (public procurement concentration, lobbyist laws, selective control and sanctions and selective public support measures). FACTORS FACILITATING BUSINESS CAPTURE IN BULGARIA, ITALY, ROMANIA AND SPAIN • The monopolisation pressure in the wholesale of fuels in Bulgaria (index 90%) is based predominantly on the suspected cartelisation of the sector, which has gone largely unnoticed by the country’s antimonopoly body, the state Commission for Protection of Competition60. In Italy, which has the second highest index value (85%), the pressure is assessed to be the result of a combination of suspected cartel and oligopolistic trends. • In the wholesale of pharmaceuticals, the monopolisation pressure in Bul­ garia is again highest among the four countries (89%) and could be at­ tributed to both monopolisation trends and the small size of the market as compared to countries whose larger markets are seen as less vulnerable to monopolisation. However, assessments for suspected cartels and oligopo­ listic trends in the market of Romania rank it second after Bulgaria. 30 State Capture Deconstructed Figure 7. General monopolisation pressure in Bulgaria is highest Construction is the most vulnerable sector to illegitimate activities Figure 9. Construction is the most vulnerable sector to illegitimate activities Figure 9. Construction is the most vulnerable sector to illegitimate activities * Specific monopolisation pressure (scores, 0-100%) Source: SceMaps, SCAD ESL 2020. 79% 60% 67% 71% 68% 62% 86% 86% 77% 73% 78% 89% Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceutical Constructuon * Specific monopolisation pressure (scores, 0-100%) Source: SceMaps, SCAD ESL 2020. Wholesale of fuels Wholesale of pharmaceutical Constructuon * Specific monopolisation pressure (scores, 0-100%) Source: SceMaps, SCAD ESL 2020. Source: SceMaps, SCAD ESL 2020. When comparing sectors, construction, and particularly its sub-sector of civil engineering, which concentrates the largest portion of public spending, appears the most vulnerable sector to state capture in all countries. Howev­ er, despite differences among the sectors, BSCP’s components reveal the ex­ istence of well-established mechanisms of state capture in all of them. BSCP also confirms the importance of privileged access to public procurement as a key element of business capture. Moreover, the state capture process, which makes such privileged access possible, often includes other mechanisms, such as selective (only targeting captors’ competitors) control and sanctions, lobbyist laws and concentration of public subsidies or grants. 61 These include: (1) privileged access to procurement, (2) laws providing illegitimate competi­ tive advantage to certain businesses, (3) selective application of control and/or sanctions, and (4) concentration of public grants and subsidies to selected companies in the sector. 62 Organisation for Economic Cooperation and Development, Anti-corruption Reforms in Eastern Europe and Central Asia: Progress and Challenges 2016-2019, 2020. 63 See: Deyong, M. et al., Corruption and public procurement, In: Ferguson G. (ed.) Global cor­ ruption: law, theory and practice, 3-rd edition, University of Victoria, 2018; Hellman, J., Jones, G., and Kaufmann D., Seize the State, Seize the Day: State Capture, Corruption, and Influence in Transition. Policy Research Working Paper No. 2444. World Bank, Washington, DC., 2000. Figure 7. General monopolisation pressure in Bulgaria is highest * scores, 0-100% Source: SceMaps, SCAD ESL 2020. 90% 85% 67% 57% 89% 71% 79% 64% 84% 68% 54% 79% Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceutical Constructuon Source: SceMaps, SCAD ESL 2020. The countries rank differently according to the other sub-component of BSCP, namely, the ineffectiveness of anti-monopoly laws, which is highest in Spain (wholesale of pharmaceuticals 64% and wholesale of fuels 50%) and in Ro­ mania (wholesale of fuels 62%), while in the other sectors and countries it remains much lower (below 46%). In the last sub-component of BSCP – the specific monopolisation pressure – Spain retains the highest values in two sectors (construction  – 89% and wholesale of pharmaceuticals 86%), while Bulgaria and Romania come sec­ ond with the highest value in a single sector each (pharmaceuticals for Roma­ nia and fuels for Bulgaria). The very high scores (above 60%) for all sectors and countries are an indication of widespread presence of such practices. Figure 8. State capture through anti-monopoly laws is highest in Spain * Share of respondents agreeing with the statement “The antimonopoly laws rather help the formation of monopolistic, oligopolistic or cartel structures than hinder them” Source: SceMaps, SCAD ESL 2020. Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceutical Constructuon 45% 39% 36% 21% 40% 26% 62% 46% 25% 50% 64% 42% * Share of respondents agreeing with the statement “The antimonopoly laws rather help the formation of monopolistic, oligopolistic or cartel structures than hinder them” Source: SceMaps, SCAD ESL 2020. Source: SceMaps, SCAD ESL 2020. Despite the high scores of all sectors, the specific monopolisation pressure index ranks construction as the only sector with scores above 70% for all Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain 31 four countries. The same trend is not observed regarding the other two BSCP sub-components (general monopolisation pressure and ineffectiveness of an­ ti-monopoly laws). These results reveal that the sector is more vulnerable to the four categories61 of illegitimate and illegal practices than the other two. four countries. The same trend is not observed regarding the other two BSCP sub-components (general monopolisation pressure and ineffectiveness of an­ ti-monopoly laws). These results reveal that the sector is more vulnerable to the four categories61 of illegitimate and illegal practices than the other two. Figure 9. State Capture and Corruption Risks in Public Procurement Public procurement, taxation, customs activities and regulatory functions, are also considered to be among the economic areas most prone to risks of corruption and conflicts of interests.62 Moreover, all forms of corruption are present in procurement, from petty or administrative corruption to political corruption and state capture.63 In procurement, the “captors” are private busi­ 32 State Capture Deconstructed ness enterprises or oligarchs controlling large groups of companies, which use different forms of corruption to influence the implementation of legislation, rules and institutional procedures in order to acquire non-competitive advan­ tages over their market rivals. The captured institutions include legal entities that spend public funds through public procurement procedures, including, national, regional and local public administrations, educational, health and social service institutions, as well as state owned enterprises obliged to com­ ply with the respective national public procurement regulations.64 ness enterprises or oligarchs controlling large groups of companies, which use different forms of corruption to influence the implementation of legislation, rules and institutional procedures in order to acquire non-competitive advan­ tages over their market rivals. The captured institutions include legal entities that spend public funds through public procurement procedures, including, national, regional and local public administrations, educational, health and social service institutions, as well as state owned enterprises obliged to com­ ply with the respective national public procurement regulations.64 The analysis of the risks of corruption-related behaviour in public procure­ ment makes use of red flag indicators based on the integration of three dif­ ferent sets of data on public procurement65, companies’ financial and owner­ ship information from public and proprietary sources66 and media articles, referring to suspicious behaviour of a particular contracting authority or company. The assessment is carried out on the basis of a combination of red flags, each of them indicating a risk situation, which might be the result of corruption or state capture and which could indicate state capture vulnera­ bilities. A single red flag is not a categorical sign of suspicious behaviour, but the accumulation of red flags for a particular company, contracting author­ ity or country indicates serious concern regarding existing problems in the procurement process on the micro (single company or contracting entity) or macro (country) level. The analysis, which is made possible through the elaboration of the web- based interactive tool, covers more than 100,000 tenders in the four countries and three selected sectors for the period 2010-2019. 64 Beyond these institutions, the captors target also regulatory and control institutions, related to the implementation of public procurement and more general competition rules, as well as the justice system. The ultimate goal is to guarantee a successful outcome in case of a possi­ ble follow-on inspection or in case the tender award decision is challenged. 65 It is based on Tenders Electronic Daily (TED) - the online version of the 'Supplement to the Official Journal' of the EU, dedicated to European public procurement, which publishes pro­ curement award notices and other tenders’ documentation. 66 Company ownership information is used to collate the data related to subsidiaries and shareholders into a single parent company. This provides a more realistic picture of the be­ haviour of economic conglomerates than when their legal entities (subsidiaries) are consid­ ered separately. 64 Beyond these institutions, the captors target also regulatory and control institutions, related to the implementation of public procurement and more general competition rules, as well as the justice system. The ultimate goal is to guarantee a successful outcome in case of a possi­ ble follow-on inspection or in case the tender award decision is challenged. 66 Company ownership information is used to collate the data related to subsidiaries and shareholders into a single parent company. This provides a more realistic picture of the be­ haviour of economic conglomerates than when their legal entities (subsidiaries) are consid­ ered separately. p g 65 It is based on Tenders Electronic Daily (TED) - the online version of the 'Supplement to the Official Journal' of the EU, dedicated to European public procurement, which publishes pro­ curement award notices and other tenders’ documentation. State Capture and Corruption Risks in Public Procurement The analysed tenders amount to more than EUR 364 billion in public money, spent by almost 3,000 contracting entities and allocated to more than 45,000 companies in Europe during this period. Construction accounts for the largest portion of public spending through procurement out of the three sectors, markedly surpassing the other two. It accounts for 58% of the three sectors in terms of awarded value in Italy, 74% in Spain and Bulgaria and 76% in Romania. The comparison between the countries, looking at the proportion of awarded value per capita annually, confirms the dominance of construction, but also reveals that Bulgaria and Romania spent between 30% to 50% more per capita annually in construc­ tion for the period 2010-2019 than Italy and Spain, despite the smaller size of their markets (respectively EUR 1,509 per capita annually in Italy, EUR 1,756 in Spain, EUR 2,631 in Romania and EUR 2,867 in Bulgaria). 33 Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Figure 10. Total value of public procurement in selected sectors 2010 – 2019 (EUR billion) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. Bulgaria Italy Romania Spain Constructuon 5.4 59.0 12.9 23.3 1.8 7.7 3.7 5.1 20.7 90.7 52.3 81.9 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 Wholesale of fuels Wholesale of pharmaceutical Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. The data shows that the number of tenders and the number of awarded com­ panies per country have increased for the period 2010-2019, which suggests that the general business and competition environment has improved. How­ ever, this is difficult to evaluate extensively due to the spread of many illegit­ imate practices, such as the use of complex networks of subsidiaries and con­ trolled companies for formal diversification of suppliers (when in reality the awarded tenders are concentrated into a small number of economic actors), or bid rigging (an illegal practice in which formally competing suppliers collude to determine the winner of a bidding process). The assessment of vulnerabilities and risks in public procurement based on the existence of red flags demonstrates that Bulgaria, Romania and partially Spain display signs of state capture and corruption in public procurement, while Italy remains a less “captured” state. State Capture and Corruption Risks in Public Procurement Despite differences among them, the review of the red flags indicates that public procurement in Bulgar­ ia and Romania is, in general, much more vulnerable to suspicious behaviour on both the side of suppliers (companies) and buyers (contracting authorities) as compared with Spain and Italy. The analysis covers the nine-year peri­ od 2011-2019 and while shorter periods could give rise to more red flags, the longer selected period, even reducing sensitivity, allows for the identification of companies with continuous (recurring) suspicious behaviour. In Bulgaria, the share of public authorities (buyers) that concentrate over 60% of the value of awarded contracts to a single supplier is about twice as much as the respective share in the other three countries (21% compared to 12% for Italy and Romania, and 10% for Spain). Bulgaria also has the largest group of buyers accounting for over 90% of the tenders of a given supplier, although the total sum of awarded contracts by these buyers is much smaller than in Spain, for example. This indicator raises red flags for both public authorities and companies, which could be further explored and investigated on a case- 34 State Capture Deconstructed by-case basis through the information and profile data of each legal entity, which is available through the web-based tool. The buyer concentration index calculates the concentration of the total value of con­ tracts, awarded by a contracting authority (the buyer), to a particular supplier for the period 2010-2019. The index represents the risk that a given buyer (contracting authority) allows particular supplier to gain competitive advantage through the use of illegal means. An index equal to 100% means that a single buyer has provided the entire sum, received from public procurement contracts by a particular supplier. Figure 11. Buyer concentration index (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. 67 The result covers only the companies ranking in the highest 20% of the ranking scale, which are assessed as being the riskiest. State Capture and Corruption Risks in Public Procurement 25 suppliers BG RO IT ES 60% 100% 70% 80% 90% Concentration percentage 478 m/ 42 suppliers 407 m/ 25 suppliers 1637 m/ 52 suppliers 1229 m/ 51 suppliers red-fagged buyers allocated amount / No of suppliers red-fagged buyers allocated amount / No of suppliers red-fagged buyers allocated amount / No of suppliers red-fagged buyers allocated amount / No of supppliers 397 m/ 37 suppliers 234 m/ 17 suppliers 828 m/ 17 suppliers 739 m/ 17 suppliers 2417 m/ 59 suppliers 2180 m/ 31 suppliers 853 m/ 19 suppliers 1072 m/ 16 suppliers 1155 m/ 50 suppliers 1194 m/ 33 suppliers 772 m/ 26 suppliers 4262 m/ 31 29 21 26 33 15 17 17 37 26 15 9 35 24 21 16 Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. The ratio of procurement exposure to employee demonstrates that for the period 2011-2019, 37 companies in Romania and 21 companies in Bulgaria received large amounts of public tenders while having a limited number of employees (hence implementation capacity) as compared to their peer com­ panies.67 In Spain, the number of respective companies is only 3, while in Italy there is not a single company that raises this red flag. In classic economic analysis, the ratio of company’s revenue per employee is a notable indicator of business efficiency. However, when this ratio is too high compared to the peer group of companies, it indicates a serious risk of misconduct, particularly Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain 35 when revenue is generated from public tenders where the rules of free market competition, based on supply and demand, are replaced by administrative decisions regarding cost and selection of winners. when revenue is generated from public tenders where the rules of free market competition, based on supply and demand, are replaced by administrative decisions regarding cost and selection of winners. The ratio of procurement exposure per employee calculates the average amount of tenders awarded per employee over a given period and ranks companies accordingly. A higher rank means that the company has a lower number of employees compared to its peers with similar revenues from public tenders. State Capture and Corruption Risks in Public Procurement This red flag represents the risk of companies with an insufficient number of employees winning tenders that require a larger workforce and, in many cases, this is combined with the undeclared - and therefore illegal - use of subcontractors. Figure 12. Procurement exposure per employee ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. 21 37 3 0 0 10 20 30 40 50 0 100,000 200,000 300,000 400,000 500,000 Bulgaria Romania Spain Italy No. of companies, ranked highest according to the ratio of awarded amounts per employee Average ammount from tenders per employee (EUR) Figure 12. Procurement exposure per employee ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. The ratio of procurement exposure to revenue calculates the share of revenue from awarded public tenders in the company's total revenue over a given period. It repre­ sents the risk that some companies depend highly on public procurement to survive and are thus more motivated to use illegal means to gain an advantage over their com­ petitors. It may also point to companies that prefer not to operate in the free market and therefore cannot be viable without the support of public money. The ratio of procurement exposure to revenue calculates the share of revenue from awarded public tenders in the company's total revenue over a given period. It repre­ sents the risk that some companies depend highly on public procurement to survive and are thus more motivated to use illegal means to gain an advantage over their com­ petitors. It may also point to companies that prefer not to operate in the free market and therefore cannot be viable without the support of public money. The procurement exposure to revenue ratio confirms that Romania and Bul­ garia are the most vulnerable out of the four countries, possessing a similar number of companies that rely primarily on public procurement for their ex­ istence. However, due to the larger value of tenders on average, Romanian companies in this group have received 63% more in terms of awarded value of contracts. This indicator reveals not only a problem regarding the depend­ ence of given companies on public procurement, but also may be an indica­ tion of the use of illegitimate means for achieving higher bid prices of their products and services. 68 As registered by SCAD-ESL (see sections above). State Capture and Corruption Risks in Public Procurement 36 State Capture Deconstructed A macro analysis of the selected red flags only partially confirms the find­ ings from the expert assessments of specific monopolisation pressure68, while at the same time contributes to the understanding of corruption and state capture risks and vulnerabilities existing in the public procurement process­ es. While experts’ assessments rank Spain first, the red flag analysis high­ lights Bulgaria and Romania as more vulnerable. However, the results of both methods present Italy as less captured. Figure 13. Procurement exposure to revenue ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. 831.7 1,312.9 61.3 0.0 Bulgaria Romania Spain Italy 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 Sum of awarded amounts from tenders (EUR mln) 0 5 10 15 20 25 30 35 No of red-fagged companies Figure 13. Procurement exposure to revenue ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. 831.7 1,312.9 61.3 0.0 Bulgaria Romania Spain Italy 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 Sum of awarded amounts from tenders (EUR mln) 0 5 10 15 20 25 30 35 No of red-fagged companies Figure 13. Procurement exposure to revenue ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. 831.7 1,312.9 61.3 0.0 Bulgaria Romania Spain Italy 0.0 200.0 400.0 600.0 800.0 1,000.0 1,200.0 1,400.0 Sum of awarded amounts from tenders (EUR mln) 0 5 10 15 20 25 30 35 No of red-fagged companies Figure 13. Procurement exposure to revenue ratio (2011 – 2019) Source: SceMaps web-based interactive tool, https://analytics.scemaps.eu, 2021. A macro analysis of the selected red flags only partially confirms the find­ ings from the expert assessments of specific monopolisation pressure68, while at the same time contributes to the understanding of corruption and state capture risks and vulnerabilities existing in the public procurement process­ es. While experts’ assessments rank Spain first, the red flag analysis high­ lights Bulgaria and Romania as more vulnerable. However, the results of both methods present Italy as less captured. A macro analysis of the selected red flags only partially confirms the find­ ings from the expert assessments of specific monopolisation pressure68, while at the same time contributes to the understanding of corruption and state capture risks and vulnerabilities existing in the public procurement process­ es. While experts’ assessments rank Spain first, the red flag analysis high­ lights Bulgaria and Romania as more vulnerable. State Capture and Corruption Risks in Public Procurement However, the results of both methods present Italy as less captured. 69 The index is a composite indicator, calculated on the basis of the experts’ assessments of each institutional enabler for a pre-defined list of public organisations with regulatory and control functions (incl. self-regulatory organisations such as industry associations) with re­ spect to the selected sectors. For more details, see: Gerganov et al., State Capture Assessment on Sectoral Level: Methodological Toolkit, Center for the Study of Democracy, 2021. Institutional Enablers of State Capture The regulatory and control authorities, as well as the relevant policies and regulations, irrespective of whether they are generally for the economy as a whole or are sector-specific, are the instruments that should ensure a trans­ parent, competitive and effective business environment. Additionally, each must develop strong cooperation with other enforcement bodies, such as anti-corruption and law enforcement agencies, in order to ensure successful outcomes in tackling corruption and state capture. When these organisations are passive or ineffective, perhaps because they are partially or fully cap­ tured, captors are able to ensure systemic privileges for themselves, priva­ tising specific government functions. The functional characteristics of these institutions, such as anti-corruption effectiveness, integrity of public officials, fairness and impartiality of decision-making and procedures, determine the second major component (in addition to BSCP) of SCAD. Known as institutional enablers, these functional characteristics determine the institutional environment in which businesses operate. The enablers af­ fect all actors in a sector and are therefore measured at the sector level, even if some of the organisations assessed have a remit for the entire economy (e.g. tax administration, customs). Enablers denote processes that could contrib­ ute to the creation of an environment that is favourable to state capture and could make institutions vulnerable to a range of corruption influences. While measuring the state capture dimensions (e.g., business capture) provides an assessment of the current status of state capture, measuring the institution­ al enablers provides an insight into the expected future dynamics of state capture processes as the enablers are structural features of the institutional framework. The SCAD approach measures four types of institutional enablers (or factors) that affect the state capture pressures and vulnerabilities on the sectoral level: • Anticorruption effectiveness - the ability of administrative structures to identify, prevent, and counteract corruption practices among officials; fi • Integrity of public officials - establishment and interiorisation of standards of behaviour, showing a consistent and uncompromising adherence to strong moral and ethical principles, including through increased account­ ability and transparency of work; • Impartiality - the ability to adequately apply rules of fairness and impar­ tiality in everyday transactions and services; i tiality in everyday transactions and services; i • Lack of bias toward specific private interests. Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain 37 State capture recognises no sectoral boundaries in public procurement The development of the computing models and algorithms, implemented in the web-based interactive tool, also revealed an unexpected challenge, which indi­ cated the need for a complex and integrated approach to the analysis of different risks and vulnerabilities associated with state capture. Initially intended to fol­ low the sector-level measurements, the analysis produced more reliable results when sectors were disregarded. One of the fundamentals of the analysis – the clusterisation of companies according to their ownership structure – makes it impossible for the results to be presented on a sector level since the companies that are included in the ownership-chain do not operate in the same sector. The clusterisation aims at overcoming the major weakness in the use of red flags, based purely on the analysis of procurement data, namely, the impossibility to see the real concentration in the procurement market hidden beyond the pres­ ence of numerous legal entities controlled by a single economic actor. Howev­ er, as the analysis reveals, very often the alleged risk companies (or red-flagged ones) belong to broader corporate groups with members working in different economic sectors. In other cases, even single companies that are not part of cor­ porate groups could implement tenders in different sectors, but awarded by a single contracting authority. In both variants, the sectoral analysis would distort the real picture, which shows the suspicious behaviour of the given company or group of companies. The reason could be that when a company uses corruption or state capture related mechanisms to obtain a non-market advantage over its competitors, it would not observe the sectoral division but will try to maximize its profit crossing the sectoral boundaries. Figure 14. Institutional environment in Bulgaria is most vulnerable to state capture Among the institutional enablers, the lack of integrity and the ineffective­ ness of anti-corruption policies have the highest scores for all sectors and all countries (i.e., represent the riskiest environmental factors for the exist­ ence of state capture). The private interest bias and the lack of impartiality in the activities of state institutions remains less significant. Among the fac­ tors determining the ineffectiveness of anti-corruption policies, external corruption pressure is the riskiest and has similar values for all countries, including Spain, despite its lowest score for the overall institutional enablers index. Finally, the assessment of the lack of impartiality (which includes sub-indicators for serving private interests, inability to sanction certain peo­ ple or companies, and braking internal rules or procedures) does not differ significantly between the sectors. Nevertheless, one of its sub-indicators (the inability of the respective organisation to sanction certain persons or com­ panies) is assessed as risky, with 2 to 3 times higher scores for all countries as compared with the other sub-indicators. Consequently, the result reveals the existence of a serious problem with persons and/or companies that are excluded from the application of general regulatory and control rules and procedures in all four countries. Figure 14. Institutional environment in Bulgaria is most vulnerable to state capture * Institutional enablers index (scores, 0 – 100%) Source: SCAD ESL 2020 35% 29% 31% 28% 33% 30% 31% 25% 36% 32% 33% 23% Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceuticals Constructuon * Institutional enablers index (scores, 0 – 100%) Source: SCAD ESL 2020 35% 29% 31% 28% 33% 30% 31% 25% 36% 32% 33% 23% Bulgaria Italy Romania Spain Wholesale of fuels Wholesale of pharmaceuticals Constructuon * Institutional enablers index (scores, 0 – 100%) * Institutional enablers index (scores, 0 – 100%) Source: SCAD ESL 2020 Among the institutional enablers, the lack of integrity and the ineffective­ ness of anti-corruption policies have the highest scores for all sectors and all countries (i.e., represent the riskiest environmental factors for the exist­ ence of state capture). The private interest bias and the lack of impartiality in the activities of state institutions remains less significant. Among the fac­ tors determining the ineffectiveness of anti-corruption policies, external corruption pressure is the riskiest and has similar values for all countries, including Spain, despite its lowest score for the overall institutional enablers index. Figure 14. Institutional environment in Bulgaria is most vulnerable to state capture Finally, the assessment of the lack of impartiality (which includes sub-indicators for serving private interests, inability to sanction certain peo­ ple or companies, and braking internal rules or procedures) does not differ significantly between the sectors. Nevertheless, one of its sub-indicators (the inability of the respective organisation to sanction certain persons or com­ panies) is assessed as risky, with 2 to 3 times higher scores for all countries as compared with the other sub-indicators. Consequently, the result reveals the existence of a serious problem with persons and/or companies that are excluded from the application of general regulatory and control rules and procedures in all four countries. Institutional Enablers of State Capture According to the overall institutional enablers index69, Bulgaria is the coun­ try within which the institutional environment is the most vulnerable and contributes to the highest risks of state capture across all sectors. However, the small differences as compared to Romania and Italy reveal that they also must significantly improve the resilience capacity of key regulatory and con­ trol institutions. 38 State Capture Deconstructed Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain 39 The MACPI (Monitoring Anti-Corruption Policy Implementation) tool assess­ es the enforcement of anti-corruption measures and policies at the level of the individual public organisation. As a result, it identifies good practices in terms of internal rules and procedures, and flags specific at-risk zones in the institutions more vulnerable to corruption pressure.70 In this case, the appli­ cation of MACPI covered nine public organisations selected as representing the types of organisations identified as fundamental for the three econom­ ic sectors based on the results of the experts’ (SCAD-ESL) assessment.71 The overall results reveal solid anti-corruption setups in most of these organisa­ tions, with some specifics that are addressed below.72 The most effective and difficult to evade anti-corruption policies are related to three groups of measures: the procedures for hiring new and managing existing personnel, the procedures for controlling budget expenditures and integrity of the staff, and the development of electronic services with a focus on reducing administrative corruption and increasing the transparency and the accountability of the organisation.73 The first group includes measures ad­ dressing the appointment of top-level management, disqualifying applicants who have been convicted (even when an appeal is pending) for crimes against the public administration, and rotation of personnel assigned to activities with high risk of corruption. The second group of measures refers to proce­ dures for control and audit of budget expenditures, as well as the verification of asset declarations, especially when they are strictly implemented and there is an external oversight institution. In particular, the control of asset decla­ rations, which is a common integrity and anti-corruption policy, could only be effective if the follow-up procedures regarding the actual checking of the declared circumstances are implemented strictly, thus ensuring effective en­ forcement in cases of misconduct. The third group of measures includes the digitisation of the services provided to the institution’s clients, which aims at reducing the need for personal contact and thus for administrative cor­ ruption. It also aims to increase and augment the transparency and account­ ability of the institution. 70 MACPI is developed as a management instrument with the main aim to provide advice to institution’s management how to improve their anti-corruption setup, based on the assess­ ment of the coverage, the implementability, the implementation and the effectiveness of an­ ti-corruption policies in a given public organisation. It could be applied also periodically to monitor the progress towards the initial benchmarking state. See: Stoyanov et al., Monitoring Anti-Corruption in Europe. Bridging Policy Evaluation and Corruption Measurement, Center for the Study of Democracy, 2015. the Study of Democracy, 2015. 71 National Revenue Agency and the Directorate for National Construction Control (Bulgaria), Chamber of Commerce of Trento and Emilia-Romagna Region (Italy), Romanian Compe­ tition Council, National Integrity Agency, and Sinaia Municipality (Romania), Valencian Anti-Fraud Agency and Government of the Region of Murcia (Spain). In Romania three in­ stitutions were included due to difficulties to secure sufficient number of responses from the Romanian Competition Council. 72 It should be noted, however, that MACPI focuses mainly on administrative corruption and is less sensitive to state capture or political corruption, even it accounts for them also at least in terms of “estimated corruption pressure”. 73 More detailed analysis and description of the specific policies and measures is available in: Gerganov, A., Monitoring Anti-Corruption Policy Implementation in high-risk sectors. Benchmark­ ing Reports of Nine Public Organisations in Bulgaria, Italy, Romania and Spain, Sofia: Center for the Study of Democracy, 2021 (forthcoming). Anti-corruption policy implementation on institutional level A critical institutional enabler that characterises the regulatory and control institutions, which are expected to ensure a transparent, competitive, fair and effective business environment, is the implementation of their internal an­ ti-corruption policies. Moreover, it is closely linked to other functional char­ acteristics related to impartiality, integrity, and procedural fairness. Often, the institutional anti-corruption setup in terms of internal rules, business processes and institutional culture covers all of these. The assessment of the specific anti-corruption policies follows the internal logic of each institution and is rarely comparable across the institutions, sectors or countries. y y 71 National Revenue Agency and the Directorate for National Construction Control (Bulgaria), Chamber of Commerce of Trento and Emilia-Romagna Region (Italy), Romanian Compe­ tition Council, National Integrity Agency, and Sinaia Municipality (Romania), Valencian Anti-Fraud Agency and Government of the Region of Murcia (Spain). In Romania three in­ stitutions were included due to difficulties to secure sufficient number of responses from the Romanian Competition Council Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain On the other end, there are less effective policies that are easy to be evade, difficult to implement, or have remained only “on paper” due to the lack of elaborated business processes regarding implemen­ tation. Examples of such measures are the declaration of gifts received on the occasion of protocol events, code of ethics or clients’ charter, information campaigns or control over “revolving door” practices. p p 73 More detailed analysis and description of the specific policies and measures is available in: Gerganov, A., Monitoring Anti-Corruption Policy Implementation in high-risk sectors. Benchmark­ ing Reports of Nine Public Organisations in Bulgaria, Italy, Romania and Spain, Sofia: Center for the Study of Democracy, 2021 (forthcoming). 40 State Capture Deconstructed The public institutions exposed to the highest corruption pressure are typ­ ically those providing oversight and inspection of clients (individuals and businesses) and public procurement. In most cases, the actual corruption pressure (i.e. officials involved in this activity report being offered a bribe in the past year) is lower than the estimated corruption pressure (i.e. officials involved in this activity estimate the possibility for being offered a bribe) for a particular activity. Having higher actual pressure than estimated pressure is usually an indication of a potential vulnerability since the real risk of cor­ ruption may not be perceived, or could even be intentionally underreported by employees. For example, the “Inspection, control, surveillance, verification and sanction procedures” activity in the Government of the Region of Murcia in Spain receives the highest actual corruption pressure ranking in the or­ ganisation (18% of the officials involved in this activity report being offered a bribe in the past year). However, the activity is ranked as having much lower estimated corruption pressure. Similarly, “Tax collection” in the Bulgarian National Revenue Agency is ranked second among all activities of the organ­ isation on actual corruption pressure (21% of the officials report being offered a bribe), but the estimated corruption pressure is again much lower. The findings of MACPI indicate that it is crucial to have more vulnerable pub­ lic services targeted by a sufficient number of highly effective, strictly imple­ mented and service-specific anti-corruption policies. When high corruption pressure activities are covered only by general policies, ranked low on their anti-corruption effectiveness, we witness a potential vulnerability in the an­ ti-corruption setup of an organisation. WHAT’S NEXT The State Capture Assessment Diagnostics demonstrates that state capture vul­ nerabilities are sizable at the national level across Europe and are particularly problematic in certain Eastern European countries. SCAD further highlights that certain sectors, such as pharmaceuticals, fuels and construction deserve special policy attention as sources of state capture vulnerabilities. The results of the piloting of the SCAD-ESL (sectoral level) and the red-flagging in public procurement (achieved by the analysis of big data) presented in the current study confirm that state capture risks in these three sectors are higher than those at the national level in all four studied countries (Bulgaria, Italy, Ro­ mania and Spain). Nevertheless, Bulgaria and partially Romania have been revealed as the countries with a higher degree of state capture vulnerability on both the sectoral and public procurement level. It is likely that the disrup­ tion of competitive market forces and the undoing of democratic checks and balances in European economies during the Covid-19 pandemic has further exacerbated state capture vulnerabilities across member states and econom­ ic sectors. The diversity of governance deficiencies and the factors affecting them confirms the need for a broader application of an integrated analytical approach to comparative assessments at the national and sectoral levels. The results of such an analysis would subsequently allow for a targeted policy response and enable the improvement of the resilience of individual public institutions. The very essence of the state capture challenge, with its corrosive impact on national regulatory and control institutions, calls for a European response. Such a response must be focused on the sectoral and sub-national level, seek­ ing to identify and unravel state capture networks across Europe’s regions. In particular, it must target those regions enjoying high levels of EU funding in which the lack of vibrant local economies and stable democratic institutions could easily lead to the concentration of market and political power and the subversion of democratic checks and balances. State capture vulnerabilities can easily lead to democratic backsliding, infiltration of organised crime into the legal economy and foreign malign influence with detrimental conse­ quences for the EU’s joint resilience. Hence, the EU’s response to state capture vulnerabilities is required to span different policy domains, integrate existing instruments, and develop new initiatives and capabilities. The current policy environment is particularly favourable for strengthening the EU’s policy response to state capture vulnerabilities. Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain Factors Facilitating Business Capture in Bulgaria, Italy, Romania and Spain For example, the above-mentioned “Inspection, control, surveillance, verification and sanction procedures” ac­ tivity is covered by only two rather general anti-corruption policies of the organisation that also aim to target 4-5 other activities. Moreover, both poli­ cies have received the lowest scores for strict control and implementation, as well as average scores for effectiveness. In contrast, the other high corruption pressure activity in the same organisation – “Public procurement” – is much better covered by 8 policies, including both specific policies directed towards this particular activity and some of the highest ranked policies in the organ­ isation in general. Where such discrepancies are found, the management of the organisation is advised to add specific and targeted anti-corruption policies specifically ded­ icated to the high-risk activity in question. For example, the high corruption pressure activities of “Control over construction documents” and “Control over construction” of the Directorate for National Construction Control in Bulgaria are covered mainly by broad and general policies related to multiple public services. A targetted policy, such as rotation or automatic random se­ lection of the employees who carry out these activities, could further improve the anti-corruption setup of the organisation. WHAT’S NEXT In 2020, the EU in­ troduced a new European rule of law mechanism aimed at securing member states’ compliance with the highest standards of democratic accountability and checks and balances. In addition, the EU launched its European Democ­ racy Action Plan to build more resilient democracies by promoting free and fair elections, strengthening media freedom and countering disinformation. It also presented its new Security Strategy with a strong focus on corruption as a tool for aggravating different security vulnerabilities, including the pro­ tection of the financial interests of the Union. Furthermore, the United States 42 State Capture Deconstructed has promoted anti-corruption as a core national security interest and a key instrument in standing up to authoritarian malign influence in the frame­ work of renewed global power competition. has promoted anti-corruption as a core national security interest and a key instrument in standing up to authoritarian malign influence in the frame­ work of renewed global power competition. Taken together, these broad policy developments target the institutional and environmental enablers of the state capture model identified by SCAD. As a next step, they require adequate enforcement instruments in order to bear fruit on the ground in the member states most vulnerable to state capture. The newly established European Public Prosecutor’s Office, for example, cannot effectively challenge entrenched state capture networks in public procure­ ment if it does not have the combined support of other EU institutions such as Europol, OLAF and DG Competition. The institutional architecture, however, has one important missing link that is particularly pertinent to tackling state capture vulnerabilities. Namely, the EU lacks a common anti-money laun­ dering agency. Establishing such a body at the EU level, capable of following money trails across member states and globally, is of critical importance for an effective strategy against state capture in Europe. In order to design effective policy instruments, the EU needs to develop a better understanding of state capture vulnerabilities and ensure the adequate monitoring of risks. The current report complements previous efforts to un­ derstand and monitor state capture and provides a useful practical frame­ work for risk assessment, which could guide EU policy and law enforcement efforts. WHAT’S NEXT The SCAD family of diagnostic instruments includes tools for moni­ toring and capacity building for tackling state capture vulnerabilities: • National level assessment (SCAD); • Sectoral level assessment (SCAD-ESL);l • Red-flagging of evidence of state capture and corruption in public pro­ curement, including market concentrations on the level of groups of com­ panies controlled by the same owner; • Institutional level anti-corruption assessment (MACPI). In Europe, the issue of state capture remains outside of mainstream policy debates, which are more focused on different forms of corruption without a systematic evaluation of the linkages between them. Adding instruments for deciphering media capture and judiciary capture to the SCAD model is needed as the next step in responding to the needs of the EU’s Rule of Law mechanism for scalable tools to undertake an integrated analysis of state cap­ ture. Short of such tools, the Union would be inadequately equipped to meet the most serious current defiance to European governance, namely, the use of the privilege of national sovereignty as a cover for abusing democracy for private gain. In many European countries, oligarchic groups insist on having complete discretion in domestic affairs while claiming the benefits of good governance at the European level. In order to expose this discrepancy – and thus challenge it – the EU must ground its policies on verifiable evidence about the specific mechanisms through which state power is being hijacked for private ends. This is exactly what the State Capture Assessment Diagnostics provides. 74 От съкращението на английски език SCAD – State Capture Assessment Diagnostics. 75 Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. EXCUTIVE SUMMARY (IN BULGARIAN LANGUAGE) Понятието „завладяване на държавата“ е в обръщение от десетилетия и описва практики, при които частни бизнес интереси манипулират държавните политики и процесите на вземане на решения в своя полза. Обикновено то се отнася до осъществяването на поредица от отделни ко­ рупционни действия на най-високите етажи на властта. Тенденциите в редица европейски и други страни показват обаче, че тази практика вече не се ограничава до нередности във функционирането на една или друга публична институция, а е прераснала в трайно институционално пове­ дение, което не се поддава на прилагането на общоприетите политики за противодействие на корупцията. В настоящия доклад са представени резултатите от прилагането на иновативния аналитичен инструмент Ди­ агностична оценка на завладяването на държавата (СКАД74), който пре­ доставя важни за управленските политики изводи за завладяването на държавата именно като системен провал на публичното управление. Теоретичен модел на завладяване на държавата Теоретичен модел на завладяване на държавата Монополизация Лобистки закони Завладяване на бизнеса Измерения на завладяването на държавата Завладяване на институциите Концентрация на преки субсидии Корупция в съдебната система Завладяване на медиите Неефективност на антимонополното законодателство Предпоставки на обществената среда Източник: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. Източник: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. От изолирани действия до системна практика Завладяването на държавата, като форма на приватизация на прави­ телствените решения и монополизация на цели икономически секто­ ри, изисква прилагането на нови аналитични инструменти в помощ на разработването на политики за добро управление. СКАД разкрива сис­ тематичното завладяване на държавни правомощия в полза на частни интереси посредством различни видове корупционни и противозакон­ ни практики75. Способите за завладяване на държавата включват овла­ дяването на правоприлагането, привилегирован достъп до публични средства, асиметричен контрол върху медийния и финансовия сектор, влияние върху вътрешната и външната политика и т.н. СКАД показва механизма, чрез който изготвянето, приемането и прилагането на нор­ мативни актове и правила е впрегнато в служба на завладяващите дър­ жавата – привилегировани субекти, ползващи се от неполагащи им се икономически и/или политически облаги. СКАД разкрива как слабостта на механизмите за управление създава условия за завладяване на държавата в четири измерения (икономика, институции, политика и черен пазар) посредством два вида предпостав­ ки. Тези предпоставки влияят върху институционалната и обществената среда, в която се осъществява управлението и по този начин улесняват завладяването на държавата (вж. схемата по-долу). 44 State Capture Deconstructed Теоретичен модел на завладяване на държавата Източник: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. Липса на прозрачност Липса на непредубеденост Предпочитание на частни интереси Неефективност на антикорупционните политики Монополизация Лобистки закони Институционални предпоставки Предпоставки на обществената среда Привилигирован достъп до обществени поръчки Привилигирован статут (селективно налагане на контрол и санкции) Концентрация на преки субсидии Неефективност на антимонополното законодателство Измерения на завладяването на държавата Завладяване на бизнеса Завладяване от черния пазар Политическо завладяване Административна корупция Завладяване на медиите Корупция в съдебната система Завладяване на институциите t p a a y i e ap eu 77 Първоначално разработен и прилаган като самостоятелен инструмент, сега МАКПИ е интегриран в методическата рамка за оценяване на завладяването на държавата на сек­ торно ниво. Вж. Стоянов, А. и др., Мониторинг на антикорупцията в Европа. Оценка на антикорупционните политики и измерване на корупцията, София: Център за изследва­ не на демокрацията, 2015. Executive Summary (in Bulgarian language) Executive Summary (in Bulgarian language) Executive Summary (in Bulgarian language) Executive Summary (in Bulgarian language) 45 новата на този анализ е интегрирането и анализа на големи бази данни за обществени поръчки и корпоративна собственост. На второ място, измерването на институционалните предпоставки за завладяването на държавата е допълнено с методика за мониторинг на прилагането на политиките за противодействие на корупцията в ключови регулаторни и надзорни институции чрез прилагане на експертни оценки. Макар и различни по характер, съчетаването на изводите от прилагането на тези два отделни изследователски инструмента върху едно и също явление позволява идентифицирането на рискове и уязвимости, които невина­ ги са откриваеми с един-единствен аналитичен инструмент. Резултатите създават условия за извършването на наблюдения и анализи, както и за консултирането на управленски решения в отделните публични инсти­ туции или дори компании. Представени са резултатите и изводите от прилагането на три взаимно допълващи се методики и съответните им изследователски инструменти (вж. схемата по-долу). • Диагностична оценка на завладяването на държавата на секторно ниво (СКАД–секторно ниво). С  този инструмент са оценени, посредством индекси, базирани на експертни оценки, рисковете от завладяване на държавата и уязвимостите на секторно ниво. Специално внимание е обърнато също върху неефективността на политиките за противодействие на корупцията, липсата на прозрачност, липсата на безпристрастност и наличието на пристрастност към частни интереси. • Диагностична оценка на завладяването на държавата на секторно ниво (СКАД–секторно ниво). С  този инструмент са оценени, посредством индекси, базирани на експертни оценки, рисковете от завладяване на държавата и уязвимостите на секторно ниво. Специално внимание е обърнато също върху неефективността на политиките за противодействие на корупцията, липсата на прозрачност, липсата на безпристрастност и наличието на пристрастност към частни интереси. • Анализ на рисковете от завладяване на държавата и корупционните практики в областта на обществените поръчки посредством „червени флагове“. Анализът се основава на интегрирани бази данни и се извършва посредством специално разработена интерактивна уеб-базирана платформа76. С платформата се прилага за пръв път триизмерен подход към анализа на рисковете от завладяване на държавата и уязвимостите, свързани както с купувачите (възлагащите органи), така и с доставчиците (компаниите). Анализът се извършва чрез интеграция на три отделни бази данни: за обществените поръчки, за финансовото състояние и собствеността на дружествата и за сигнали от медиите за подозирани нередности, свързани с обществените поръчки. Оценката се извърша въз основа на червени флагове, като всеки един флаг обозначава рискова ситуация, възникнала в резултат на корупция или завладяване на държавата. • Мониторинг на прилагането на политики за противодействие на корупцията (МАКПИ)77. 76 https://analytics.scemaps.eu. Измерване на завладяването на държавата в икономическите сектори Разработен въз основа на анализи на корупцията и завладяването на държавата в няколко европейски страни през последното десетилетие, СКАД измерва резултатите и последствията от завладяването на бизне­ са, както и факторите или предпоставките, определящи институцио­ налната и обществена среда. Освен това инструментът създава условия за по-подробно изследване на пътищата за оказване на влияние върху отделни публични институции, икономически сектори или стопански организации, което на свой ред подпомага усъвършенстването на съот­ ветните институционални и секторни политики за противодействие. На­ стоящия доклад представя изводите от анализа на основните измерения на завладяването на бизнеса и определящите институционални предпос­ тавки на секторно ниво в няколко икономически отрасъла (строителство, търговия на едро с горива и търговия на едро с лекарствени средства) в четири европейски държави (България, Испания, Италия, и Румъния.) За целите на изследването на завладяването на държавата на секторно равнище в СКАД методиката са добавени два важни елемента. На първо място, предвид важността на привилегирования достъп до обществени поръчки като част от способите за завладяване на държавата чрез биз­ неса, в доклада са анализирани рисковете от завладяване на държавата и корупционните практики в областта на обществените поръчки. В ос­ Executive Summary (in Bulgarian language) Executive Summary (in Bulgarian language) Този инструмент е използван за определянето на уязвимостите и евентуалните опасности, свързани с наличието на риск от корупционни практики по високите етажи на властта в отделните публични институции (идентифицирани посредством СКАД – секторно ниво като ключови за регулирането на 46 State Capture Deconstructed Оценка на завладяването на държавата на секторно ниво – концепция и изследователски инструменти. Източник: Център за изследване на демокрацията, 2021. Липса на прозрачност Липса на непредубеденост Предпочитание на частни интереси Неефективност на антикоруп- ционните политики Лобистки закони Привилигирован достъп до обществени поръчки Концентрация на преки субсидии Измерения на завладяването на държавата Завладяване на бизнеса МАКПИ СКАД секторно ниво Анализ на рисковете чрез „червени флагове“ Монополизация Привилигирован статут (селективно налагане на контрол и санкции) Неефективност на антимонополното законодателство Институционални предпоставки отделните сектори), от една страна, и липсата на антикорупционни политики за преодоляване на тези рискове, от друга. Оценката посредством СКАД на завладяването на държавата на нацио­ нално ниво осигурява ценна информация за уязвимите области в ико­ номиката като цяло. От друга страна специфичните секторни инстру­ менти на СКАД - секторно ниво имат по-голямо практическо значение на равнището на икономическите сектори поради техните специфични характеристики. Освен това, източниците на информация (включително експертните знания и похвати, използвани в инструмента МАКПИ), а също и уязвимостите и слабостите на политиките, са различни за отдел­ ните сектори и съответно оценките на секторно ниво осигуряват по-на­ деждни резултати. Оценка на завладяването на държавата на секторно ниво – концепция и изследователски инструменти. Източник: Център за изследване на демокрацията, 2021. Липса на прозрачност Липса на непредубеденост Предпочитание на частни интереси Неефективност на антикоруп- ционните политики Лобистки закони Привилигирован достъп до обществени поръчки Концентрация на преки субсидии Измерения на завладяването на държавата Завладяване на бизнеса МАКПИ СКАД секторно ниво Анализ на рисковете чрез „червени флагове“ Монополизация Привилигирован статут (селективно налагане на контрол и санкции) Неефективност на антимонополното законодателство Институционални предпоставки завладяването на държавата на секторно ниво – концепция и изследователски инструменти. Монополизация МАКПИ СКАД секторно ниво Източник: Център за изследване на демокрацията, 2021. отделните сектори), от една страна, и липсата на антикорупционни политики за преодоляване на тези рискове, от друга. отделните сектори), от една страна, и липсата на антикорупционни политики за преодоляване на тези рискове, от друга. Оценката посредством СКАД на завладяването на държавата на нацио­ нално ниво осигурява ценна информация за уязвимите области в ико­ номиката като цяло. Executive Summary (in Bulgarian language) Executive Summary (in Bulgarian language) От друга страна специфичните секторни инстру­ менти на СКАД - секторно ниво имат по-голямо практическо значение на равнището на икономическите сектори поради техните специфични характеристики. Освен това, източниците на информация (включително експертните знания и похвати, използвани в инструмента МАКПИ), а също и уязвимостите и слабостите на политиките, са различни за отдел­ ните сектори и съответно оценките на секторно ниво осигуряват по-на­ деждни резултати. отделните сектори), от една страна, и липсата на антикорупционни политики за преодоляване на тези рискове, от друга. Оценката посредством СКАД на завладяването на държавата на нацио­ нално ниво осигурява ценна информация за уязвимите области в ико­ номиката като цяло. От друга страна специфичните секторни инстру­ менти на СКАД - секторно ниво имат по-голямо практическо значение на равнището на икономическите сектори поради техните специфични характеристики. Освен това, източниците на информация (включително експертните знания и похвати, използвани в инструмента МАКПИ), а също и уязвимостите и слабостите на политиките, са различни за отдел­ ните сектори и съответно оценките на секторно ниво осигуряват по-на­ деждни резултати. отделните сектори), от една страна, и липсата на антикорупционни политики за преодоляване на тези рискове, от друга. Оценката посредством СКАД на завладяването на държавата на нацио­ нално ниво осигурява ценна информация за уязвимите области в ико­ номиката като цяло. От друга страна специфичните секторни инстру­ менти на СКАД - секторно ниво имат по-голямо практическо значение на равнището на икономическите сектори поради техните специфични характеристики. Освен това, източниците на информация (включително експертните знания и похвати, използвани в инструмента МАКПИ), а също и уязвимостите и слабостите на политиките, са различни за отдел­ ните сектори и съответно оценките на секторно ниво осигуряват по-на­ деждни резултати. 78 Stoyanov, A., Gerganov, A., and Yalamov, T., State Capture Assessment Diagnostics, Sofia: Center for the Study of Democracy, 2019. EXCUTIVE SUMMARY (IN ITALIAN LANGUAGE) Il concetto di “cattura dello stato” (state capture) è da molto tempo utilizzato per descrive le pratiche delle imprese private che manipolano le politiche e i processi decisionali del governo a loro favore. Di solito si riferisce all’attu­ azione di una serie di singoli atti di corruzione al più alto livello governati­ vo. Tuttavia, le tendenze in un certo numero di paesi, sia europei che non, mostrano che questa pratica non si limitata più alle irregolarità nel funzion­ amento di una determinata istituzione pubblica, ma si è trasformata in un comportamento istituzionale permanente e resistente alle politiche anticor­ ruzione standardizzate e generali. Nel presente rapporto sono presentati i ri­ sultati dell’implementazione dell’innovativo strumento analitico State Capture Assessment Diagnostics – SCAD (Valutazione diagnostica della cattura dello stato) a livello settoriale, che fornisce risultati rilevanti sulle policy in tema di cattura dello stato, caratterizzando questo fenomeno come un fallimento sistemico della governance pubblica. Dalle azioni isolate alla pratica sistematica La nuova realtà, in cui la cattura dello stato è di fatto una privatizzazione su larga scala delle decisioni di governo e la monopolizzazione di interi settori economici, richiede l’applicazione di nuovi strumenti analitici al fine di soste­ nere lo sviluppo di politiche di buon governo. SCAD rivela lo sfruttamento sistematico e costante dei poteri di governo a favore di interessi privati ​at­ traverso vari tipi di atti corruttivi e illeciti78. Le modalità per catturate lo stato includono la padronanza dell’applicazione della legislazione, l’accesso privi­ legiato ai fondi pubblici, il controllo asimmetrico sul settore dei media e quel­ lo finanziario, l’impatto sulla politica interna ed estera, ecc. SCAD mostra il meccanismo attraverso il quale la preparazione, l’adozione e l’applicazione di atti normativi e regolatori viene imbrigliata al servizio di quelli che catturano lo stato – soggetti privilegiati, che godono di immeritati vantaggi economici e/o politici. SCAD rivela come la debolezza dei meccanismi di gestione crei le condizioni per la cattura dello stato in quattro direzioni (economia, istituzioni, politica e mercato nero) attraverso due facilitatori (o fattori abilitanti). Questi ulti­ mi si riferiscono alle caratteristiche istituzionali e ambientali che incidono sul sistema di governance consentendo o facilitando la cattura dello stato (vedi il diagramma sotto). 48 State Capture Deconstructed Schema per la cattura dello stato Fonte: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. Fattori istituzionali che contribuiscono la cattura dello stato Fattori ambientali che contribuiscono alla cattura dello stato Corruzione e inefcacia delle politiche anticorruzione Media Cattura del settore imprenditoriale Cattura istituzionale Cattura del mercato nero Cattura politica Monopolizzazione Leggi sul lobbismo Status privilegiato (controllo selettivo e sanzioni) Concentrazione dei sussidi pubblici diretti IInefcacia delle leggi antitrust Mancanza di trasparenza Mancanza di imparzialità Pregiudizio a favore di interessi privati Corruzione amministrativa Corruzione nel sistema giudiziario Accesso privilegiato agli appalti pubblici Direzioni cattura dello stato Schema per la cattura dello stato Monopolizzazione Pregiudizio a favore di interessi privati Concentrazione dei sussidi pubblici diretti Corruzione nel sistema giudiziario Media Corruzione amministrativa IInefcacia delle leggi antitrust Fonte: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. te: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnosti https://analytics.scemaps.eu. 80 Originariamente sviluppato e implementato come strumento autonomo, il MACPI è ora inte­ grato nel quadro metodologico per valutare la cattura dello stato a livello settoriale. Si veda: Stoyanov A. et al, Monitoring Anti-Corruption in Europe. Bridging Policy Evaluation and Corrup­ tion Measurement, Sofia: Center for the Study of Democracy, 2015. 79 https://analytics.scemaps.eu. Specializzazione dello strumento: focalizzazione alla cattura di settori Sviluppato sulla base di analisi della corruzione e della cattura dello stato in diversi paesi europei che coprono un intero decennio, SCAD misura i risul­ tati e le conseguenze dello state capture, nonché i facilitatori istituzionali e ambientali a livello nazionale. Inoltre, lo strumento consente di approfon­ dire come vengano colpite le singole istituzioni pubbliche, i settori economici e le organizzazioni imprenditoriali, contribuendo al perfezionamento delle relative politiche istituzionali e settoriali. In questo rapporto sono presentati i risultati dell’analisi, realizzata valutando a livello settoriale le principali dimensioni della cattura dello stato e le caratteristiche istituzionali che la definiscono in diversi settori economici (edilizia, commercio all’ingrosso di combustibili e medicinali) e in quattro paesi europei (Bulgaria, Italia, Roma­ nia e Spagna). Ai fini del nuovo livello (settoriale) di analisi, sono stati aggiunti alla metod­ ologia due elementi importanti. In primo luogo, data l’importanza dell’acces­ so privilegiato agli appalti pubblici nell’ambito delle modalità per la cattura dello stato da parte dei privati, il rapporto analizza sulla base dell’integrazi­ one di big data i rischi di cattura dello stato e le pratiche corruttive nel cam­ po degli appalti pubblici. In secondo luogo, la misurazione dei facilitatori istituzionali attraverso indici basati su valutazioni di esperti è stata integra­ ta da una metodologia per il monitoraggio dell’attuazione delle politiche Executive Summary (in Italian language) Executive Summary (in Italian language) 49 anticorruzione nelle principali istituzioni di regolamentazione e vigilanza, sempre basata sulla valutazione degli esperti. Sebbene di natura diversa, la combinazione dei risultati di queste due analisi consente di identificare rischi e vulnerabilità non sempre rilevabili con un unico strumento analitico. Inol­ tre, i risultati creano le condizioni per condurre osservazioni e analisi robuste, nonché per fornire consigli relativamente alle decisioni gestionali a livello di singole istituzioni pubbliche o di aziende. I risultati sono stati ottenuti applicando tre metodologie tra loro complemen­ tari e i rispettivi strumenti di ricerca (si veda lo schema sotto). • State Capture Assessment Diagnostics on Economic Sector Level (SCAD-ESL) (Valutazione diagnostica della cattura dello stato a liv­ ello settoriale). Tramite questo strumento sono stati valutati, utilizzando indici basati su valutazioni di esperti, i rischi e le vulnerabilità di cattura dello stato a livello settoriale. Specializzazione dello strumento: focalizzazione alla cattura di settori Particolare attenzione è rivolta anche all’inef­ ficacia delle politiche anticorruzione, alla mancanza di integrità, alla man­ canza di imparzialità e alla presenza di pregiudizi a favore degli interessi privati.tt • State Capture Assessment Diagnostics on Economic Sector Level (SCAD-ESL) (Valutazione diagnostica della cattura dello stato a liv­ ello settoriale). Tramite questo strumento sono stati valutati, utilizzando indici basati su valutazioni di esperti, i rischi e le vulnerabilità di cattura dello stato a livello settoriale. Particolare attenzione è rivolta anche all’inef­ ficacia delle politiche anticorruzione, alla mancanza di integrità, alla man­ canza di imparzialità e alla presenza di pregiudizi a favore degli interessi privati. • Analisi dei rischi di cattura dello stato e di pratiche corruttive nel campo degli appalti pubblici attraverso “segnali d’allarme” (red flags). L’analisi si basa su dati integrati e viene eseguita utilizzando una piattaforma web interattiva appositamente sviluppata.79 La piattaforma utilizza per la pri­ ma volta un approccio tridimensionale all’analisi dei rischi e delle vulner­ abilità di cattura dello stato legati sia agli acquirenti (l’amministrazione aggiudicatrice) che ai fornitori (le aziende). L’analisi viene effettuata attra­ verso l’integrazione di dati sugli appalti pubblici, di informazioni sulla condizione finanziaria e sulla proprietà delle aziende e di segnali dai me­ dia per sospette irregolarità relative agli appalti pubblici. La valutazione viene effettuata sulla base di specifici segnali d’allarme, ciascuno dei quali indica una situazione di rischio che potrebbe essere legata a episodi di corruzione o cattura dello stato. • Monitoring Anticorruption Policy Implementation (MACPI) (Monitor­ aggio dell’attuazione delle politiche anticorruzione)80. Questo strumen­ to è stato utilizzato per identificare le vulnerabilità e le potenziali minacce legate al rischio di pratiche corruttive ad alto livello nelle singole istituzi­ oni pubbliche (identificate tramite la SCAD-ESL come chiave per la regol­ amentazione dei singoli settori), da un lato, e la mancanza di politiche an­ ticorruzione per far fronte a questi rischi, dall’altro. 50 State Capture Deconstructed Se da un lato la valutazione della cattura dello stato a livello nazionale attraverso la SCAD fornisce preziose informazioni sulle aree vulnerabili dell’economia nel suo complesso, dall’altro gli strumenti settoriali sono di maggiore importanza pratica al livello dei settori economici per le loro caratteristiche specifiche. Inoltre, le fonti di informazione (comprese le conoscenze specialistiche e le tecniche utilizzate nel MACPI), nonché le vulnerabilità e le debolezze delle politiche, variano da settore a settore e, di conseguenza, le valutazioni a livello settoriale forniscono risultati più affidabili e robusti. Specializzazione dello strumento: focalizzazione alla cattura di settori Valutazione della cattura dello stato a livello settoriale – concetto e strumenti di ricerca. Fonte: Center for the Study of Democracy 2021. Fattori istituzionali che contribuiscono la cattura dello stato Corruzione e inefcacia delle politiche anticorruzione Cattura del settore imprenditoriale Monopolizzazione Leggi sul lobbismo Status privilegiato (controllo selettivo e sanzioni) Concentrazione dei sussidi pubblici diretti IInefcacia delle leggi antitrust Mancanza di trasparenza Mancanza di imparzialità Pregiudizio a favore di interessi privati Accesso privilegiato agli appalti pubblici Direzioni cattura dello stato MACPI SCAD-ESL valutazioni di esperti Analisi dei rischi – "segnali d’allarme" Valutazione della cattura dello stato a livello settoriale – concetto e strumenti di ricerca. Valutazione della cattura dello stato a livello settoriale  concetto e strumenti di ricerca. Fattori istituzionali che contribuiscono la cattura dello stato Corruzione e inefcacia delle politiche anticorruzione Cattura del settore imprenditoriale Monopolizzazione Leggi sul lobbismo Status privilegiato (controllo selettivo e sanzioni) Concentrazione dei sussidi pubblici diretti IInefcacia delle leggi antitrust Mancanza di trasparenza Mancanza di imparzialità Pregiudizio a favore di interessi privati Accesso privilegiato agli appalti pubblici Direzioni cattura dello stato MACPI SCAD-ESL valutazioni di esperti Analisi dei rischi – "segnali d’allarme" Monopolizzazione SCAD-ESL valutazioni di esperti Fonte: Center for the Study of Democracy 2021. Fonte: Center for the Study of Democracy 2021. Se da un lato la valutazione della cattura dello stato a livello nazionale attraverso la SCAD fornisce preziose informazioni sulle aree vulnerabili dell’economia nel suo complesso, dall’altro gli strumenti settoriali sono di maggiore importanza pratica al livello dei settori economici per le loro caratteristiche specifiche. Inoltre, le fonti di informazione (comprese le conoscenze specialistiche e le tecniche utilizzate nel MACPI), nonché le vulnerabilità e le debolezze delle politiche, variano da settore a settore e, di conseguenza, le valutazioni a livello settoriale forniscono risultati più affidabili e robusti. 81 Stoyanov, A., Gerganov, A., and Yalamov, T., State Capture Assessment Diagnostics, Sofia: Center for the Study of Democracy, 2019. EXCUTIVE SUMMARY (IN ROMANIAN LANGUAGE) Noţiunea “capturarea statului” a fost folosită de mult timp pentru a descrie practici prin care diferitele interese private ale mediului de afaceri manipulează politicile de stat şi procesele de luare a deciziilor, în propriul beneficiu. De obicei, se referea la o serie întreagă de acte de corupţie la nivel înalt, în mediul guvernamental. Tendinţele din multe ţări europene, dar şi din alte state, arată că aceste practici nu se mai limitează doar la simple deviații în funcţionarea diferitelor instituţii publice, ci au devenit un comportament instituţional durabil, rezistent la aplicarea politicilor standard, generale pentru combaterea corupţiei. În prezentul raport sunt prezentate rezultate obţinute în urma aplicării unor instrumente inovatoare de analiză la nivel sectorial, precum Diagnosticul privind evaluarea capturării statului (SCAD), care oferă constatări relevante de politici publice despre capturarea statului, caracterizând-o ca eşecul sistematic al administrării publice. De la accident la practica sistematică Realitatea nouă, în care capturarea statului poate fi caracterizată ca privatizare de facto de amploare a deciziilor guvernamentale şi o monopolizare a unor întregi sectoare economice, necesită dezvoltarea unor instrumente noi de analiză, care să vină în ajutorul elaborării politicilor de bună guvernare. SCAD arată exploatarea sistematică şi constantă a puterii guvernamentale în beneficiul unor interese private81, care implică diferite tipuri de acte de corupţie şi activitate nelegitimă. Modalităţile de capturare a statului includ controlul asupra aplicării legislaţiei, acces privilegiat la fonduri publice, control asimetric asupra sectorului mass media şi a celui financiar, influență asupra politicii interne şi externe a statului etc. SCAD ne arată mecanismele prin care elaborarea, adoptarea şi aplicarea actelor normative şi a altor reglemenări la nivel guvernamental, funcţionează în beneficiul unui număr mic de entități care au intenția de a captura statul; adică în favoarea unor actori privilegiaţi care beneficiază de beneficii economice şi/sau politice care nu li se cuvin. SCAD dezvăluie modul în care capturarea de stat este facilitată de mecanisme de guvernanță slabe prin evidențierea a patru dimensiuni (afaceri, instituționale, politice și piața neagră) și două tipuri de facilitatori, care se referă la caracteristicile instituționale și de mediu care afectează sistemul de guvernare, permițând sau facilitarea capturării (vezi figura de mai jos). 52 State Capture Deconstructed Schema capturării statului Sursa: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. Facilitatori instituționali Facilitatori de mediu Corupţia şi inefcienţa politicilor anticorupție Mass media Dimensiunile capturării statului Capturarea afacerilor Capturarea instituțională Capturarea pieței neagră Capturarea politică Monopolizare Legi susținute de lobbyiști Statut privilegiat (control şi sancţiuni selective) Concentrarea unor subvenţii directe Inefciența legilor anti-monopol Lipsa de transparenţă Lipsa de imparţialitate Comportamentul părtinitor față de interesele private Corupție administrativa Corupţia în sistemul judiciar Acces privilegiat la achiziţii publice Schema capturării statului Lipsa de imparţialitate Capturarea instituțională Concentrarea unor subvenţii directe Corupţia în sistemul judiciar Corupție administrativa Inefciența legilor anti-monopol Sursa: Stoyanov, Gerganov, and Yalamov, State Capture Assessment Diagnostics, Center for the Study of Democracy, 2019. https://analytics.scemaps.eu. 83 Iniţial a fost elaborat şi aplicat ca un instrument de sine stătător, iar acum a fost integrat în cadrul metodologic de evaluare a capturii statului la nivel de sector. Vezi Stoyanov A. et al, Monitoring Anti-Corruption in Europe. Bridging Policy Evaluation and Corruption Measurement, Sofia: Center for the Study of Democracy, 2015. 82 https://analytics.scemaps.eu. Dezvoltarea instrumentului: concentrarea asupra capturii sectoriale Bazată pe o analiză de zece ani a corupției și capturării statului în mai multe țări europene, SCAD măsoară rezultatele și efectele capturării afacerilor, precum și facilitatorii instituționali și de mediu la nivel național. În plus, instrumentul permite o examinare mai atentă a modului în care sunt afectate instituțiile publice, sectoarele economice și organizațiile de afaceri individuale, sporind astfel eficacitatea politicilor instituționale și sectoriale respective. Acest raport prezintă concluziile unei astfel de examinări prin evaluarea la nivel sectorial a dimensiunii esențiale a capturării afacerilor și a caracteristicilor instituționale care o facilitează în mai multe sectoare economice (construcția și comerțul cu ridicata al combustibililor și al produselor farmaceutice) din patru țări europene (Bulgaria, Italia, România și Spania). Noua metodologie la nivel sectorial adaugă două elemente importante. În primul rând, luându-se în calcul importanţa accesului privilegiat la achiziţii publice ca parte a dimensiunii capturării afacerilor, în cadrul raportului sunt analizate riscurile de capturare a statului şi practicile corupte din domeniul achiziţiilor publice, pe baza analizei big data. Apoi, măsurarea factorilor instituţionali prin evaluări ale experților bazate pe indecși, este completată şi de o metodologie care implică monitorizarea aplicării politicilor anticorupție în cadrul unor instituţii cheie cu rol de reglementare şi de supraveghere, identificate prin evaluări făcute de experţi. Cu toate că au un Executive Summary (in Romanian language) Executive Summary (in Romanian language) Executive Summary (in Romanian language) Executive Summary (in Romanian language) 53 caracter diferit, combinarea concluziilor celor două cercetări, legate de acelaşi fenomen, ne dă posibilitatea să identificăm riscurile şi punctele vulnerabile, care nu întotdeauna pot fi depistate printr-un singur instrument de analiză. De asemenea, rezultatele creează posibilități pentru observarea şi analizarea fenomenului, dar și pentru consiliere la nivelul managamentului din cadrul unei organizații publice sau companii. De asemenea, rezultatele creează posibilități pentru observarea şi analizarea fenomenului, dar și pentru consiliere la nivelul managamentului din cadrul unei organizații publice sau companii. Concluziile sunt rezultate ale aplicării unui număr de trei metodologii complementare și a instrumentelor lor de cercetare corespunzătoare (vezi schema de mai jos): • Diagnosticul de Evaluare a Capturării Statului la Nivel de Sectoare Economice (SCAD–ESL). Prin acest instrument au fost evaluate, cu ajuto­ rul indecșilor bazați pe evaluări făcute de experţi, riscurile de capturare şi punctele vulnerabile la nivel de sector. Instrumentul analizează cu atenție ineficienţa politicilor anticorupție, lipsa de integritate, de imparţialitate şi comportamentul părtinitor față de interesele private • Analiza riscurilor de capturare a statului, a practicilor corupte din do­ meniul achiziţiilor publice, prin intermediul factorilor de risc (red flags), bazați pe date integrate şi implementat printr-o platformă web interac­ tivă, special dezvoltată82. Platforma deschide drumuri printr-o abordare tridimensională pentru analiza riscurilor și vulnerabilităților legate de capturarea statului atât din perspectiva cumpărătorilor (autorități contrac­ tante), cât și a furnizorilor (companii), combinând date privind achizițiile publice, informații financiare și legate de proprietate ale companiei și un sistem de alertă media, care identifică presupuse cazuri de abateri legate de achiziții. Evaluarea se efectuează pe baza unei combinații de factori de risc, fiecare indicând o situație de risc care ar putea fi rezultatul corupției sau al capturării statului.i p • Monitorizarea aplicării politicilor anticorupție (MACPI)83 identifică vulnerabilitățile și decalajele potențiale dintre practicile de corupție cu risc ridicat din instituțiile publice individuale (identificate ca esențiale pentru reglementarea sectoarelor prin SCAD-ESL) și disponibilitatea poli­ ticilor anticorupție care abordează aceste riscuri. Apoi, evaluează ușurin­ ța implementării, implementarea efectivă și aplicarea ulterioară a acestor politici. 54 State Capture Deconstructed În timp ce evaluarea capturării statului la nivel național prin aplicarea SCAD oferă cunoștințe valoroase despre zonele vulnerabile din întreaga economie, instrumentele sale specifice sectoriale au o relevanță practică mai mare la nivelul sectoarelor economice datorită caracteristicilor lor particulare. Sursa: Center for the Study of Democracy 2021. EXCUTIVE SUMMARY (IN SPANISH LANGUAGE) La noción de captura del Estado lleva circulando desde hace décadas y describe aquellas prácticas en las que intereses empresariales privados manipulan las políticas del Estado, así como los procesos de toma de decisiones, a su favor. Por lo general, se trata de la práctica de una serie de actuaciones corruptivas a alto nivel gubernamental. No obstante, las tendencias en varios países europeos y en terceros países demuestran que esta práctica ya no se está limitando a las irregularidades en el funcionamiento de una u otra institución pública, sino que se ha transformado en una conducta institucional permanente que no está sometida a las políticas universalmente admitidas para contrarrestar la corrupción. En este informe se presentan los resultados de la aplicación de la herramienta analítica innovadora, denominada Evaluación de Diagnóstico para la Captura del Estado (SCAD, por sus siglas en inglés ). Esta ofrece unas conclusiones relevantes acerca de la captura del Estado precisamente como un fracaso sistemático de la gestión pública. Executive Summary (in Romanian language) Executive Summary (in Romanian language) De asemenea, multe dintre sursele de informații (inclusiv cunoștințele și know- how-ul experților care iau parte la aplicarea instrumentului MACPI), precum și vulnerabilitățile și lacunele de politici publice, diferă în funcție de sectoare și, prin urmare, evaluarea sectorială produce rezultate mai robuste și mai fiabile. Evaluarea capturii statului la nivel de sector – conceptul şi instrumentele de cercetare Sursa: Center for the Study of Democracy 2021. Facilitatori instituționali Corupţia şi inefcienţa politicilor anticorupție Dimensiunile capturării statului Capturarea afacerilor Monopolizare Legi susținute de lobbyiști Statut privilegiat (control şi sancţiuni selective) Concentrarea unor subvenţii directe Inefciența legilor anti-monopol Lipsa de transparenţă Lipsa de imparţialitate Comportamentul părtinitor față de interesele private Acces privilegiat la achiziţii publice MACPI SCAD ESL evaluarea experților Red-fags analiza de risc Evaluarea capturii statului la nivel de sector – conceptul şi instrumentele de cercetare Facilitatori instituționali Corupţia şi inefcienţa politicilor anticorupție Dimensiunile capturării statului Capturarea afacerilor Monopolizare Legi susținute de lobbyiști Statut privilegiat (control şi sancţiuni selective) Concentrarea unor subvenţii directe Inefciența legilor anti-monopol Lipsa de transparenţă Lipsa de imparţialitate Comportamentul părtinitor față de interesele private Acces privilegiat la achiziţii publice MACPI SCAD ESL evaluarea experților Red-fags analiza de risc Evaluarea capturii statului la nivel de sector – conceptul şi instrumentele de cercetare S C f h S d f D 2021 Facilitatori instituționali Corupţia şi inefcienţa politicilor anticorupție Dimensiunile capturării statului Capturarea afacerilor Monopolizare Legi susținute de lobbyiști Statut privilegiat (control şi sancţiuni selective) Concentrarea unor subvenţii directe Inefciența legilor anti-monopol Lipsa de transparenţă Lipsa de imparţialitate Comportamentul părtinitor față de interesele private Acces privilegiat la achiziţii publice MACPI SCAD ESL evaluarea experților Red-fags analiza de risc Evaluarea capturii statului la nivel de sector – conceptul şi instrumentele de cercetare Monopolizare Lipsa de imparţialitate Inefciența legilor anti-monopol SCAD ESL evaluarea experților Sursa: Center for the Study of Democracy 2021. Sursa: Center for the Study of Democracy 2021. În timp ce evaluarea capturării statului la nivel național prin aplicarea SCAD oferă cunoștințe valoroase despre zonele vulnerabile din întreaga economie, instrumentele sale specifice sectoriale au o relevanță practică mai mare la nivelul sectoarelor economice datorită caracteristicilor lor particulare. De asemenea, multe dintre sursele de informații (inclusiv cunoștințele și know- how-ul experților care iau parte la aplicarea instrumentului MACPI), precum și vulnerabilitățile și lacunele de politici publice, diferă în funcție de sectoare și, prin urmare, evaluarea sectorială produce rezultate mai robuste și mai fiabile. 84 Stoyanov, A., Gerganov, A. y Yalamov, T., State Capture Assessment Diagnostics, Sofia: Centro de Investigación de la Democracia, 2019. Desde las actuaciones aisladas hasta la práctica sistemática La nueva realidad, en la cual la captura del Estado prácticamente representa ya la privatización a gran escala de las decisiones gubernamentales y la monopolización de sectores económicos en su totalidad, requiere la aplicación de nuevas herramientas de análisis que contribuyan al desarrollo de políticas de buena gestión. SCAD revela la explotación sistemática y permanente de los poderes gubernamentales a favor de intereses privados mediante diversos tipos de acciones corruptivas y contrarias a la ley84. Estos métodos incluyen la captura de la aplicación de las leyes, el acceso privilegiado a recursos públicos, el control asimétrico sobre los sectores mediático y financiero, la influencia sobre la política nacional e internacional, etc. SCAD muestra el mecanismo a través del cual la elaboración, la aprobación y la aplicación de las disposiciones legales y de las normas se han sometido a favor de aquellos que capturan el Estado: sujetos privilegiados que disfrutan de beneficios económicos y/o políticos. SCAD revela la manera en que la debilidad de los mecanismos de gestión va creando las condiciones para capturar el Estado en cuatro direcciones (economía, instituciones, política y mercado negro) a través de dos tipos de factores que la favorecen (enablers). Estos factores ejercen influencia sobre el entorno institucional y social donde se realiza la gestión, facilitando así la captura del Estado (véase el esquema más abajo). 56 State Capture Deconstructed Esquema para la captura del Estado Fuente: Stoyanov, Gerganov y Yalamov, State Capture Assessment Diagnostics, Centro de Investigación de la Democracia, 2019. Factores institucionales que favorecen la CE Factores del entornoque favorecen la CE Corrupción e inefciencia de las políticas anticorruptivas Medios de comunicación Captura del Estado direcciones Captura del sector de los negocios Captura institucional Captura de mercado negro Captura política Monopolización Leyes creadas por grupos de presión Categoría privilegiada (control selectivo y sanciones) Concentración de las subvenciones públicas directas Inefcacia de las leyes antimonopólicas Falta de transparencia Falta de imparcialidad Parcialidad hacia los intereses privados Corrupción administrativa Corrupción en el sistema judicial Acceso privilegiado a la contratación pública Esquema para la captura del Estado Monopolización Acceso privilegiado a la contratación pública Captura institucional Corrupción en el sistema judicial Medios de comunicación Corrupción administrativa Inefcacia de las leyes antimonopólicas Factores del entornoque favorecen la CE Fuente: Stoyanov, Gerganov y Yalamov, State Capture Assessment Diagnostics, Centro de Investigación de la Democracia, 2019 nte: Stoyanov, Gerganov y Yalamov, State Capture Assessment Diagnostics Especialización de la herramienta: enfoque sobre la captura de los sectores Elaborada sobre la base del análisis de la corrupción y la captura del Estado en varios países europeos que abarcan toda una década, SCAD mide los resultados y las consecuencias de la captura de las empresas, así como los factores que determinan el entorno institucional y social. Además, la herramienta va creando las condiciones para una investigación más detallada sobre las formas de ejercer influencia sobre determinadas instituciones públicas, sectores u organizaciones de la economía, que, por su parte, contribuye al perfeccionamiento de las políticas institucionales y sectoriales correspondientes. En este informe se presentan precisamente las conclusiones de esta investigación. Las dimensiones principales de la captura de las empresas y las características institucionales determinantes han sido analizadas en varios sectores de la economía ( construcción, comercio mayorista de combustibles y medicamentos) y en cuatro países europeos (Bulgaria, Italia, Rumanía y España). Con miras a la importancia del acceso privilegiado a las contrataciones públicas, en este informe se han analizado los riesgos para la captura del Estado y las prácticas corruptivas en el ámbito de la contratación pública. Para ello, e n la base de este análisis está la integración de grandes bases de datos. En segundo lugar, la medición de los factores institucionales que la favorecen, a través de índices basados en evaluaciones de expertos, se ha Executive Summary (in Spanish language) Executive Summary (in Spanish language) 57 completado con la metodología del monitoreo de la aplicación de políticas anticorrupción en instituciones clave de regulación, supervisión, contratación e inspección. Una vez más gracias a las valoraciones de expertos. A pesar de que ambas difieren en su carácter, la combinación de las conclusiones de estas dos investigaciones permite identificar riesgos y vulnerabilidades que no siempre se pueden descubrir con una única herramienta . Además, los resultados van creando las condiciones apropiadas para realizar observaciones y análisis posteriores, así como para el asesoramiento sobre las soluciones de gestión y de política interna que se adoptarán a nivel de cada una de las instituciones públicas o incluso de las compañías. Se presentan los resultados y las conclusiones de tres métodos que se complementan y sus respectivas herramientas de investigación (véase el esquema más abajo). • Evaluación de Diagnóstico de la Captura del Estado a Nivel Sectorial (SCAD-ESL, por sus siglas en inglés). https://analytics.scemaps.eu. 86 Inicialmente desarrollado y aplicado como una herramienta independiente, ahora el MAPCC está integrado en el marco metodológico para la evaluación de la captura del Estado a nivel sectorial. Véase Stoyanov A. et al., Monitoring Anti-Corruption in Europe. Bridging Policy Eval­ uation and Corruption Measurement, Sofía: Centro de Investigación de la Democracia, 2015. 85 https://analytics.scemaps.eu. Especialización de la herramienta: enfoque sobre la captura de los sectores Con esta herramienta se han evalu­ ado los riesgos de captura y las vulnerabilidades a nivel sectorial mediante índices basados en evaluaciones de expertos. Se ha prestado una especial atención también a la ineficacia o ineficiencia de las políticas anticorrup­ ción, la falta de ética y la ausencia de imparcialidad. • Análisis de los riesgos de captura del Estado y las prácticas corruptivas en el ámbito de las contrataciones públicas mediante “banderas rojas”. El análisis está fundamentado en bases de datos integradas y se realiza a través de una plataforma interactiva, disponible una página web públi­ ca85. Aquí se aplica, por primera vez, una perspectiva tridimensional en el análisis de los riesgos de captura del Estado y las vulnerabilidades rel­ acionadas tanto con los compradores (las autoridades contratantes) como con los proveedores (las compañías). Un examen que se realiza mediante el cruzado de datos de licitaciones públicas, información financiera y de propiedad de las sociedades y alertas de los medios de comunicación so­ bre irregularidades en el proceso de contratación. La evaluación mediante banderas rojas, señalando cada una de ellas una situación de riesgo de corrupción o de captura del Estado. • Monitoreo de la Aplicación de Políticas para Contrarrestar la Corrup­ ción ( MACPI, por sus siglas en inglés)86. Esta herramienta ha sido utiliza­ da para determinar las vulnerabilidades y los posibles riesgos de prácticas corruptivas a alto nivel en cada institución pública (identificadas mediante la SCAD-ESL como claves para la regulación de cada uno de los sectores), por una parte, y, por otra parte, la falta de políticas anticorrupción para superar estos riesgos. 58 State Capture Deconstructed Mientras que, por una parte, la evaluación de la captura del Estado a nivel nacional mediante SCAD asegura una información valiosa sobre los ámbitos vulnerables de la economía en su totalidad, las herramientas sectoriales específicas, por otra parte, tienen una mayor importancia práctica a nivel sectorial , debido a sus características específicas. Además, las fuentes de información (incluidos los conocimientos y las prácticas de expertos que se emplean en la herramienta MACPI ), así como las vulnerabilidades y las debilidades de las políticas anticorrupción, son diferentes para cada uno de los sectores, y, respectivamente, las evaluaciones a nivel sectorial aseguran resultados más fiables. Evaluación de la captura del Estado a nivel sectorial: planteamiento y herramientas de investigación Fuente: Centro de Investigación de la Democracia (CID), 2021. Especialización de la herramienta: enfoque sobre la captura de los sectores Factores institucionales que favorecen la CE Corrupción e inefciencia de las políticas anticorruptivas Captura del Estado direcciones Captura del sector de los negocios Monopolización Leyes creadas por grupos de presión Categoría privilegiada (control selectivo y sanciones) Concentración de las subvenciones públicas directas Inefcacia de las leyes antimonopólicas Falta de transparencia Falta de imparcialidad Parcialidad hacia los intereses privados Acceso privilegiado a la contratación pública MACPI SCAD ESL en evaluaciones de expertos Análisis de los riesgos – “banderas rojas” Evaluación de la captura del Estado a nivel sectorial: planteamiento y herramientas de investigación Factores institucionales que favorecen la CE Corrupción e inefciencia de las políticas anticorruptivas Captura del Estado direcciones Captura del sector de los negocios Monopolización Leyes creadas por grupos de presión Categoría privilegiada (control selectivo y sanciones) Concentración de las subvenciones públicas directas Inefcacia de las leyes antimonopólicas Falta de transparencia Falta de imparcialidad Parcialidad hacia los intereses privados Acceso privilegiado a la contratación pública MACPI SCAD ESL en evaluaciones de expertos Análisis de los riesgos – “banderas rojas” Evaluación de la captura del Estado a nivel sectorial: planteamiento y herramientas de investigación Monopolización Falta de imparcialidad SCAD ESL en evaluaciones de expertos Fuente: Centro de Investigación de la Democracia (CID), 2021. Fuente: Centro de Investigación de la Democracia (CID), 2021. Mientras que, por una parte, la evaluación de la captura del Estado a nivel nacional mediante SCAD asegura una información valiosa sobre los ámbitos vulnerables de la economía en su totalidad, las herramientas sectoriales específicas, por otra parte, tienen una mayor importancia práctica a nivel sectorial , debido a sus características específicas. Además, las fuentes de información (incluidos los conocimientos y las prácticas de expertos que se emplean en la herramienta MACPI ), así como las vulnerabilidades y las debilidades de las políticas anticorrupción, son diferentes para cada uno de los sectores, y, respectivamente, las evaluaciones a nivel sectorial aseguran resultados más fiables.
https://openalex.org/W2582161424
https://europepmc.org/articles/pmc5273847?pdf=render
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Equity aspects of the Primary Health Care Choice Reform in Sweden – a scoping review
International journal for equity in health
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* Correspondence: bo.burstrom@ki.se 1Department of Public Health Sciences, Equity and Health Policy Research Group, Karolinska Institutet, SE 171 77 Stockholm, Sweden Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Equity aspects of the Primary Health Care Choice Reform in Sweden – a scoping review Bo Burström1*, Kristina Burström1,2, Gunnar Nilsson3, Göran Tomson4, Margaret Whitehead1,5 and Ulrika Winblad6 Abstract Background: Good health and equal health care are the cornerstones of the Swedish Health and Medical Service Act. Recent studies show that the average level of health, measured as longevity, improves in Sweden, however, social inequalities in health remain a major issue. An important issue is how health care services can contribute to reducing inequalities in health, and the impact of a recent Primary Health Care (PHC) Choice Reform in this respect. This paper presents the findings of a review of the existing evidence on impacts of these reforms. Methods: We reviewed the published accounts (reports and scientific articles) which reported on the impact of the Swedish PHC Choice Reform of 2010 and changes in reimbursement systems, using Donabedian’s framework for assessing quality of care in terms of structure, process and outcomes. Results: Since 2010, over 270 new private PHC practices operating for profit have been established throughout the country. One study found that the new establishments had primarily located in the largest cities and urban areas, in socioeconomically more advantaged populations. Another study, adjusting for socioeconomic composition found minor differences. The number of visits to PHC doctors has increased, more so among those with lesser needs of health care. The reform has had a negative impact on the provision of services for persons with complex needs. Opinions of doctors and staff in PHC are mixed, many state that persons with lesser needs are prioritized. Patient satisfaction is largely unchanged. The impact of PHC on population health may be reduced. Conclusions: The PHC Choice Reform increased the average number of visits, but particularly among those in more affluent groups and with lower health care needs, and has made integrated care for those with complex needs more difficult. Resource allocation to PHC has become more dependent on provider location, patient choice and demand, and less on need of care. On the available evidence, the PHC Choice Reform may have damaged equity of primary health care provision, contrary to the tenets of the Swedish Health and Medical Service Act. This situation needs to be carefully monitored. Keywords: Equity, Inequalities, Health care need, Primary Health Care Choice Reform, Quality of care, Reimbursement system, Resource allocation © The Author(s). Burström et al. International Journal for Equity in Health (2017) 16:29 DOI 10.1186/s12939-017-0524-z Burström et al. International Journal for Equity in Health (2017) 16:29 DOI 10.1186/s12939-017-0524-z Burström et al. International Journal for Equity in Health (2017) 16:29 DOI 10.1186/s12939-017-0524-z Open Access Open Access The Swedish welfare state and health care system y Swedish welfare services, including health care services, were developed after World War II, with the aim of creating a comprehensive public system for provision of services, of high quality and universally accessible for all [5, 6]. The notion of providing good quality care services to the whole population has been regarded as part of the public welfare system in Sweden, providing high quality health care, schools, elder care and other social services to the entire population [5]. A universal welfare system, such as the Swedish health care system, requires the loyalty of the middle class in order to gain legitimacy and be sus- tained [7]. This in turn means that the services have to be of high quality to satisfy all users, an idea referred to by Rothstein [8] as “the high-quality standardized solution”. If successful, the system could then as intended promote egalitarianism and social integration. The proportion of GPs and level of resources going to PHC in Sweden is lower compared to other high-income countries [9]. However, in a recent Swedish government investigation, a well-functioning PHC was mentioned as “probably the single most important activity by which the health care system can contribute to improve equity in health” ([10] p 375). In international comparison, the average number of outpatient visits (to PHC as well as to specialist out- patient services) is considerably lower in Sweden than in other countries. In Sweden (and Finland) the average number of visits is about three per person and year, compared to about six for OECD countries [9]. This may be due to the organization of health care in Sweden, where a greater number of visits are done by nurses, and to the fact that Swedish health care is dominated by hospital based care. Nevertheless, access to outpatient care is considered to be lower in Sweden than in other countries. g g The Swedish health care system is tax funded and the responsibility for it is decentralized to county councils and regions, which collect taxes for the purpose [5]. As other parts of the welfare system, the health care system is based to a great extent on egalitarian principles, and the national level provides legislation and guidelines for health care. The Swedish welfare state and health care system The main objective for Swedish health care, as expressed in the Health and Medical Services Act [2] from 1982, is good health in the entire population and health on equal terms, and equitable care based on need. The bulk of health services have been operated by public providers, but in recent years the proportion of private (for-profit) providers has increased, particularly in out- patient care and PHC [5]. Abstract 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Burström et al. International Journal for Equity in Health (2017) 16:29 Page 2 of 10 Burström et al. International Journal for Equity in Health (2017) 16:29 Page 2 of 10 Background and comprehensive care, ranging from health promotion, disease prevention and curative care to rehabilitation. PHC deals with the whole population, the whole indi- vidual, the entire disease panorama, and the disease process over the entire life course of individuals. This distinguishes PHC from secondary and tertiary care, which provide important services at specific occasions of patients’ diseases. In many countries, including Sweden, primary health care (PHC) is the basis of the health care system and contributes in an important way to the improvement of health in the population [1]. Good health and equal health care are the cornerstones of the Swedish Health and Medical Service Act [2]. Recent studies show that the average level of health, measured as longevity, im- proves in Sweden, among men and women [3]. However, social inequalities in health remain a major issue, new health divides are surfacing and the Government has recently commissioned an investigation into how in- equalities in health can be reduced [4]. An important issue is how health care services can contribute to reducing inequalities in health, and the impact of re- cent reforms in PHC in this respect. This paper pre- sents the findings of a review of the existing evidence on impacts of these reforms, as well as identifying the gaps in the current literature that need to be addressed by new empirical studies. Public PHC centres in Sweden have been established in a planned manner in local residential catchment areas to serve the population, and typically include 4–10 general practitioners (GPs), nurses, other paramedical professionals (physiotherapists, occupational therapists, podiatrists). In addition many PHC centres provide maternal and child health services. PHC also includes outreach services, not least through district nurses, to patients who need such services, and interacts with other public authorities [5]. PHC also offers on-site medical care services to residents in local nursing homes. Although not a gate keeper to secondary care as in some other countries, PHC is the first point of contact for most patients in Sweden who end up in secondary care [5]. Results The review resulted in 6 scientific articles and 9 reports, which are presented in Table 1. The main objectives of the PHC Choice Reform were to increase patients’ choice of PHC provider, expand the provision of privately provided health care and increase quality and innovation through competition among providers [12]. An overview of the effects of the PHC Choice Reform based on Donabedian’s framework and different reimbursement systems is presented in Table 2. Donabedian’s framework for assessing quality of care [16], which distinguishes three aspects of quality in care: structure, process and outcomes, may be used as a point of departure for the analysis. In Donabedian’s framework, structure refers to the settings in which care occurs, including facilities, equipment and monetary resources, human resources and organizational structure such as staff organization and methods of reimbursement. Process describes what is done in health care, including the patient’s seeking care as well as the practitioner’s activities in diagnosing and treating the patient. Outcome denotes the effects of care on health status of patients and popula- tions, also including the patient’s satisfaction of care [16]. Choice reform and market orientation in primary health care In recent years market orientation has increased in the Swedish health care system as a whole. A national law on freedom of choice by citizens was passed in 2008 [11], to enable citizens to choose among providers in different sectors, including health and social care [12]. In 2010 an amendment was made to the Health and Medical Services Act, mandating the regions and county councils to allow citizens to choose their PHC provider, and to allow private providers of PHC to freely establish practices, if they met certain defined criteria. The The core function of Swedish PHC is to be the first health care contact for the population, to provide holistic Page 3 of 10 Burström et al. International Journal for Equity in Health (2017) 16:29 Page 3 of 10 objectives of this PHC Choice Reform were to increase patient choice, expand the provision of private health care to increase access to care, and to increase quality and innovation through competition among providers [11–13]. process and outcome of PHC. In particular, the reform may influence: 1) The establishment of new PHC clinics and resource allocation between PHC clinics, 2) The organization and implementation of PHC and how different patient groups are prioritized, and 3) The im- pact of PHC on population health. The law on freedom of choice was enforced by a centre-right government and there have been differing ideological views on the benefits of the PHC Choice Re- form. The PHC Choice Reform implies a shift from an egalitarian towards a libertarian ideology in health care [14]. An analysis of policymakers’ arguments when the PHC Choice Reform was legislated focused on whether and how the PHC Choice Reform harmonizes with the emphasis on equity in the Health and Medical Services Act, which population groups will actually benefit from the reform, and ultimately how the reform may impact on the role of PHC on population health and inequalities in health [15]. The study concluded that because health inequalities is one of the main challenges, the impact of health care reforms on equity should receive more atten- tion in policy making [15]. The aim of this study was to review and analyze the evidence regarding the equity impact of the PHC Choice Reform in Sweden. Methods We did a scoping review of the published accounts (scientific articles and reports) which reported on the impact of the Swedish PHC Choice Reform of 2010 and changes in reimbursement systems, from 2008 to September 2016. From a search on PubMed and Web of Science we found six scientific articles. We also searched for “grey literature” including publications from relevant public agencies in Sweden concerning the PHC Choice Reform and found nine publications, three with a nationwide focus (one with in-depth analysis of data from three county councils), two covering three county councils and four reports were based solely on data from Stockholm County Council where the reform was intro- duced already in 2008, before it was legislated nationally in 2010. Theoretically, the reform may impact in different ways on equity aspects of PHC. On the one hand, access to care may be increased for all by allowing free establishment of providers; on the other hand, the free establishment may result in providers choosing where to establish, and reduce political opportunities for deliberate need-based resource allocation between PHC centres. In addition, the Choice Reform may impact on the role and assignment of PHC and thereby the prioritization of patients and the work of GPs. The organization of work and prioritization of patients in PHC is further affected by the type of financial reimbursement system employed, and how different PHC activities are incentivized. The reform has also subse- quently been implemented differently in different county councils [14]. However, there is little scientific evidence on the impact of the reform. The results of the review are organized according to Donabedian’s framework of structure, process and outcome. The results are summarized in a narrative manner. Effects on the structure of PHC – new facilities and reimbursement Effects on the establishment of PHC facilities Since 2010, over 270 new private PHC practices have been established throughout the country, operating for profit [17]. In 2014 the Swedish National Audit Office presented an investigation of the PHC Choice Reform [18]. Their report concluded that the number of PHC clinics had increased in 20 out of 21 county councils, but that the new establishments had primarily located in the largest cities and urban areas, in socioeconomically more advantaged populations [18]. In view of the emphasis on equity in Swedish health care policy, one important research question is how the PHC Choice Reform and increased market orientation will affect PHC in terms of equity aspects on structure, Burström et al. International Journal for Equity in Health (2017) 16:29 Page 4 of 10 Table 1 List of reviewed publications Publication Ref. no. Year Area(s) Data Focus Results Scientific articles Beckman, Anell [23] 2013 Region Skåne Population register data Process of care – PHC visits Visits increased more among high-income than low income earners Agerholm et al [25] 2015 Stockholm County council Population register data, public health survey data Process of care – PHC visits Visits increased more among person with lesser needs; less among those with greater needs Glenngård [32] 2013 Region Halland, Skåne, Västra Götaland Patient survey data Outcome – Patient satisfaction Satisfaction with primary care higher in areas with low level of social deprivation and in smaller practices Maun et al [27] 2013 Gothenburg Interviews with 24 PHC managers Process of care – doctors’ views Prioritisation conflicts among doctors between patients with different needs and demands. Chronically ill patients were crowded out. Hollman et al [28] 2014 Gothenburg Interviews with PHC district nurses Process of care – nurses’ views Reimbursement system emphasizes doctors and plays down nurses’ role. Negative for job satisfaction and work environment Isaksson et al [17] 2016 Nationwide Area socioeconomic composition of population in relation to established clinics Structure – establishment of new practices New centres located in areas with fewer old adults living alone and fewer single parents. No significant effects of income, percentage immigrants, education unemployment “Grey literature” Rehnberg et al [21] 2008 Stockholm County council Population register data Visits, productivity, resource allocation Increase in visits and in productivity overall. Organization of health care, role of PHC Effects on the structure of PHC – new facilities and reimbursement Resources decreased in areas with greater need Glenngård [31] 2012 Region Halland, Region Skåne, Region Västra Götaland Patient survey data Outcome - Patient satisfaction Satisfaction with primary care higher in areas with low level of social deprivation and in smaller practices Swedish Association of Local Authorities and Regions [19] 2012 Nationwide Survey among 360 PHC managers Doctors’ views on reform and reimbursement systems Dissatisfaction with reimbursement systems, leading to prioritization of patients with lesser needs Johansson [30] 2012 Stockholm County Council Survey among PHC doctors and nurses Health promotion in PHC Negative impact on health promotion because of lack of reimbursement Dahlgren et al [24] 2013 Stockholm County Council Population register data Visits, patient satisfaction, new practices Increase in visits for all but more among high income earners. Patient satisfaction generally not affected, but lower among patients with greater needs. New practices spread out. Mohmand [29] 2014 Stockholm County Council Interviews with 6 PHC doctors Process of care – doctors’ views PHC reform makes patients to be customers Reimbursement system prioritises those with lesser needs National Audit Office [18] 2014 Nationwide, three county councils in-depth Population register data, interviews Structure – establishment of new practices More new practices in wealthy larger urban areas, interviews suggesting practitioners not establishing in areas with greater need Myndigheten för vårdanalys [26] 2015 Stockholm County Council, Region Västra Götaland, Region Östergötland Population register data Process of care – PHC visits Increase in visits among all, stronger among high income earners. Higher increase among person with no diagnosis indicating higher health care need. Government investigation “Efficient care” [10] 2016 Nationwide Meetings, interviews, documents Organization of health care, role of PHC PHC very important to the whole health care system, should be first line for all. PHC Choice Reform has made cooperation around patients with complex needs more difficult. Suggest legislation for separate organization of PHC for these patients. Increase in visits and in productivity overall. Resources decreased in areas with greater need Satisfaction with primary care higher in areas with low level of social deprivation and in smaller practices Increase in visits and in productivity overall. Effects on the structure of PHC – new facilities and reimbursement Resources decreased in areas with greater need Satisfaction with primary care higher in areas with low level of social deprivation and in smaller practices Dissatisfaction with reimbursement systems, leading to prioritization of patients with lesser needs Process of care – doctors’ views Structure – establishment of new practices Process of care – PHC visits PHC very important to the whole health care system, should be first line for all. PHC Choice Reform has made cooperation around patients with complex needs more difficult. Suggest legislation for separate organization of PHC for these patients. Burström et al. International Journal for Equity in Health (2017) 16:29 Page 5 of 10 Table 2 Overview of potential and observed effects of the PHC Choice reform and reimbursement systems on structure, process and outcome in PHC in Sweden PHC Choice Reform Reimbursement system based on fee-for service Comments - impact on equity and need-based care Structure - Access, resources Number of practices Increased Less increase in disadvantaged areas Practice distribution Providers’ choice determines practice distribution Reduced political influence on distribution by need, may cause maldistribution Resource allocation Patients’ choice determines resource allocation between practices Short visits are incentivised = more income Reduced political influence on resource allocation by need GP’s work environment Patients become customers - change in professional focus Many short visits are incentivised Priority on those with lesser needs Process - Delivery of health care Number of visits to PHC Increased Increased Greater increase for those with lesser needs Prioritisation of patients Patients as customers Focus on short visits by healthier patients More demand-driven care. Less focus on those with greater need Integrated care More complex to achieve integration, competition Integrated care not incentivised More difficult for those in need of integrated care Holistic care De-limited, differentiated PHC assignments (e.g. ENT, gynaecology, child health) One visit, one problem (short itemized visits) Itemized care not beneficial for those with complex needs Inter-professional care Focus on doctors Less teamwork doctors and nurses No benefit for those in need of inter-professional care Outcomes – impact on health Health among those with complex needs Coordination and integration more difficult Counteracts holistic care Potentially adverse effects on those with greater needs Treatment impact Reduced focus on prevention, more emphasis on cure Focus on short visits - curative care for self-limiting diseases Increase in preventable health problems? Effects on the structure of PHC – new facilities and reimbursement Population health Focus only on listed individuals limits population impact Less emphasis on health promotion and on collaboration with other agencies Reduces PHC impact on population health In contrast, another recent study [17] which adjusted for effects of county council regulation found that PHC clinics established after the PHC Choice Reform were located in areas with fewer older adults living alone as well as fewer single parents, but that no significant effects were noted for mean income, percentage of immi- grants, education, unemployment and children <5 years. The study concluded that there were some negative effects on geographical equity, but that these were relatively minor [17]. county councils, but most have a mix of capitation (an annual lump sum per listed individual), and fee-for- service (payment per visit), and a smaller portion of pay- for-performance (connected to meeting certain set targets) [19]. The capitation part may also be adjusted for need of health care, by taking into account the socioeconomic composition or the burden of disease in the population, which is the case in most county councils. In most county councils the capitation part constitutes most (about 80%) of the total reimbursement [19]. In terms of reimbursement systems, Stockholm County Council is an outlier and changed its system of resource allocation markedly with the introduction of the reforms. Since the inception of a purchaser/provider model in health care in the 1990s, the county council for many years operated a need-based resource allocation system in order to distribute resources to match the differing needs of health care in the populations of different geographic areas. The system combined need-adjusted capitation (75%), taking age and socioeconomic composition of the population into account, and fee-for-service (25%), and Council An important aspect of the choice reform is that the location of clinics and the patient’s choice of provider to a large extent determine the allocation of resources in PHC, as resources follow the patient. In addition, the design of the reimbursement system for PHC may further impact on resource allocation. The design of Swedish reimbursement systems in PHC vary between different Burström et al. International Journal for Equity in Health (2017) 16:29 Page 6 of 10 Page 6 of 10 productivity in general, and a decrease in resources in areas with greater need.). Another study from the Scania (Skåne) Region [23] found that access to PHC increased in all groups, but particularly among high income earners. Studies from Stockholm county council have shown varying results. Overall there has been a 35% increase in the number of visits per adult person from 2005 to 2012 [24]. The number of visits per person has increased in all areas, more among high income earners than among low income earners, but more among those with low education than among those with high education [24]. These studies did not adjust for health care needs. However, another study which made this adjustment found that the rate of increase in the number of visits was significantly lower among persons with greater needs (particularly women) and among men born outside Sweden who live in disadvantaged areas [25]. was in operation until 2007. It resulted in considerable extra resources being allocated to disadvantaged areas, in order to match their greater need of health care [20]. g With the implementation of the Choice Reform in Stockholm County Council 2008, this needs-based resource allocation system was abandoned. A new re- imbursement system was introduced, with the stated intention of creating equal terms for all providers of PHC (rather than equal terms for patients, as in the previous system). Reimbursement became predominantly based on demand (fee-for-service) (60%), and partly (40%) on the number of listed patients in the population (capitation). The capitation was weighted only by age (higher for persons aged 65 years and above) [21]. The notion of creating equal terms for all providers was criticized, as the variation in burden of disease and socioeconomic composition of listed patients creates very different conditions for PHC providers, and would need to be compensated for [22]. Council The resulting effect on resource allocation was considerable for PHC clinics in disadvantaged areas, one clinic lost more than 30% of its resources from 1 year to the next. This system was in place from 2008 to 2015, when it was replaced in 2016 by a capitation-dominated (60%) system, partly weighted for socioeconomic composition. A recent study of three county councils [26] found an increase from 47 to 55% between 2005 and 2012 in Stockholm county council in the proportion of patients making one or more visits to the doctor. In Region Östergötland the proportion was 47% over the same time; in Region Västra Götaland the proportion was 55%. There was an increase in the number of visits in all three county councils, most pronounced in Stockholm County Council where the average number of visits was higher than in the other county councils. In general, groups with low education or low income had a rela- tively higher number of visits than other groups [26] (as would be expected from their higher level of healthcare need), but it is not clear in this study if the higher num- ber of visits in more disadvantaged groups matched their higher level of need. Effects on the process of PHC – the delivery of PHC Effects on the number of visits Effects on the number of visits When assessing the outcomes of the PHC Choice Re- form, different outcomes should be considered. One fre- quently used outcome is change in number of visits. It should be noted, however, that average number of visits to primary care may not be a conceptually sound meas- ure of access to care. First, it is not clear whether an increase in visits is a good or a bad outcome in terms of the health of the population. An increase, for instance, could indicate an increase in morbidity in the popula- tion, which the increase in visits may or may not match. Second, more disadvantaged groups are in greater need of health services because of their greater preva- lence of ill-health and poorer recovery. They often have higher rates of primary care visits than their more afflu- ent counterparts, but even so, their higher rates of visits may still not match their higher level of need. Assess- ments of equity of access by socio-economic status, therefore, have to adjust for the higher health care need of more disadvantaged groups before assessing if access is equitable and whether inequalities in access have changed over time. Only a few studies make this latter adjustment. These two provisos need to be borne in mind when interpreting the findings of the following studies. When assessing the outcomes of the PHC Choice Re- form, different outcomes should be considered. One fre- quently used outcome is change in number of visits. It should be noted, however, that average number of visits to primary care may not be a conceptually sound meas- ure of access to care. First, it is not clear whether an increase in visits is a good or a bad outcome in terms of the health of the population. An increase, for instance, could indicate an increase in morbidity in the popula- tion, which the increase in visits may or may not match. Effects on the work of doctors and nurses Effects on the work of doctors and nurses Few studies have reported on how the PHC Choice reform and altered reimbursement systems have affected the delivery of PHC, the GP work environment and prioritization of patients. A national survey among 360 public and private PHC managers in Sweden in 2012 [19] found that only 16% of the respondents considered that the current reimbursement system to a great extent promoted the priorities they wanted to work for. The proportion was higher in county councils where capita- tion reimbursement was weighted by socioeconomic factors and morbidity. Nine out of ten respondents in Stockholm County Council stated that the reimburse- ment system incentivized short visits. The proportion of respondents who agreed that the current reimbursement system supported a prioritization of patients with great health care needs was lowest in Stockholm County Council. There were considerable differences in responses between public and private PHC managers. For instance, at a national level, 70% of public managers agreed with the Second, more disadvantaged groups are in greater need of health services because of their greater preva- lence of ill-health and poorer recovery. They often have higher rates of primary care visits than their more afflu- ent counterparts, but even so, their higher rates of visits may still not match their higher level of need. Assess- ments of equity of access by socio-economic status, therefore, have to adjust for the higher health care need of more disadvantaged groups before assessing if access is equitable and whether inequalities in access have changed over time. Only a few studies make this latter adjustment. These two provisos need to be borne in mind when interpreting the findings of the following studies. A first study from Stockholm County Council in 2008 [21] reported both increased number of visits and Burström et al. International Journal for Equity in Health (2017) 16:29 Page 7 of 10 Page 7 of 10 statement that the current principles of reimbursement risks crowding out patients with greater health care needs, compared to nearly 54% of private managers. Corre- sponding figures in Stockholm county council were 89% and nearly 61%, respectively. Nationally, only 20% of all PHC managers agreed that the current principles of reimbursement support a health promoting and preventive way of work [19]. Effects on the work of doctors and nurses into two different organizations: one according to the current PHC Choice Reform and another organization for elderly persons with complex health care needs, which should be exempted from the mandatory PHC choice regulations [10]. Effects on the outcomes of PHC Effects on patient satisfaction Patient satisfaction has been investigated in several studies after the PHC Choice Reform. One study [31], based on patient survey data from three county councils (Region Scania, Region Halland and Region Västra Götaland), found that the patient perceived quality was lower in larger cities and in clinics with a greater proportion of the listed having more difficult socioeconomic circumstances, but higher among patients with greater need of health care. Private clinics had higher patient ratings than public clinics, but were to a greater extent located in more affluent areas. Patients at clinics with a greater number of listed patients were less satisfied than those with fewer listed patients [31]. Similar results were shown in another report [32]. An interview study with 24 managers of publicly owned PHC centres in Gothenburg in 2013 found that the reform was perceived as a rapid change, enforced through finan- cial incentives, and that prioritization conflicts arise between patient groups with different needs, demands and levels of empowerment [27]. While the average number of visits per patient increased, chronically ill patients were considered to be crowded out by healthier and more verbally demanding patients. An interview study among district nurses in western Sweden [28] in 2013 found that the focus on economic benefit may limit the cooperation and exchange of experi- ences within and between different care units. This could in turn have a negative impact on the quality of care due to competition between different care providers. The re- imbursement system emphasized many short doctor visits and the role of nurses was played down. Underused resources and restrictions on nurses had a negative impact on their job satisfaction and the working environment, and may have affected the quality of care as a result [28]. Effects on population health Before the PHC Choice Reform, the responsibility for health of the population in the catchment area lay with the local PHC clinic. With the reform, the assignment of PHC was limited to the listed individuals, which may have an important impact on the role of PHC in improving public health on a population level. Health promotion may be carried out through collaboration with other local agencies such as schools, social services, employment agency or non-governmental organizations, in working with improving health-related behaviors requiring community-wide action, for instance increasing physical activity or reducing smoking. As this is no longer part of the assignment of PHC reimbursed by the county council, there is a risk that such collaboration activities are no longer seen as the responsibility of the local PHC clinic, as found in one study in Stockholm County Council [26]. Another small in-depth interview study among six GPs in Stockholm County Council in 2013 [29]) dis- tinguished between effects of the PHC Choice Reform, which resulted in patients becoming customers rather than patients, and effects of the fee-for-service reim- bursement system, which put the focus on performing many short visits among patients with lesser needs, in order to generate income. The prioritization of pa- tients with lesser needs was perceived not to be in line with the intentions of the Health and Medical Service Act [29]. A study of how the PHC Choice Reform had affected health promotion and prevention in PHC in Stockholm County Council found that financial incentives were geared towards producing many visits, at the expense of health promotion and preventive activities, for which there was not time and no reimbursement [30]. The government investigation which found that the PHC Choice Reform had increased the difficulties in achieving integrated care among elderly with complex needs [10] suggests that PHC is not organized optimally with respect to elderly patients with complex needs. Achieving integrated care for persons with greater health care needs is another goal of PHC and requires organized collaboration between PHC and secondary and tertiary health care services, as well as with municipal social services which are responsible for residential care of elderly. A recent government investigation [10] concluded that the PHC Choice Reform has not been conducive, but rather an obstacle, to achieving integrated care. The in- vestigation even suggested that PHC should be divided Effects on the structure of PHC In order to follow up the impact of the PHC Choice Re- form, different indicators may be studied. In terms of the impact of the PHC Choice Reform on the structure of PHC, it is evident that the reform has increased the number of PHC clinics and the average number of visits to PHC, but it is debated whether that is a good Burström et al. International Journal for Equity in Health (2017) 16:29 Page 8 of 10 Page 8 of 10 indicator of improvement in access to health care in its wider sense. The evidence reviewed in this paper indi- cates that increases in number of visits have not been uniform across the population. The National Audit Of- fice concluded that more new clinics had established in already well served areas, and in interviews PHC providers indicated they were unwilling to establish practices in areas with high levels of need for care, even if reimburse- ment systems were to take patients’ need of care into account (17). However, the study by Isaksson et al con- cluded that there were some, but only minor, negative effects on equity (16). It is difficult to measure the supply and access to care in correct ways, and on a meaningful area level. population in disadvantaged areas will receive more resources than the population in better-off areas. This was the case in the former need-based reimbursement system in Stockholm County Council in place until 2008, in line with the intentions of the Swedish Health and Medical Services Act [1]. However, if resources are distributed only by popula- tion size (B), the disadvantaged area will receive less re- sources in relation to the level of need in that area, and the population in the better-off area will receive more resources in relation to the area’s level of need. This situ- ation corresponds to the capitation part of the fee-for- service dominated reimbursement system with no socio- economic weighting, disregarding population differences in need. The National Audit Office report suggests that PHC providers have been choosing their patients, rather than patients choosing their PHC provider, which is actually the reverse of what the PHC Choice Reform was designed to do. Effects on the structure of PHC If this conclusion holds, it would be an example of Julian Tudor Hart’s classical ‘inverse care law’ about the operation of market forces in health care, as stated in his 1971 seminal Lancet paper [33]: “The availability of health care tends to vary inversely with the need for it in the population. This inverse balance oper- ates more completely where health care is most exposed to market forces, and less so with less exposure.” If resources are allocated by demand for health care services (C), the population in the better-off area is likely to get more resources than the population in the disadvan- taged area, because their demand for services is greater, in spite of their relatively lesser need of health care ser- vices [34]. This situation may represent the effect of the fee-for-service component in the reimbursement sys- tem in Stockholm County Council, which generates an increase in the number of visits, as fee-for-service in- centivizes many short visits, generated both by demand of patients and by doctors prioritizing such visits. In this manner, an adverse outcome of the PHC Choice Reform and a demand-oriented reimbursement system, may be a reallocation of resources away from areas with greater need to areas with less health care needs, as evidenced in Stockholm County Council [29]. A crucial impact of the PHC Choice Reform, as evidenced particularly in Stockholm County Council, is a change in resource allocation to the detriment of areas with greater health care needs, through the combined effect of the PHC Choice Reform itself, the fee-for- service dominated reimbursement system without socio- economically weighted capitation which was in place 2008 −2015, and a difference in health-seeking behavior and demand for health care between different areas. Funding This study was partly supported by a research grant (2014-4763) from the Swedish Research Council for Health, Working Life and Welfare. Conclusion In conclusion, the evaluative evidence is sparse and incomplete. The studies to date indicate that the PHC Choice Reform, as implemented by some county councils, predominantly Stockholm, increased access to PHC and increased the average number of visits to PHC, but seems to have particularly benefitted those in more affluent groups and with lower health care needs. In addition, it has made integrated care for those with complex needs more difficult. Among GPs and nurses in PHC there are mixed opinions about the reform. Some consider that persons with greater needs are not prioritized; others are more positive. Resource allocation to PHC has become more dependent on provider location, patient choice and demand, and less on need of care. In view of the more restricted assignment there is also a risk of a reduced impact on population health of PHC. On the available evidence, the PHC Choice Reform may have damaged equity of primary health care provision, contrary to the tenets of the Swedish Health and Medical Service Act. This situation needs to be carefully monitored and countered where necessary. Further studies are needed to follow up the long-term impacts of the reform on the structure, process and outcomes of PHC in Sweden and how different types of reimbursement systems may moderate these impacts. Authors’ contributions BB reviewed the articles and reports quoted in the article and wrote the first and final drafts of the article. KB, GN, GT, MW and UW discussed the findings, critically reviewed and revised previous drafts of the article and approved the final draft. All authors read and approved the final manuscript. Effects on process – delivery of PHC The evidence suggests that the PHC Choice Reform has increased the number of visits to PHC. However, some reports indicate a greater increase among groups with lesser health care needs than among those with greater health care needs. According to some studies the reform appears to have had a negative impact on the process An illustration is given in Fig. 1, comparing two areas with the same population size, but with different composition of the population with respect to need. If resources are allocated according to need (A), the Fig. 1 Effects of resource allocation by different principles (a need, b population, c demand) on two populations of the same size with different levels of need (adapted from Hung et al. [37] Burström et al. International Journal for Equity in Health (2017) 16:29 Page 9 of 10 greater among those with lesser needs of care [28, 30], in line with an earlier review study of choice reforms in health care in European countries [36]. and delivery of PHC, as evidenced by the studies of PHC managers and nurses [19, 23–25]. These studies provide an account of how employees in publicly owned PHC clinics perceive the PHC Choice Reform and changes in reimbursement systems, with a shift in prioritization of patients towards those with lesser needs, and an in- creased focus on the role of the doctor. As indicated in the national survey of PHC managers, those working in privately owned PHC clinics were more positive to the reform and changes in reimbursement systems than those working in publicly owned clinics [19]. This is an important aspect to study in further detail. Availability of data and materials Data sharing not applicable to this article as no datasets were generated or analysed during the current study. Acknowledgements W k l d We acknowledge comments on previous versions of the manuscript by Janne Agerholm, Karin Josefsson and Sofie Vengberg. We are grateful for comments by anonymous reviewers. Abbreviations GP: General Practitioner; PHC: Primary Health Care p The interaction between the PHC Choice Reform and simultaneous changes in the reimbursement system pro- vides further difficulties in interpreting the findings of evaluations. The greater increase in the number of visits observed in Stockholm County Council, for example, may be related to the fee-for-service based reimbursement system. In most of the reviewed studies, the number of patient visits to doctors was the measured outcome. How- ever, as this measure depends on the reimbursement system, it may have severe shortcomings. As indicated in some of the referenced studies, the available statistics on the number of visits do not reflect the content or quality or potential effect on health of the visit. As the goal of health care is to improve health, there is a need to go beyond mea- sures of productivity, such as number of visits, to actually measuring the impact on health status improvement, by using patient-reported outcomes [35]. In Stockholm county council there is anecdotal evidence that previously longer visits were divided into several shorter, in order to gain revenue, because of the reimbursement system which gave the same amount for a short as for a longer visit. In some of the studies the increase in number of visits has been Competing interests p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Effects on outcomes of PHC One key finding concerns the impact on integrated care: that the PHC Choice Reform is considered by providers to be an obstacle in organizing integrated care, particu- larly for elderly with complex needs of care [10]. This is important not least because elderly with complex needs of care are a large and important group of patients in PHC, and the finding is in stark contrast to the inten- tions of the Health and Medical Services Act [1]. The PHC Choice Reform and change in reimbursement system was also considered by doctors and nurses in Stockholm county council to have reduced the emphasis on health promotion and prevention, because of the focus to produce many visits and generate income to the clinic [26]. The responsibility of PHC for population oriented health promotion activities has also been re- duced, because of the focus only on listed patients after the reform, which may further reduce the population health impact of PHC [26]. Abbreviations GP: General Practitioner; PHC: Primary Health Care Abbreviations GP: General Practitioner; PHC: Primary Health Care References Om välfärdsstatens moraliska och politiska logik [What should the state do?] Stockholm: SNS Förlag, 2004. (In Swedish) 9. OECD. OECD health data 2015. http://www.oecd-ilibrary.org/social-issues- migration-health/data/oecd-health-statistics/oecd-health-data-health-care- utilisation_data-00542-en. Accessed 26 Jan 2017 31. Glenngård AH. Vad påverkar patientupplevd kvalitet i primärvården? [What affects patient perceived quality of care in primary care?]. Stockholm: Myndigheten för vårdanalys, 2012. http://www.vardanalys.se/Global/ Rapporter%20pdf-filer/2012/R1_2012_Vad_paverkar_patientupplevd.pdf. Accessed 10 Sept 2016 (In Swedish) 10. Regeringen. Effektiv vård [Effective/efficient care]. SOU 2016:2. Government investigation. 11. Government of Sweden. Lag om valfrihetssystem (Freedom of Choice Act) 2008/09:29. Stockholm: Ministry of Social Affairs, Government of Sweden, 2008. (In Swedish) 32. Glenngård AH. Is patient satisfaction in primary care dependent on structural and organizational characteristics among providers? Findings based on data from the national patient survey in Sweden. Health Econ Policy Law. 2013;8:317–33. 12. Ekman B, Wilkens J. A literature review of the regional implementation of the Swedish government’s health care reforms on choice and privatization. Heal Econ Rev. 2015;5:39. 33. Hart JT. The inverse care law. Lancet. 1971;297:405–12. 34. Burström B. Market-oriented, demand-driven health care reforms and equity in health and health care utilization in Sweden. Int J Health Serv. 2009;39:271–85. 13. Anell A. Choice and privatisation in Swedish primary care. Health Econ Policy Law. 2011;6:549–69. 35. Appleby J, Devlin N, Parkin D. Using patient reported outcomes to improve health care. Oxford: Wiley Blackwell; 2016. 35. Appleby J, Devlin N, Parkin D. Using patient health care. Oxford: Wiley Blackwell; 2016. 14. Maynard A. Beware of the libertarian wolf in the clothing of the egalitarian sheep: an essay on the need to clarify ends and means. In: McIntyre D, Mooney G, editors. The economics of health equity. Cambridge: Cambridge University Press; 2007. 36. Fotaki M, Roland M, Boyd A, McDonald R, Scheaff R, Smith L, Fotaki M, Roland M, Boyd A, McDonald R, Scheaff R, Smith L. What benefits will choice bring to patients? Literature review and assessment of implications. J Health Serv Res Policy. 2008;13:178–84. doi:10.1258/ jhsrp.2008.007163. 15. Fredriksson M, Blomqvist P, Winblad U. The trade-off between choice and equity: Swedish policymakers’ arguments when introducing patient choice. J European Social Policy. 2012;23:192–209. 37. Hung PM, Dzung TV, Dahlgren G, Tuan T. Vietnam: Efficient, equity-oriented financial strategies for health. In: Evans T, Whitehead M, Diderichsen F, Bhuiya A, Wirth M, editors. Challenging inequities in health. From ethics to action. Oxford: Oxford University Press; 2001. p. 296–306. 16. Donabedian A. Author details 1 f 20. Stockholm county council. 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Accessed 10 Sept 2016 (In Swedish) 1Department of Public Health Sciences, Equity and Health Policy Research Group, Karolinska Institutet, SE 171 77 Stockholm, Sweden. 2Department of Learning, Informatics, Management and Ethics, Health Outcomes and Economic Evaluation Research Group, Karolinska Institutet, Stockholm, Sweden. 3Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden. 4Department of Learning, Informatics, Management and Ethics, Karolinska Institutet, Stockholm, Sweden. 5 1Department of Public Health Sciences, Equity and Health Policy Research Group, Karolinska Institutet, SE 171 77 Stockholm, Sweden. 2Department of Learning, Informatics, Management and Ethics, Health Outcomes and Economic Evaluation Research Group, Karolinska Institutet, Stockholm, Sweden. 3Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden. 4Department of Learning, Informatics, Management and Ethics, Karolinska Institutet, Stockholm, Sweden. 5Department of Public Health and Society, Institute of Psychology, Health and Society University of Liverpool, Liverpool, UK. 6Department of Public Health and Caring Sciences, Health Services Research, Uppsala University, Uppsala, Sweden. 21. Rehnberg C, Janlöv N, Khan J. Uppföljning av Vårdval år 2008 [Follow-up of PHC Choice Reform Stockholm 2008]. http://dok.slso.sll.se/CES/FHG/Jamlik_ halsa/Rapporter/Aldre_rapporter/vardval-stockholm-uppfoljning.2009_6.pdf. Accessed 10 Sept 2016 (In Swedish) 22. Halldin J. Vårdval Stockholm skapar en orättfärdig och ojämlik vård [Stockholm PHC Choice Reform creates inequitable care]. http://www. lakartidningen.se/Functions/OldArticleView.aspx?articleId=13558. Accessed 10 Sept 2016 (In Swedish) 23. Beckman A, Anell A. Changes in health care utilisation following a reform involving choice and privatisation in Swedish primary care: a five-year follow-up of GP-visits. BMC Health Serv Res. 2013;13:452. Received: 21 September 2016 Accepted: 20 January 2017 follow-up of GP-visits. BMC Health Serv Res. 2013;13:452. 24. Dahlgren C, Brorsson H, Svereus S, Rehnberg C. Fem år med husläkarsystemet inom Vårdval Stockholm [Five years with choice in primary care in Stockholm]. Stockholm: Karolinska institutet; 2013 (Report), . (In Swedish). 25. Agerholm J, Bruce D, Ponce de Leon A, Burström B. Equity impact of a choice reform and change in reimbursement system in primary care in Stockholm County Council. BMC Health Serv Res. 2015;15:420. doi:10.1186/s12913-015-1105-8. Ethics approval This study was a review and did not require ethics approval. Page 10 of 10 Page 10 of 10 Page 10 of 10 Burström et al. International Journal for Equity in Health (2017) 16:29 Burström et al. International Journal for Equity in Health (2017) 16:29 Burström et al. International Journal for Equity in Health (2017) 16:29 References 1. Macinko J, Starfield B, Shi L. The contribution of primary care systems to health outcomes within OECD countries 1970–1998. Health Serv Res. 2003;38:831–65. 26. Myndigheten för Vårdanalys. Vårdval och jämlik vård inom primärvården. En jämförande studie mellan tre landsting före och efter vårdvalets införande. [PHC Choice Reform and equitable care in primary health care. A comparative study of three county councils.] Stockholm: Myndigheten för vårdanalys, 2015. (Rapport 2015:6) (In Swedish) 2. Hälso- och sjukvårdslagen [Health and Medical Services Act]. SFS 1982:763. (In Swedish) 3. Public Health Agency of Sweden. Folkhälsan i Sverige. Årsrapport 2014 [Public health in Sweden. Annual Report 2014]. https://www. folkhalsomyndigheten.se/pagefiles/17825/Folkhalsan-i-Sverige-arsrapport-2014. pdf. Accessed 4 Mar 2016 (In Swedish) 3. Public Health Agency of Sweden. Folkhälsan i Sverige. Årsrapport 2014 [Public health in Sweden. Annual Report 2014]. https://www. folkhalsomyndigheten.se/pagefiles/17825/Folkhalsan-i-Sverige-arsrapport-2014. pdf. Accessed 4 Mar 2016 (In Swedish) 27. Maun A, Nilsson K, Furåker C, Thorn J. Primary healthcare in transition – a qualitative study of how managers perceived a system change. BMC Health Serv Res. 2013;13:382. 4. Socialdepartementet. 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In: Pavolini E & Guillén A.M. (eds.). Health Care Systems in Europe under Austerity: Institutional Reforms and Performance. Palgrave Macmillan Publishers, 2013. 30. Johansson H. Vårdval Stockholm, Husläkarverksamheten och det hälsofrämjande arbetet. En studie med utgångspunkt från vårdens professioner [Choice in PHC in Stockholm: PHC and health promoting work]. Umeå: Umeå universitet, Folkhälsa och klinisk medicin/ epidemiologi och global hälsa, 2012. (In Swedish) 7. Esping-Andersen G. The three worlds of welfare capitalism. Polity Press, 1990. 7. Esping-Andersen G. The three worlds of welfare capitalism. Polity Press, 1990. 8. Rothstein B. Vad bör staten göra? References The quality of care – how can it be assessed? JAMA. 1988;260:1743–8. 17. Isaksson D, Blomqvist P, Winblad U. Free establishment of primary care providers: effects on geographical equity. BMC Health Serv Res. 2016;16:28. 18. Riksrevisionen [National Audit Office]. Primärvårdens styrning – efter behov eller efterfrågan? [Primary health care governance – based on need or demand?]. Stockholm: Riksrevisionen, 2014. (RiR 2014:22). (In Swedish) 19. Swedish Association of Local Authorities and Regions (SKL). Vårdval i primärvården [Patient choice in primary care]. http://webbutik.skl.se/ bilder/artiklar/pdf/7164-826-6.pdf?issuusl=ignore. Accessed 20 Dec 2012 (In Swedish)
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ROLE OF URIC ACID IN CARDIAC CHANGES IN OVERWEIGHT AND OBESE CHILDREN.
Zenodo (CERN European Organization for Nuclear Research)
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ROLE OF URIC ACID IN CARDIAC CHANGES IN OVERWEIGHT AND OBESE CHILDREN. 402 Schusterova I1, Bánovcinova A2, Takacova J3, Takac L3, Artemiou P 4 and Leenen FHH.5. 1. 1th Department of Cardiology, East Slovak Institute of Cardiovascular Diseases, Kosice, Slovakia. 2. Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, P.J. Šafárik University in Košice, Slovakia 3. University of Veterinary Medicine and Pharmacy, Kosice, Slovakia. 4. The National Institute of Cardiovascular Disease, Bratislava, Slovakia. 5. Hypertension Unit, University of Ottawa Heart Institute, Ottawa, Canada (retired) …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 08 August 2019 Final Accepted: 10 September 2019 Published: October 2019 Key words:- Childhood obesity, left ventricle, Uric acid, cardiometabolic risk Background: High serum uric acid (UA) levels appear to contribute to an increase in blood pressure (BP) in obese adolescents and may amplify the cardiac volume overload effects of obesity with pressure overload. Association between UA and ejection fraction (EF) is still controversial. Some studies showed negatively correlations, others did not found any correlation. So far, no data have been published regarding the influence of UA levels on structural and functional changes of the LV in children with obesity. Objective: The aim of our study was to assess the influence of UA levels on LV structure and function in overweight/obese children. Study groups and methods: In 25 (mean age 13.0 ± 2.3) overweight/obese subjects and 24 lean controls, BP, fasting plasma glucose, insulin, and UA were measured. LV structural and functional parameters were measured by echocardiography. Results: In overweight/obese children UA correlates with LV diastolic volumes and LV systolic function but not with LVM whereas in children without obesity UA correlates with LVM and LV diastolic function. Conclusion: The present study demonstrates a positive correlation of UA with EF and left atrium and LV volume in volume overload due to obesity in children. These conclusions require further investigation. Copy Right, IJAR, 2019,. All rights reserved. …………………………………………………………………………………………………….... Introduction:- Obesity is associated with a shortened life expectancy mainly because of increased risk for cardiovascular (CV) disease. Uric acid (UA) levels have been shown to be a good marker for metabolically unhealthy obesity i.e. those with features of metabolic syndrome and elevated CV risk factors in adolescence and adulthood (1). High UA levels are a risk factor for hypertension in adults (2). Several studies have assessed the relationship between UA levels and LV mass (LVM) in adults with hypertension (3,4). ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(10), 402-410 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/9843 DOI URL: http://dx.doi.org/10.21474/IJAR01/9843 ROLE OF URIC ACID IN CARDIAC CHANGES IN OVERWEIGHT AND OBESE CHILDREN. The development of LV hypertrophy (LVH) in hypertensive adults with high UA levels can at least partially explain the elevated CV risk observed in Corresponding Author:-Banovcinova A. Address:- Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, P.J. Šafárik University in Košice Slovakia Objective: The aim of our study was to assess the influence of UA levels on LV structure and function in overweight/obese children. g Study groups and methods: In 25 (mean age 13.0 ± 2.3) overweight/obese subjects and 24 lean controls, BP, fasting plasma glucose, insulin, and UA were measured. LV structural and functional parameters were measured by echocardiography. Results: In overweight/obese children UA correlates with LV diastolic volumes and LV systolic function but not with LVM whereas in children without obesity UA correlates with LVM and LV diastolic function. Conclusion: The present study demonstrates a positive correlation of UA with EF and left atrium and LV volume in volume overload due to obesity in children. These conclusions require further investigation. Copy Right, IJAR, 2019,. All rights reserved. Copy Right, IJAR, 2019,. All rights reserved. Copy Right, IJAR, 2019,. All rights reserved. 40 …………………………………………………………………………………………………….. Introduction:- Obesity is associated with a shortened life expectancy mainly because of increased risk for cardiovascular (CV disease. Uric acid (UA) levels have been shown to be a good marker for metabolically unhealthy obesity i.e. thos with features of metabolic syndrome and elevated CV risk factors in adolescence and adulthood (1). High UA levels are a risk factor for hypertension in adults (2). Several studies have assessed the relationshi between UA levels and LV mass (LVM) in adults with hypertension (3,4). The development of LV hypertroph (LVH) in hypertensive adults with high UA levels can at least partially explain the elevated CV risk observed i Corresponding Author:-Banovcinova A. Address:- Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, P.J. Šafárik University in Košice, Slovakia Methods:- This was an observational study conducted in the Department of Metabolic Disease of the Pediatric Clinic at the Children Hospital, Kosice. All participants were referred for evaluation of obesity. Twenty five Caucasian overweight/ obese subjects (13.0 ± 2.3 years of age, 9 female) with Body Mass Index (BMI) ≥ 85 percentile for age and gender were included in the study and were compared with 24 lean healthy subjects (12.9 ± 3.4 years of age, 12 female). Subjects with secondary causes of obesity were excluded and none were taking medications or had a history of cardiovascular disease. Age- and gender-matched children were children with BMI< 85th percentile for age and gender (11) BMI was calculated as weight (kg) divided by the square of height (m) and BMI percentiles and waist circumference (WC) were measured according to WHO recommendations (12). Blood pressure (BP) was measured with a standard mercury sphygmomanometer and a cuff appropriate for the size of the child’s upper right arm. Systolic and diastolic BP were measured three times after 10 min rest in the supine position, and the average of the 3 measurements was calculated. Echocardiographic examination was done with a Vivid 5 echocardiograph, using the 3.5MHz probe S611, by the same cardiologist. The echocardiographic examination included a comprehensive 2-D examination, colour and spectral Doppler examination and complete examination in M-mode acquired from 2-D projection. The techniques used to measure LV inner diameter, enddiastolic and endsystolic interventricular septal thickness (IVS dias, IVS sys) and LV posterior wall thickness (PWTh dias, PWTh sys) comply with the recommendations of American Society of Echocardiography. Pressure half time from blood flow Doppler evaluation (PHT), LV volume in systole and diastole, myocardial performance index (MPI), LV mass (LVM), LV mass indexed to body height2.7 (LVMIV), left atrial (LA) M mode, ejection fraction (EF), stroke volume (SV) were calculated. The recommendations of the American Society of Echocardiography were used to determine diastolic function and its individual indexes (13). The pulse Doppler examination of flow through the mitral valve was used to obtain the following parameters: peak early transmitral filling wave velocity (E-wave), peak late transmitral filling wave velocity (A-wave), deceleration time of early diastolic filling (DT), and isovolumic relaxation time (IVRT). Fasting blood samples were drawn after 12 h overnight fast. Plasma glucose was measured enzymatically using a Siemens ADVIA. Methods:- Fasting serum insulin was measured by a sandwich ECLA method using a Roche Modular Analytics E170 analyzer. UA was measured by photometric kinetic method using the Siemens ADVIA biochemical autoanalyzer. The homeostasis model assessment of insulin resistance (HOMA index) was calculated according to the standard formula (14). Introduction:- Obesity is associated with a shortened life expectancy mainly because of increased risk for cardiovascular (CV) disease. Uric acid (UA) levels have been shown to be a good marker for metabolically unhealthy obesity i.e. those with features of metabolic syndrome and elevated CV risk factors in adolescence and adulthood (1). High UA levels are a risk factor for hypertension in adults (2). Several studies have assessed the relationship between UA levels and LV mass (LVM) in adults with hypertension (3,4). The development of LV hypertrophy (LVH) in hypertensive adults with high UA levels can at least partially explain the elevated CV risk observed in p g Address:- Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, P.J. Šafárik U i it i K ši Sl ki 402 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 these patients, as LVH is a strong predictor of CV disease (5). Allopurinol caused regression of LVM in patients with type 2 diabetes and LVH (6). High UA levels also appear to contribute to an increase in BP in obese adolescents (7) and may therefore amplify the cardiac volume overload effects of obesity with pressure overload. these patients, as LVH is a strong predictor of CV disease (5). Allopurinol caused regression of LVM in patients with type 2 diabetes and LVH (6). High UA levels also appear to contribute to an increase in BP in obese adolescents (7) and may therefore amplify the cardiac volume overload effects of obesity with pressure overload. UA may also affect LV function. Experimental studies have shown that high UA leads to cardiac fibrosis and LV diastolic dysfunction (8). Hyperuricemia was associated with worse cardiac function, such as increased right atrial pressure and decreased cardiac index in patients with primary pulmonary hypertension, and increased atrial pressures in patients with ischemic heart disease or dilated cardiomyopathy in small case series (9). Increased serum UA levels may contribute to cardiac dysfunction through effects on endothelial function and inflammation. UA inhibits nitrogen oxide (NO) production by vascular endothelial cells and their proliferation and migration (10). So far, no data have been published regarding the influence of UA levels on structural and functional changes of the LV in obese children. The aim of our study was to assess the relation of UA levels and structural and functional changes of the LV in children with obesity compared to lean controls. Anthropometric, clinical and biochemical parameters Anthropometric, clinical and biochemical parameters p p Body weight (BW), BMI, BMI percentile, BMI Z-score, WC, systolic and diastolic BP, HOMA index and insulin levels were all significantly higher in overweight/obese subjects compared to lean controls. UA tended (p=0.16) to be higher in overweight/obese subjects (Table 1). Correlations of uric acid and other biochemical parameters with echocardiographic parameters UA correlated with BW in both lean controls (r=0.67, p<0.01), and in overweight/obese children (r=0. In lean controls, UA positively correlated with IVS dias, IVS sys and LVM, and negatively with A wave (Table S2). In overweight/obese children UA correlated with LA area (Table S2), LV diastolic volume, SV and EF (Table S2). Statistical significant differences between the two study groups were only found for the correlation between UA and SV (Figure 1, Figure 2). HOMA did not correlate with any of LV functional parameters in obese and lean controls. Only in lean controls plasma glucose correlated with diastolic functional parameters (data not shown). Statistical analysis:- y Data were processed using methods of descriptive and inductive statistics, depending on the type and number of variables monitored. For the purpose of inductive statistics, we assumed that our data represent a random sample of the relevant population. The first step was a one-dimensional analysis, the tabulation of all monitored variables using frequency tables. The second step was a two-dimensional analysis, the assessment of pairs of monitored variables. To compare numerical and categorical variables, analysis of variance was used to determine the statistical significance of differences, if the distribution of variables was normal. All calculations were performed using version 8.0 of statistical Package for the Social Sciences software. The significance level was set to the traditional p< 0.05. 403 Int. J. Adv. Res. 7(10), 402-410 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 Echocardiographic parameters Regarding structural echocardiographic parameters (Table 2a), overweight/obese subjects had significantly higher IVS dias, LV dias, LV sys and LA 2D area, LVM and LVMIV. Functional diastolic echocardiographic parameters showed increased A wave and IVRT, and significantly decreased DT and PHT in overweight/obese children (Table 2b). LV volumes and SV were also significantly higher in overweight/obese children (Table 2b). In both overweight/obese and lean control groups structural echocardiographic parameters correlated with BW and BMI. Only in obese children WC correlated with IVS dias, LV dias, PWTh dias, and with LVM (Table S1). Only overweight/obese children showed negative correlations of E wave with BW, WC and BMI (Table S1). Most functional systolic echocardiographic parameters correlated with BW and BMI but only in overweight/obese children correlations were present between WC and LV and SV volumes (Table S1). In both lean controls and overweight/obese children there were significant positive correlations between systolic BP and structural echocardiographic parameters and LV volume dias and SV (Table S1). Diastolic BP followed the same pattern (data not shown). Discussion:- This appears to be the first study focusing on the relation of UA levels and echocardiographic parameters of cardiac structure and function in overweight/obese children. We demonstrate that in overweight/obese children UA correlates with LV diastolic volumes and LV systolic function but not with LVM whereas in children without obesity UA correlates with LVM and LV diastolic function. Obesity represents a chronic volume overload for the heart and - as expected - significant increases in LA and LV volumes and LVM were observed in obese children (15, 16). Studies analyzing the relationship between UA levels and LVM in adults have shown variable results. A positive correlation between UA levels and LVM was identified in several studies in adults (17) but not in other studies in adult patients with hypertension (3,5). Participants of Framingham Offspring Cohort, in the highest serum UA quartile had significantly higher LV wall thickness, LV end-diastolic diameter and LVM, compared with those in the lowest quartile (18). Conflicting conclusions regarding the relationship between UA levels and LVM may be partially explained by the methodology used (3). Experimentally, UA induces cardiomyocyte growth and interstitial fibrosis of the heart, partially by activation of the renin-angiotensin system (RAS) (19). The RAS may cause LVH and cardiac fibrosis through an increase in BP, a direct effect of angiotensin II on myocardial myocytes and indirectly via aldosterone (20). UA may also increase LVM by inducing endothelial dysfunction and proliferation of smooth muscle cells by increased activity of xanthine oxidase and oxidative stress (21). Increased oxidative stress may increase degradation of endothelium-derived NO and cause impairment of vascular tone regulation (22). UA also may activate inflammatory mediators, and stimulate mitogen-activated protein kinases, which may contribute to cardiac hypertrophy (23). These mechanisms may function differently in obese children, because in our group of children a correlation between LVM and UA was 404 Int. J. Adv. Res. 7(10), 402-410 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 present only in non-obese children. Moreover, the normal/mildly elevated UA levels correlated with BW, and when taking BW into account UA levels did not correlate with LVM in either group, suggesting that UA levels did not directly affect the heart. Impact of UA on LVM may require a longer duration of obesity, more significant hyperuricemia and/or longer duration (1). Higher serum UA levels might also contribute to cardiac dysfunction. Conflict Of Interest The authors declare no conflict of interests Discussion:- Patients with congestive heart failure (HF) and increasing filling pressures had significantly higher serum UA levels and serum UA levels correlated with echocardiographic parameters of diastolic function (24). Patients with gouty tophi had a significantly thicker LV interventricular septum and posterior wall and increased LA volume index reflecting LV diastolic dysfunction (25) Hyperuricemia is an independent predictive factor for LV diastolic dysfunction also in patients with chronic kidney disease (26). Findings of these studies are consistent with our results in lean controls in whom serum UA correlated with parameters of LV structure and LV diastolic function but not with LV volumes. On the other hand, this is the first study showing a correlation between UA and LV volumes due to obesity induced volume overload. Codoñer-Franch et al, (27) showed a higher concentration of NO production markers, which correlated with the increased markers of oxidative stress in obese children. We suggest that increased levels of NO in early stages of obesity in childhood could be involved in different effects of UA on LV structure and subsequent diastolic function at this age. Association between serum UA and EF is still controversial. Some studies showed negatively correlations (28, 29) others did not found any correlation (26, 27, 28). In the present study a positive correlation was observed between UA and EF, (p<0.05 in the presence of obesity). Tavil et al used issue Doppler imaging for the evaluation of myocardial performance index (MPI) (30). MPI in patients with arterial hypertension and hyperuricemia was significantly higher than in hypertensive patients without hyperuricemia. Systolic and diastolic BP were significantly higher in obese in the present study and BP may play an important role in the relationship between UA and EF. Conclusion:- The present study demonstrates a positive correlation of UA with EF and LA and LV volume in volume overload due to obesity in children. We did not confirm an influence of UA on LVM and on LV diastolic function in the presence of obesity. These conclusions require further investigation involving a larger cohort of children. Approx word count = 1,917, Max allowed = 3,000 words Acknowledgement:- Table 2a:-Structural echocardiographic parameters Parameter Overweight/Obese (BMI ≥ 85 percentile) N=25 Lean control (BMI < 85 percentile) N=24 p- value IVS dias (cm) 0.91 ± 0.20 0.75 ± 0.18 < 0.01 IVS sys (cm) 1.44 ± 0.40 1.30 ± 0.26 ns PWTh dias (cm) 0.82 ± 0.15 0.73 ± 0.16 0.06 PWTh sys (cm) 1.40 ± 0.29 1.37 ± 0.32 ns LV dias (cm) 4.85 ± 0.56 4.30 ± 0.50 < 0.01 LV sys (cm) 3.11 ± 0.75 2.62 ± 0.53 < 0.05 LVM (g) 149 ± 10 54 ± 43 < 0.01 LVMIV (g/m2.7) 39.6 ± 28.3 10.1 ± 8.3 <0.001 LA 2D area (cm2) 14.7 ± 3.5 12.4 ± 2.7 < 0.05 Data are shown as mean ± SD, ns- not significant, LA = Left atrium, LV = Left ventricular; IVS dias - Enddiastolic interventricular septum thickness; LV dias Enddiastolic diameter; PWTh dias - Enddiastolic posterior wall thickness; IVS sys - Endsystolic interventricular septum thickness; LV sys - Endsystolic diameter PWTh sys - Endsystolic posterior wall thickness; LVM- LV mass; LVMIV LV mass indexed to body height2.7. Table 2 b :-functional LV systolic and diastolic echocardiographic parameters Overweight/Obese (BMI ≥ 85 percentile) N=25 Lean control (BMI < 85 percentile) N=24 p- value LV diastolic parameters A wave (m/s) 0.60 ± 0.10 0.51 ± 0.11 <0.01 E wave (m/s) 1.02 ± 0.23 0.96 ± 0.11 ns IVRT (ms) 177.5 ± 70.4 132.7 ± 34.0 < 0.001 DT (ms) 143.1 ± 55.7 186.3 ± 75.1 <0.05 PHT (ms) 42.01 ± 6.3 54.5 ± 22.1 <0.05 LV systolic parameters he mean ± standard deviation (SD), ns- not significant BMI, body mass index; HOMA index, assessment of insulin resistance; BP, blood pressure. Data are shown as the mean ± standard deviation (SD), ns- not significant BMI, body mass index homeostasis model assessment of insulin resistance; BP, blood pressure. Acknowledgement:- g Schusterova I. conceived experiments, Schusterova I. and Leenen FHH. carried out a data analyses and interpretation. Tohatyova A. carried out a literature review and data collection. Artemiou P. carried out generation of figures, Takacova J. and Takac L. statistically analyzed data. All authors were involved in writing the paper and ha final approval of the submitted and published version. This research was supported by the Grant project of the Ministry of Education, science, research and sport of the SR („Origin and pathogenesis of obesity in relation to non-traditional obesity risk factors“ ) Table 1:-Anthropometric, clinical and biochemical characteristic Variables Overweight/Obese (BMI ≥ 85 percentile) N=25 Lean control (BMI < 85 percentile) N=24 p- value Age (years) 13.0 ± 2.3 12.9 ± 3.4 ns Body weight (kg) 72.3 ± 19.6 50.5 ± 14.3 < 0.01 Height (cm) 164 ± 16 161 ± 13 ns 405 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 BMI (kg/m2) 27.4 ± 3.5 18.8 ± 3.1 < 0.01 BMI percentile 94.3 ± 3.5 36.2 ± 2.8 < 0.01 BMI Z-score 2.09 ± 0.51 -0.38 ±1.01 < 0.01 Waist circumference (cm) 95.5 ± 12.6 73.1 ± 8.8 < 0.01 Uric acid (umol/l) 321 ± 67 282 ± 69 ns Glucose (mg/dl) 89 ± 9.1 89 ±8.2 ns Insulin (IU/L) 15.4 ±7.2 8.0±4.2 < 0.05 HOMA-index 3.5 ± 2.0 1.9±1.9 <0.01 Systolic BP (mmHg) 133 ± 16 120 ± 17 <0.05 Diastolic BP (mmHg) 80 ± 11 70 ± 7 <0.01 Data are shown as the mean ± standard deviation (SD), ns- not significant BMI, body mass index; HOMA index, homeostasis model assessment of insulin resistance; BP blood pressure Waist circumference (cm) 95.5 ± 12.6 73.1 ± 8.8 < 0.01 Uric acid (umol/l) 321 ± 67 282 ± 69 ns Glucose (mg/dl) 89 ± 9.1 89 ±8.2 ns Insulin (IU/L) 15.4 ±7.2 8.0±4.2 < 0.05 HOMA-index 3.5 ± 2.0 1.9±1.9 <0.01 Systolic BP (mmHg) 133 ± 16 120 ± 17 <0.05 Diastolic BP (mmHg) 80 ± 11 70 ± 7 <0.01 Data are shown as the mean ± standard deviation (SD), ns- not significant BMI, body mass index; HOMA index, homeostasis model assessment of insulin resistance; BP, blood pressure. Acknowledgement:- 7(10), 402-410 ISSN: 2320-5407 LV volume dias (cm3) 118.5 ± 34.7 85.3 ± 21.7 < 0.01 LV volume sys (cm3) 44.1 ± 9.0 36.8 ± 11.0 < 0.05 EF (%) 61 ± 9 56 ± 10 0.09 SV (ml) 74 ± 30 49 ± 19 < 0.01 MPI 0.14 ± 0.52 0.21 ± 0.22 ns Data shown as mean ± SD, ns- not significant wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity; IVRT - isovolumic relaxation time; DT – deceleration time of early LV volume dias (cm3) 118.5 ± 34.7 85.3 ± 21.7 < 0.01 LV volume sys (cm3) 44.1 ± 9.0 36.8 ± 11.0 < 0.05 EF (%) 61 ± 9 56 ± 10 0.09 SV (ml) 74 ± 30 49 ± 19 < 0.01 MPI 0.14 ± 0.52 0.21 ± 0.22 ns Data shown as mean ± SD, ns- not significant wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity; IVRT - isovolumic relaxation time; DT – deceleration time of early diastolic filling; PHT - pressure half time from blood flow Doppler evaluation; MPI - Myocardial performance index; EF - Ejection fraction; SV - Stroke volume. LV volume dias- LV volume in diastole; LV volume sys- LV volume in systole. Data shown as mean ± SD, ns- not significant wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity; IVRT - isovolumic relaxation time; DT – deceleration time of early diastolic filling; PHT - pressure half time from blood flow Doppler evaluation; MPI - Myocardial performance index; EF - Ejection fraction; SV - Stroke volume. LV volume dias- LV volume in diastole; LV volume sys- LV volume in systole. Table S1 (Supporting Table) Correlations of LV structural and LV functional diastolic and systolic echocardiographic parameters and with Blood Pressure (BP) and anthropometric parameters. Lean control Overweight/Obese Parameter Body Weight BMI Syst BP Body Weight Waist Circum. Acknowledgement:- Table 2a:-Structural echocardiographic parameters Parameter Overweight/Obese (BMI ≥ 85 percentile) N=25 Lean control (BMI < 85 percentile) N=24 p- value IVS dias (cm) 0.91 ± 0.20 0.75 ± 0.18 < 0.01 IVS sys (cm) 1.44 ± 0.40 1.30 ± 0.26 ns PWTh dias (cm) 0.82 ± 0.15 0.73 ± 0.16 0.06 PWTh sys (cm) 1.40 ± 0.29 1.37 ± 0.32 ns LV dias (cm) 4.85 ± 0.56 4.30 ± 0.50 < 0.01 LV sys (cm) 3.11 ± 0.75 2.62 ± 0.53 < 0.05 LVM (g) 149 ± 10 54 ± 43 < 0.01 LVMIV (g/m2.7) 39.6 ± 28.3 10.1 ± 8.3 <0.001 LA 2D area (cm2) 14.7 ± 3.5 12.4 ± 2.7 < 0.05 Data are shown as mean ± SD, ns- not significant, LA = Left atrium, LV = Left ventricular; IVS dias - Enddiastolic interventricular septum thickness; LV dias Enddiastolic diameter; PWTh dias - Enddiastolic posterior wall thickness; IVS sys - Endsystolic interventricular septum thickness; LV sys - Endsystolic diameter PWTh sys - Endsystolic posterior wall thickness; LVM- LV mass; LVMIV LV mass indexed to body height2.7. Table 2 b :-functional LV systolic and diastolic echocardiographic parameters Overweight/Obese (BMI ≥ 85 percentile) N=25 Lean control (BMI < 85 percentile) N=24 p- value LV diastolic parameters A wave (m/s) 0.60 ± 0.10 0.51 ± 0.11 <0.01 E wave (m/s) 1.02 ± 0.23 0.96 ± 0.11 ns IVRT (ms) 177.5 ± 70.4 132.7 ± 34.0 < 0.001 DT (ms) 143.1 ± 55.7 186.3 ± 75.1 <0.05 PHT (ms) 42.01 ± 6.3 54.5 ± 22.1 <0.05 LV systolic parameters 406 Int. J. Adv. Res. ISSN: 2320-5407 ns- not significant. LA = Left atrium, LV = Left ventricular; IVS dias - Enddiastolic interventricular septum thickness; LV dias Enddiastolic diameter; PWTh dias - Enddiastolic posterior wall thickness; IVS sys - Endsystolic interventricular septum thickness; LV sys - Endsystolic diameter PWTh sys - Endsystolic posterior wall thickness; LVM- LV mass; LVMIV -LV mass indexed to body height2.7, E wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity velocity; EF - Ejection fraction; SV - Stroke volume. LV volume dias - LV volume in diastole; LV volume sys- LV volume in systole BMI- body mass index, In lean controls, Waist Circumference showed no correlations ns- not significant. LA = Left atrium, LV = Left ventricular; IVS dias - Enddiastolic interventricular septum thickness; LV dias Enddiastolic diameter; PWTh dias - Enddiastolic posterior wall thickness; IVS sys - Endsystolic interventricular septum thickness; LV sys - Endsystolic diameter PWTh sys - Endsystolic posterior wall thickness; LVM- LV mass; LVMIV -LV mass indexed to body height2.7, E wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity velocity; EF - Ejection fraction; SV - Stroke volume. LV volume dias - LV volume in diastole; LV volume sys- LV volume in systole BMI- body mass index, In lean controls, Waist Circumference showed no correlations Table S2:-(Supporting Table) Correlations between UA and LV structural and LV diastolic and systolic echocardiographic parameters Lean control Overweight/Obese IVS dias (cm) r=0.55 p< 0.05 ns IVS sys (cm) r=0.70 p< 0.01 ns LV dias (cm) p=0.07 ns LVM (g) r=0.56 p< 0.05 ns LVMIV (g/m2.7) p=0.055 ns LA 2D area (cm2) p=0.08 r=0.45 p< 0.05 A wave (m/s) r= -0.60 p< 0.05 ns E wave (m/s) ns ns EF (%) ns r=0.42 p< 0.05 SV (ml) ns r=0.59 p< 0.01 LV dias volume (cm3) p=0.10 r=0.59 p< 0.01 S2:-(Supporting Table) Correlations between UA and LV structural and LV diastolic and systolic rdiographic parameters Table S2:-(Supporting Table) Correlations between UA and LV structural and LV diastolic and echocardiographic parameters ns - not significant. Acknowledgement:- BMI Syst BP IVS dias (cm) r=0.60 p< 0.01 r=0.44 p< 0.05 ns r=0.51 p< 0.05 p=0.05 p=0.07 ns IVS sys (cm) r=0.72 p<0.001 r=0.64 p< 0.01 ns r=0.48 p< 0.05 p=0.05 p< 0.01 r=0.56 ns PWTh dias (cm) r=0.66 p<0.001 r=0.55 p< 0.01 p=0.055 r=0.59 p< 0.01 p=0.05 ns r=0.55 p<0.05 PWTh sys (cm) r=0.57 p< 0.01 r=0.49 p< 0.05 ns r=0.44 p< 0.05 p=0.06 ns ns LV dias (cm) r=0.68 p<0.001 p=0.06 r=0.61 p<0.05 r=0.58 p< 0.01 p=0.05 ns r=0.58 p<0.05 LV sys (cm) r=0.48 p< 0.05 ns r=0.72 p<0.01 ns ns ns ns LVM (g) r=0.76 p<0.001 r=0.54 p< 0.05 r=0.57 p<0.05 r=0.72 p<0.001 r=0.55 p< 0.05 p=0.05 r=0.62 p<0.01 LVMIV (g/m2.7) r=0.52 p=0.07 ns ns ns ns ns LA 2D area (cm2) r=0.71 p<0.001 r=0.56 p< 0.01 ns r=0.59 p< 0.01 r=0.47 p< 0.05 ns p=0.06 E wave (m/s) ns ns ns r= -0.49 p< 0.05 r= -0.47 p< 0.05 r= -0.45 p< 0.05 ns A wave (m/s) ns ns ns ns ns p=0.06 ns EF (%) r=0.55 p< 0.05 p=0.06 ns r=0.52 p< 0.05 ns p=0.05 r=0.58 p<0.05 SV (ml) r=0.79 p<0.001 r=0.69 p< 0.01 r=0.65 p<0.05 r=0.62 p<0.001 r=0.44 p<0.001 r=0.41 p< 0.01 r=0.58 p<0.001 LV sys volume (cm3) ns ns ns r=0.68 p<0.001 r=0.56 p< 0.05 ns ns LV dias volume (cm3) r=0.82 p<0.001 r=0.74 p<0.001 r=0.68 p<0.05 r=0.88 p<0.001 r=0.80 p<0.001 r=0.57 p< 0.01 r=0.77 p<0.001 S1 (Supporting Table) Correlations of LV structural and LV functional diastolic and systolic rdiographic parameters and with Blood Pressure (BP) and anthropometric parameters. 407 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 ISSN: 2320-5407 LA = Left atrium, LV = Left ventricular; IVS dias - Enddiastolic interventricular septum thickness; LV dias Enddiastolic diameter; IVS sys - Endsystolic interventricular septum thickness, LVM- LV mass; LVMIV LV mass indexed to body height2.7, E wave - peak early transmitral filling wave velocity velocity; A wave - peak late transmitral filling wave velocity ; EF - Ejection fraction; SV - stroke volume, LV volume dias- LV volume in diastole. Figure 1:-Correlations between UA and LV structural echocardiographic parameters Figure 1:-Correlations between UA and LV structural echocardiographic parameters None of the correlations differed between the 2 groups. Red dots - Overweight and Obese, Blue dots - Lean control, IVS dias - Enddiastolic interventricular septum thickness, LVM- LV mass, LA-Left atrium. Figure 2:-Correlations between UA and LV functional diastolic and systolic echocardiographic parameters igure 1: Correlations between UA and L g p p None of the correlations differed between the 2 groups. Red dots - Overweight and Obese, Blue dots - Lean control, IVS dias - Enddiastolic interventricular septum thickness, LVM- LV mass, LA-Left atrium. Figure 2:-Correlations between UA and LV functional diastolic and systolic echocardiographic parameters 408 Int. J. Adv. Res. 7(10), 402-410 ISSN: 2320-5407 Difference between study groups in correlation of SV and UA: p<0.05. Other correlations did not differ between the 2 groups. Red dots - Overweight and Obese, Blue dots - Lean control, A wave - Peak late transmitral filling wave velocity velocity; EF - Ejection fraction; SV - stroke volume, LV volume dias- LV volume in diastole. Difference between study groups in correlation of SV and UA: p<0.05. Other correlations did not differ between the 2 groups. Red dots - Overweight and Obese, Blue dots - Lean control, A wave - Peak late transmitral filling wave velocity velocity; EF - Ejection fraction; SV - stroke volume, LV volume dias- LV volume in diastole. References:- e e e ces: 1. Gustafsson D, Unwin R. The pathophysiology of hyperuricaemia and its possible relationship to cardiovascular disease, morbidity and mortality. BMC Nephrol 2013; 29:14:1642 2. Sundstrom J , Sullivan L, D’Agostino RB, et al. Relations of Serum Uric Acid to Longitudinal Blood Pressure Tracking and Hypertension Incidence. Hypertension 2005;45:28-33 2. Sundstrom J , Sullivan L, D’Agostino RB, et al. Relations of Serum Uric Acid to Longitudinal Blood Pressure Tracking and Hypertension Incidence. Hypertension 2005;45:28-33 3. Mule G, Nardi E, Costanzo M, et al. Metab Absence of an independent association between serum uric acid and left ventricular mass in Caucasian hypertensive women and men. Nutrition & Cardiovascular Diseases 2013; 23:715-722 4. 4. Iwashima Y, Horio T, Kamide K, et al. Uric acid, left ventricular mass index, and risk o disease in essential hypertension. Hypertension 2006;47:195-202 5. Tsioufis C, Chatzis D, Vezali E, et al. The controversial role of serum uric acid in essential hypertension: relationships with indices of target organ damage. J Hypertension 2005;19:211-217 5. Tsioufis C, Chatzis D, Vezali E, et al. The controversial role of serum uric acid in essential hypertension: relationships with indices of target organ damage. J Hypertension 2005;19:211-217 6. Szwejkowski BR, Gandy SJ, Rekhraj S, et al. Allopurinol reduces left ventricular mass in patients with type 2 diabetes and left ventricular hypertrophy. J Am Coll Cardiol. 2013 Dec 17;62(24):2284-93 6. Szwejkowski BR, Gandy SJ, Rekhraj S, et al. Allopurinol reduces left ventricular mass in patients with type 2 diabetes and left ventricular hypertrophy. J Am Coll Cardiol. 2013 Dec 17;62(24):2284-93 yp p y 7. Soletsky B, Feig DI. 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Am J Dis Child 1991;145:259-263 12. American Academy of Pediatrics. National cholesterol education program: Report of the exper cholesterol levels in children and adolescents. Pediatrics 1992;89:525-584 13. Schiller NB, Shah PM, Crawford M, et al. American Society of Echocardiography Committee on Standards Subcommitee on Quantitation of Two-dimensional Echocardiograms. Recommendations for quantitation of the left ventricle by two- dimensional echocardiography. J Am Soc Echocardiogr 1989; 41:126-131 14. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28:412-419 15. Schusterova I, Saligova J, Potocnakova L, et al. Impact of obesity and overweight on left ventricle mass, systolic and diastolic function in children and adolescents. Structured summary. Circulation 2008;118: 417 409 ISSN: 2320-5407 Int. J. Adv. Res. 7(10), 402-410 Int. J. Adv. Res. 7(10), 402-410 16. Schusterova I, Saligová J, Potocnákova L, et al. Lipids disorders, obesity and left ventricle mass and geometry in children and adolescents. Structured summary. Atherosclerosis Suppl 2005;6:140 16. Schusterova I, Saligová J, Potocnákova L, et al. Lipids disorders, obesity and left ventricle mass and geometry in children and adolescents. Structured summary. Atherosclerosis Suppl 2005;6:140 17. Mitsuhashi H, Yatsuya H, Matsushita K, et al. Uric acid and left ventricular hypertrophy in Japanese men. Circ y pp 17. Mitsuhashi H, Yatsuya H, Matsushita K, et al. Uric acid and left ventricular hypertrophy in J 2009;73:67-72 18. Krishnan E, Hariri A, Dabbous O, et al. Hyperuricemia and the echocardiographic measures of myocardial dysfunction. Congest Heart Fail. 2012 May-Jun;18(3):138-43 E, Hariri A, Dabbous O, et al. Hyperuricemia ardiographic measures of myocardial dysfunction Congest Heart Fail 2012 May Jun;18(3):138 43 raphic measures of myocardial dysfunction. 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Elevated serum uric acid levels are associated with diastolic dysfunction in patients with dilated cardiomyopathy. Am Heart J 2002;143(6):1107-1111 25. Pan KL, Lin JC, Lin CL, et al. The effects of gout on left atrial volume remodelling: a prospective echocardiographic study. Rheumatology 2014;53:867-874 26. Gromadziński L, Januszko-Giergielewicz B, Pruszczyk P. Hyperuricemia is an Independent Predictive Factor for Left Ventricular Diastolic Dysfunction in Patients with Chronic Kidney Disease. Adv Clin Exp Med 2015;24: 47–54 27. Codoner-Franch P, Tavarez-Alonso S, Estal R, Megias-Vericat J, et al. Nitric oxide production is increased in severly obese children and related to markers of oxidative stress and inflammation. Atherosclerosis 2011;215:475-480 28. Amin A, Vakilian F, Majid Maleki M. Serum Uric Acid Levels Correlate with filling Pres Heart Failure. Congest Heart Fail 2011;17:79-83 akilian F, Majid Maleki M. Serum Uric Acid Levels Correlate with filling Pressures in Systolic e. Congest Heart Fail 2011;17:79-83 29. Yazicioğlu MV, Avci A, Açar G, et al. Elevated uric acid and functional mitral regurgitation in dilated cardiomyopathy. Eur Rev Med Pharmacol Sci 2012;16:1637-1641 y y 30. Tavil Y, Kaya MG, Sen N, et al. Assessment of left ventricular systolic and diastolic function by tissue Doppler analysis in patients with hypertension with or without hyperuricemia. Blood Press Monit 2008;13:79-84. 410
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Hair growth-promoting effect of Geranium sibiricum extract in human dermal papilla cells and C57BL/6 mice
BMC complementary and alternative medicine
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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 DOI 10.1186/s12906-017-1624-4 Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 DOI 10.1186/s12906-017-1624-4 Open Access Hair growth-promoting effect of Geranium sibiricum extract in human dermal papilla cells and C57BL/6 mice William A. Boisvert1†, Miri Yu2†, Youngbin Choi2, Gi Hee Jeong2, Yi-Lin Zhang2, Sunghun Cho3, Changsun Choi2, Sanghyun Lee3 and Bog-Hieu Lee2* * Correspondence: lbheelb@cau.ac.kr †Equal contributors 2Department of Food and Nutrition, College of Biotechnology and Natural Resources, Chung-Ang University, Anseong 17546, Korea Full list of author information is available at the end of the article Abstract Background: Geranium sibiricum L. has been used as a medicinal plant to treat diarrhea, bacterial infection, and cancer in Bulgaria, Peru, and Korea. However, its hair growth-promoting effect was not investigated so far. This study examined the effects of Geranium sibiricum L. extract (GSE) on hair growth, using in vitro and in vivo models. Methods: Antioxidant, proliferation and migration assay of GSE was performed with human dermal papilla cells (hDPCs). Hair-growth promoting effect was measured in animal model. Relative expression of interleukin-1, vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor beta 1 was determined by real time RT-PCR. Expression of Ki-67 and stem cell factor were analyzed by immunohistochemistry. Results: GSE treatment proliferated and migrated human dermal papilla cells (hDPCs) more than treatment of 10 μM minoxidil. GSE significantly stimulated the expression of Ki-67 protein and the mRNA levels of hepatocyte growth factor and vascular endothelial growth factor in hDPCs. Topical application of 1,000 ppm GSE for 3 weeks promoted more significant hair growth on shaved C57BL/6 mice than did 5% minoxidil. The histological morphology of hair follicles demonstrated an active anagen phase with the induction of stem cell factor. GSE treatment significantly reduced the number of mast cells and the expression of transforming growth factor beta 1 in mouse skin tissues. Conclusions: These results demonstrated that GSE promotes hair growth in vitro and in vivo by regulating growth factors and the cellular response. Conclusions: These results demonstrated that GSE promotes hair growth in vitro and in vivo by regulating growth factors and the cellular response. Keywords: Hair loss, Antioxidant, Geranium sibiricum L, Human dermal papilla cells, Transforming grow oss, Antioxidant, Geranium sibiricum L, Human dermal papilla cells, Transforming growth factor beta 1 Background Plant phenolics and flavonoid are recently of interest, since these compounds possess antioxidation, anti- inflammatory, anti-microbial, and anti-carcinogenic properties [9]. Geranium sibiricum L., which belongs to the Geraniaceae family of plants, grows in China, Japan, Korea, and some European countries. While it is used as a food ingredient in Russia and Turkey, it has been used as a medicinal plant to treat diarrhea, bacterial infection, and cancer in Bulgaria, Peru, and Korea [10]. The extract and phenolic compounds from G. sibiricum showed high antioxidant capacity in 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, superoxide radical scavenging, nitric oxide scavenging, β-carotene-linoleic acid bleaching, and reducing power [11]. As several pharmacological studies of G. sibiricum have shown anti-inflammatory, anti-bacterial, anti- diarrheal effect and anti-gastric ulcer action [12–15], it is widely used in cosmetic industry nowadays. Shim et al. [16] has reported that ethanol extract of Geranium sibiri- cum L. decreased expression of interleukin (IL)-1β, COX- 2 and inducible nitric oxide synthase (iNOS) in PMACI stimulated HMC-1 cells. IL-1β and COX-2 are known as potent inhibitors of hair growth in vitro and in vivo. Inui et al. [17] has also found that dihydrotestosterone (DHT), contributing to androgenic alopecia, increases iNOS from occipital dermal papilla cells and suggested that iNOS and NO are downstream effectors of androgen receptors. However, the effects of G. sibiricum extract (GSE) on hair growth have not been studied so far. Therefore, the study aimed to investigate whether the topical treatment of GSE could promote hair growth in vitro and in vivo models by regulating the expression of growth factors and inflamma- tory cytokines. HPLC separation of corilagin and gallic acid for quali- tative and quantitative analysis was performed using a reverse phase system. Discovery® C18 (4.6 × 250 mm, 5 μm) column was used with a mobile phase consisting of a gradient system of water containing 0.2% acetic acid and ACN (90:10 to 60:40 for 30 min). UV detection was conducted at 270 nm. The injection volume was 10 μl and the flow rate was 1 mL/min. All injections were performed in triplicate. Corilagin and gallic acid were weighed and dissolved in MeOH to obtain a stock stand- ard solution (1.0 mg/mL). Solutions of aqueous corilagin and gallic acid were prepared at concentrations of 0.0001, 0.001, 0.01, 0.1, and 1 mg/mL for the construction of a calibration curve. The contents of the analysis were determined from the corresponding calibration curves. Background in men and can lead to the birth of deformed baby if used by pregnant women. In efforts to find natural sub- stances that are less toxic than minoxidil and finaste- ride, previous studies have screened about 1,000 plant extracts for hair growth or hair loss-preventing effects [5, 6]. Among the natural extracts, Allium cepa extract and Ziziphus jujuba extract were found to promote hair growth [5, 6], with the antioxidant capacity of each ex- tract being concluded as the contributing factor. Hair loss is defined as a state in which hair does not exist at a typical area or less hair regrowth is observed in the area [1]. In modern society, hair loss occurs via genetic reasons as well as external factors such as environmental pollution, work stress, and alteration of hormone secretion [1]. Minoxidil and finasteride are the only chemicals approved by the US Food and Drug Administration to treat hair loss [2–4]. However, both these chemicals have serious adverse effects such as weight gain, edema, angina pectoris, and hypogonadism All living organisms are constantly challenged by a diversity of exogenous- and endogenous stressors, which induce biological responses to protect or adapt to stressors. The systemic biological response of the organism to stressor induces stress response through activation of hypothalamic-pituitary-adrenal axis (HPA) by proinflammatory cytokines to increase circulating * Correspondence: lbheelb@cau.ac.kr †Equal contributors 2Department of Food and Nutrition, College of Biotechnology and Natural Resources, Chung-Ang University, Anseong 17546, Korea Full list of author information is available at the end of the article Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 2 of 9 Page 2 of 9 Page 2 of 9 GSE was dissolved in dimethylsulfoxide (DMSO). Water, acetonitrile (ACN), and methanol (MeOH) used in this research were of HPLC grade, and all other reagents were of analytical grade. Corilagin and gallic acid were provided by Professor Sam Sik Kang, Seoul National University, Korea. They were used as standard chem- ical for high pressure liquid chromatography (HPLC) analysis. HPLC chromatograms were recorded with a Waters Breeze system (Massachusetts, USA) equipped with a Waters 1525 binary HPLC pump and 2489 sys- tem UV/VIS detector. glucocorticoids and catecholamines [7]. The growing body of evidence now supports that a wide range of neuropeptides, neurotransmitters, and neurohormones modulating systemic stress responses can indeed alter hair growth, indicating that hair follicles represent an important target for stressors [8]. Antioxidative activity of GSE y The analysis of total polyphenol content was determined using Folin-Denis method. Point five milliliter of GSE prepared 50% methanol at concentration of 0.2 mg/mL was mixed with 0.5 mL of Folin-Denis reagent (Fluka, Buchs, Switzerland) and was added to 0.5 mL of 10% sodium carbonate solution after 3 min at room temperature. The mixture was kept at room temperature for 1 h and then the absorbance was measured at 760 nm using 7315 UV spectrophotometer (JENWAY, Staffordshire, United Kingdom). Total polyphenol con- tent was expressed as mg of tannic acid (Yakuri Pure Chemicals Co., Ltd., Kyoto, Japan) equivalent (TAE). Background The calibration functions of the compound were calculated using the peak area (Y), concentration (X, μg/10 μl), and mean values (n = 3) ± standard devi- ation (SD). Preparation of GSE and HPLC analysis EDA % ð Þ ¼ ABScontrol−ABSsample ABScontrol  100 EDA % ð Þ ¼ ABScontrol−ABSsample ABScontrol  100 Animal care and in vivo experiment for hair growth Fifteen male 4-week-old C57BL/6 mice were pur- chased from Central Lab, Animal Inc. (Seoul, Korea). The mice were acclimated to their surroundings for 2 weeks to lead to the early onset of the telogen phase in mice hair cycles. Mice were randomly assigned to the experimental groups with 5 mice per group and were singly housed in cages to minimize the loss of the topically applied extracts by contact with other mice. All animals were bred in a laminar airflow room with 12 h of artificial light and darkness for 1 week. Animal facility was maintained at 22 ± 1 °C room temperature and a relative humidity of 55 ± 10%. All experimental procedures were approved by the Animal Care Committee of the Chung-Ang University (ap- proval no: 13–0058). Proliferation and migration assay of human dermal papilla cells (hDPCs) Cell proliferation was determined by Cell Counting Kit- 8 (CCK-8) (Sigma, St. Louis, MO, USA). hDPCs were seeded at a density of 5 × 103 cells/well of a 96-well microplate. After incubation for 24 h, the cells were cultured with 100 μL of serum-free Dulbecco’s modified Eagle’s medium (DMEM, Sigma) for 24 h and then treated with 9.8, 19.5, 39.1, 78.1, 156.3 μg/mL of GSE in DMEM. Serum-free DMEM medium and triton X- 100 (Sigma, St. Louis, MO, USA) were used as negative control (NC) and blank, respectively. Boyera et al. [19] has found that micromolar concentrations of minoxidil stimulated proliferation in both human keratinocytes of epidermal and hair follicle origin cell types and in all culture conditions, whereas millimolar concentrations inhibited cell growth. Similarly, treatment concentra- tion of minoxidil was examined for hair growth effect with less toxicity in our preliminary study. Thus, 10 μM minoxidil was used as positive control (PC) in vitro assay. After treatment for 24 h, 10 μL of CCK-8 solu- tion was added to each well, the cells were then incu- bated at 37 °C for 3 h. The absorbance was measured at 450 nm (test wavelength) and 650 nm (reference wave- length) by microplate spectrophotometer (Epoch Multi- Volume Spectrophotometer System, BioTek, VT, USA). The measured absorbance was used to determine cell proliferation by the following equation. Experimental groups contained NC, PC, and GSE group. Mice were randomly separated into three groups with five mice per group. In order to synchronize the stage of hair growth, back skin of all experimental animals were artificially shaved. Topical treatments on the shaved back skin were applied daily with 1% di- methyl sulfoxide (NC group), 5% minoxidil (dissolved into 1% DMSO) (PC group), or GSE dissolved in 1% DMSO with the final concentration of 1,000 ppm. Hair growth of each mouse was measured and photographed every week for 3 weeks after shave. Preparation of GSE and HPLC analysis Preparation of GSE and HPLC analysis GSE was purchased from a Korea Pla GSE was purchased from a Korea Plant Extract Bank at Korea Research Institute of Bioscience & Biotechnology (KRIBB, Daejeon, Korea). G. sibiricum L. used in this study were collected from Mountain Gamak in Paju-si, Korea. Botanical samples were authenticated by Professor Shin-Ho Kang from Department of Natural Medicine Resources in Semyung University. The air-dried plant materials were ground into fine powder and extracted with 100% methanol. After filtration of total extract, the extract was evaporated to dryness in vacuum and weighed. The recovery rate was 6.7%. y To determine the total flavonoid content, 0.5 mL of the 0.2 mg/mL dissolved in methanolic GSE was mixed with 5 mL of diethylene glycol (Duksan Pure Chemicals, Ansan, Korea). After 5 min at room temperature, the mixture was added to 0.5 mL of 1 N NaOH (Duksan Pure Chemicals, Ansan, Korea) and allowed to stand for 1 h in 37 °C water bath. The absorbance was measured at 420 nm using 7315 UV spectrophotometer (JENWAY, Staffordshire, United Kingdom). Total flavonoid content Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 3 of 9 Page 3 of 9 Page 3 of 9 was expressed as mg of naringin (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) equivalent (NE). 200 μL pipette tip and the cell debris was removed by washing the cells with sterile PBS. hDPCs were treated with 9.8, 19.5, 39.1, 78.3 μg/mL of GSE. Serum-free DMEM medium and 10 μM minoxidil were used as NC and PC, respectively. After incubation for 24 h, hDPCs were fixed with 4% paraformaldehyde, washed two times with phosphate buffered saline (PBS). The cells were stained with hematoxylin (Sigma, St. Louis, MO, USA) and images were taken using a Leica 500 optical microscope (Leica, Wetzlar, Germany) at a magnifica- tion of 40 ×. The method was conducted as described by Blois [18] with some modifications to determine DPPH scavenging activity of GSE. 0.5 mL of diluted to 0.2 mg/ mL GSE was mixed with 3 mL of 0.2 mM methanolic DPPH solution. 0.5 ml of 100% methanol was used as a carrier control. After 30 min in the dark at room temperature, the absorbance of the mixture was measured at 517 nm by a 7315 UV spectrophotometer (JENWAY, Staffordshire, United Kingdom). The electron donating ability (EDA) was calculated by the following equation. Immunohistochemistry of Ki-67 and stem cell factor hDPC or mouse skin, 1 μg of total RNA was treated with DNase I (Invitrogen). cDNA of mRNA was synthesized using cDNA synthesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quantitative real-time polymerase chain reaction (qPCR) was conducted with Piko-real 96 real-time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA) and performed for 45 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s. cDNA was ampli- fied using Maxima SYBR Green/ROX qPCR Mater Mix 2X (Thermo Fisher Scientific Inc., Waltham, MA, USA). Primers were used as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5′-GGA AGG TGA AGG TCG GAG TC-3′ (forward); GAPDH, 5′-CTC AGC CTT GAC GGT GCC ATG-3′ (reverse); VEGF, 5′-CTT TAG AGA TCA GCC CAA CC-3′ (forward); VEGF, 5′-CTA CCC AGA GGG AAG AAA TAA C-3′ (reverse); HGF, 5′-AGA AAT GCA GCC AGC ATC AT-3′ (forward); HGF 5′- CAC ATG GTC CTG ATC CAA TC-3′ (reverse), TGF- β1; 5′-GCC CTG GAC ACC AAC TAT TG-3′ (forward); TGF-β1; 5′-GTC CAG GCT CCA AAT GTA GG-3′ (reverse). Immunohistochemistry of Ki-67 and stem cell factor To confirm the effect of GSE on hDPCs proliferation, the immunohistochemistry for Ki-67 was performed with some modification described in previous study [21]. hDPCs were seeded at a density of 2 × 103 cells/ well of an 8-well chamber slide and incubated until grow to 80-90% confluence in 10% FBS DMEM medium. After culture with serum-free DMEM medium for 24 h, the cells were added to each treat- ment and incubated for 24 h; serum-free DMEM (NC), 10 μM minoxidil (PC), 19.5 μg/mL of GSE. The cells were fixed for 20 min with 4% paraformaldehyde, washed three times with PBS. Endogenous alkaline phosphatase was quenched with levamisole and 10% normal goat serum was used for blocking non-specific reaction. Monoclonal mouse anti-human Ki-67 antibody (DAKO, Glostrup, Denmark) at 1:100 was incubated for 1 h at room temperature. Polyclonal goat anti-mouse im- munoglobulins/alkaline phosphatase (DAKO, Glosturp, Denmark) was applied for 1 h. Positive signals were visual- ized with freshly prepared fast red (Vector Laboratories Inc., Burlingame, California, USA). Images were taken using a Leica DM 500 optical microscope (Leica, Wetzlar, Germany) at a magnification of 200 ×. mRNA relative expression of cytokines p y In vitro model, hDPCs were plated on 6-well plate (3 × 104) and incubated until grow to 80-90% conflu- ence in 10% FBS DMEM medium. After culture with serum-free DMEM medium for 24 h, The cells were added to each treatment and incubated for 24 h; serum-free DMEM (NC), 10 μM minoxidil (PC), 19.5 μg/mL of GSE. Total RNA was extracted using RNeasy Mini kit (Qiagen, Dusseldorf, Germany) ac- cording to manufacturer’s instruction. In vivo model, mouse skins of each group were collected after 3 weeks of depilation. Total RNA of skin tissue was extracted using RNeasy Mini kit (Qiagen) following the manu- facturer’s instruction. Active components of GSE Contents of GSE were analyzed by HPLC (Fig. 1). The corilagin and gallic acid contents of GSE were quanti- fied using the linear regression equation. The linear regression data from the extracts had a good linear relationship and the resulting equation was valid over the relevant concentration range. The linear calibration equations were Y = 917496X + 24289 and Y = 2000000X + 91670, where Y is peak area and X is concentration of cor- ilagin and gallic acid. The correlation coefficients (r2) of corilagin and gallic acid were 0.9998 and 0.9997, respect- ively. The contents of corilagin and gallic acid were deter- mined as 47.33 ± 1.99 and 3.31 ± 0.33 mg/g of GSE, respectively (Fig. 1). Statistical analysis Data are presented as mean ± standard error (SE). One- way analysis of variance (ANOVA) and Duncan’s mul- tiple range test was performed using SPSS program for windows, version 21.0 (SPSS Inc., Chicago, USA). Statis- tical significance was defined as p < 0.05. In order to examine the expression of the stem cell factor in skin tissues, immunohistochemistry was per- formed by standard ABC protocol. Mouse anti-stem cell factor antibody (Santa cruz, Dallas, TX, USA) at 1:100 dilutions was incubated. Secondary antibody used with polyclonal goat anti-mouse immunoglobulins/horse rad- ish peroxidase (DAKO, Glosturp, Denmark). For the substrate of horse radish peroxidase, 3,3’-diaminobenzi- dine (DAB; Roche Diagnostics GmbH, Mannheim, Germany) was used to visualize the positive signal on tissues. Each well was mounted with coverslip using aqueous mounting media (Vector laboratories). Histopathology of hair growth Dorsal skin tissues from each mouse were excised and fixed in 4% neutral formalin for 24 h. They were embed- ded in paraffin after routine tissue processing. For the histopathogic evaluation of hair follicles, 4 μm tissue section was stained with standard hematoxylin and eosin (H&E) protocol. For the counting of mast cells, toluidine blue stain (Sigma, St. Louis, MO, USA) was performed on skin tissues by manufacturer’s protocol. The morph- ology of hair follicles, the number of mast cells, and expression of stem cell factor (SCF) were evaluated by veterinary pathologists. Cell proliferation % ð Þ ¼ ABSsample‐ABSblank   450nm‐ ABSsample‐ABSblank   650nm ABScontrol‐ABSblank ð Þ450nm‐ ABScontrol‐ABSblank ð Þ650nm  100 Migration assay was conducted as describe by previous study [20]. hDPCs were plated on 8-well chamber slide (Nalge Nunc International, Naperville, Illinois, USA) and grown to 90% confluence in 10% FBS DMEM medium. The cells were then scratched in straight line with Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 4 of 9 Page 4 of 9 mRNA Expression of cytokines in hDPCs The relative expression levels of VEGF, HGF, and TGF- β1 in GSE-treated hDPCs were measured by real-time RT-PCR (Fig. 3c). Compared with NC group, GSE sig- nificantly upregulated mRNA expression of HGF and VEGF in hDPCs by 5.85-fold and 3.75-fold, respectively (p < 0.05). Although TGF-β1 mRNA expression of GSE was not significantly different with NC group, PC group significantly increased mRNA expression of TGF-β1 higher than GSE and NC group (p < 0.05). Proliferation and migration of hDPCs Hair comprises at least fifteen distinct types of cells where the dermal papilla cell (DPC) is a primary cell type that regulates hair growth. Thus, determining the proliferation and migratory effects of GSE on hDPCs is necessary. The proliferative and migratory effects of GSE (9.8-156.3 ppm) were measured in hDPCs (Fig. 2a). The treatment of 19.5 ppm of GSE showed the highest prolif- eration rate of hDPCs. The proliferation rate of hDPCs was even higher in 19.5 ppm of GSE than PC by 132.7% vs. 120.5%. The concentration of 19.5-39.1 ppm GSE was significantly higher in proliferation rate than NC (p < 0.05). It was observed that GSE exhibited toxicity (cell viability less than 85%) above the concentration of 156.3 ppm. Antioxidative activity In order to compare the antioxidant capacity of GSE, its total polyphenol and flavonoid contents and EDA were measured. Total polyphenol, flavonoid and EDA of GSE were 305.9 mg TAE/g extract, 104 mg NE/g extract and 76.6%, respectively. For analyzing the expression of hepatocyte growth fac- tor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta 1 (TGF-β1) of Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 5 of 9 Proliferation and migration of hDPCs Hair comprises at least fifteen distinct types of cells the NC and PC groups (2.7 and 5.3 cells, respectively) (Fig. 3b). Fig. 1 HPLC chromatograms of corilagin (a), gallic acid (b), and GSE (c) Fig. 1 HPLC chromatograms of corilagin (a), gallic acid (b), and GSE (c) Fig. 1 HPLC chromatograms of corilagin (a), gallic acid (b), and GSE (c) the NC and PC groups (2.7 and 5.3 cells, respectively) (Fig. 3b). Proliferation and migration of hDPCs Histopathology and cytokine expression of depilated mice NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil; GSE: hDPCs treated with 19.5 ppm GSE Fig. 3 Changes in the levels of Ki67 protein, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta 1 (TGF-β1) in human dermal papilla cells (hDPCs) induced by GSE. a Immunohistochemistry of Ki67. b Numbers of Ki67- positive cells. c Relative expression levels of HGF, VEGF, and TGF-β1 in GSE-treated hDPCs. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil; GSE: hDPCs treated with 19.5 ppm GSE was most strongly expressed at the cytoplasm of skin epidermis and dermal papillary cells of hair follicles in GSE-treated group compared to the NC and PC groups (Fig. 4b). The induction of SCF in the in vivo model suggested that hDPCs underwent the regeneration process of hair follicles. not been studied so far. Therefore, we investigated whether the topical treatment of GSE could promote hair growth, using in vitro and in vivo models. The mRNA expression of cytokines in mouse skins was measured by qPCR. The expression of HGF, VEGF, and TGF-β1 was normalized with GAPDH housekeep- ing gene and presented as fold change. The real-time RT-PCR results showed that 1,000 ppm GSE treatment significantly downregulated VEGF, HGF, and TGF-β1 mRNA expression in the mouse skin (Fig. 4d). g g This study confirmed the corilagin and gallic acid as active components of GSE in HPLC chromatogram as both were identified from G. sibiricum [13]. Other study also reported the maximum content of gallic acid in G. sibiricum collected in Liaoning Province, China [22]. The antioxidant activity of corilagin and gallic acid was shown by scavenging various radicals effectively [23, 24]. Anti-inflammatory activity and anti-cancer effect of corilagin and gallic acid were also addressed in several studies [25–28]. Furthermore, gallic acid could prevent allergic reaction by blocking histamine release and pro-inflammatory cytokine expression in mast cell [26]. Histopathology and cytokine expression of depilated mice On the other hand, the migration rate of the PC group was 59%, whereas that of 19.5 ppm GSE-treated group was 402%, indicating 19.5 ppm GSE-treated group had the highest migration rate among all the treatment groups (p < 0.05) (Fig. 2b). The morphology of the hair follicles, number of mast cells, and expression of SCF were evaluated by veterinary pathologists (Fig. 4b). From the histological analysis, it was observed that the depth of the hair follicle was dee- per, and the length of the hair follicular shaft was longer in the GSE group than those in the NC and PC groups (Fig. 4b). The number of mast cells was significantly lower in the GSE group (1.75 mast cells) than in the NC (6.0 mast cells) and PC (7.5 mast cells) groups (Fig. 4b, c). Mast cells were located in dermal connective tissue. SCF Immunohistochemistry localized the expression of Ki-67 protein on hDPCs treated with GSE (Fig. 3a). Positive signal of Ki-67 was observed only in nucleus of hDPCs. Based on the histomorphologic analysis, Ki-67 protein expression was significantly elevated in the GSE-treated group (average 13.4 cells) compared with Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 6 of 9 Fig. 2 Effect of GSE on the proliferation and migration of human dermal papilla cells (hDPCs) (a) Proliferation of hDPCs was analyzed by the Cell Counting Kit-8 assay, and values are shown as the mean with SEM. b Migration effect of GSE was measured by the scratch migration assay, and the alteration of the scratch line width was converted into a percentage. NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil Fig. 3 Changes in the levels of Ki67 protein, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta 1 (TGF-β1) in human dermal papilla cells (hDPCs) induced by GSE. a Immunohistochemistry of Ki67. b Numbers of Ki67- positive cells. c Relative expression levels of HGF, VEGF, and TGF-β1 in GSE-treated hDPCs. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. Histopathology and cytokine expression of depilated mice Therefore, it seemed that the action of two major components of GSE contributed the promoting of hair growth by reducing inflammation in vitro and in vivo model. Histopathology and cytokine expression of depilated mice NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil; GSE: hDPCs treated with 19.5 ppm GSE Fig. 2 Effect of GSE on the proliferation and migration of human dermal papilla cells (hDPCs) (a) Proliferation of hDPCs was analyzed by the Cell Counting Kit-8 assay, and values are shown as the mean with SEM. b Migration effect of GSE was measured by the scratch migration assay, and the alteration of the scratch line width was converted into a percentage. NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil Fig. 2 Effect of GSE on the proliferation and migration of human dermal papilla cells (hDPCs) (a) Proliferation of hDPCs was analyzed by the Cell Counting Kit-8 assay, and values are shown as the mean with SEM. b Migration effect of GSE was measured by the scratch migration assay, and the alteration of the scratch line width was converted into a percentage. NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil Fig. 2 Effect of GSE on the proliferation and migration of human dermal papilla cells (hDPCs) (a) Proliferation of hDPCs was analyzed by the Cell Counting Kit-8 assay, and values are shown as the mean with SEM. b Migration effect of GSE was measured by the scratch migration assay, and the alteration of the scratch line width was converted into a percentage. NC: negative control of hDPCs treated with Dulbecco’s modified Eagle’s medium; PC: positive control of hDPCs treated with 10 μM minoxidil Fig. 3 Changes in the levels of Ki67 protein, hepatocyte growth factor (HGF) l d h li l h f (VEGF) d f i Fig. 3 Changes in the levels of Ki67 protein, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta 1 (TGF-β1) in human dermal papilla cells (hDPCs) induced by GSE. a Immunohistochemistry of Ki67. b Numbers of Ki67- positive cells. c Relative expression levels of HGF, VEGF, and TGF-β1 in GSE-treated hDPCs. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. Discussion NC: negative control of C57BL/6 mice skin treated with dimethyl sulfoxide; PC: positive control of C57BL/6 mice skin treated with 5% minoxidil; GSE: C57BL/6 mice skin treated with 1,000 ppm GSE Fig. 4 Hair growth-stimulating effect of GSE in an in vivo model. a Observation of C57BL/6 mice hair growth for 3 weeks. b Histological analysis of hair follicles, mast cells, and stem cell factor expression on the back skin of C57BL/6 mice. c The numbers of mast cells were counted by toluidine blue staining. d Expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1) in the back skin of C57BL/6 mice. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. NC: negative control of C57BL/6 mice skin treated with dimethyl sulfoxide; PC: positive control of C57BL/6 mice skin treated with 5% minoxidil; GSE: C57BL/6 mice skin treated with 1,000 ppm GSE NE/g extract, respectively. Their values were signifi- cantly higher than those of Lespedeza cuneata G. Don (228.9 mg TAE/g extract, 90.2 mg NE/g extract) which was selected by screening the highest antioxidant extract from many plant extracts [29]. Moreover, the electron- donating ability of GSE (76.6%) was not only higher than the synthetic antioxidants butylated hydroxyanisole (69.2%) and butylated hydroxytoluene (45.6%) but also comparable to α-tocopherol (75.1%) and Lespedeza cuneata G. Don (75.7%) [29, 30]. Naito et al. [31] has re- ported that lipid peroxides can induce apoptosis of hair follicle cells and lead to the early onset of catagen phase in murine since it can produce reactive oxygen species (ROS) causing apoptosis of hair matrix and epithelial cells by stimulating the expression of p53. Plant extracts with high DPPH radical scavenging activity may prevent hair loss presumably by protecting hair follicle cell from hydroperoxides. suggested that GSE treatment activates cell proliferation of hDPCs by maintaining and stimulating the active phases of cell growth, since the Ki67 marker is expressed only in the active phases of cell growth, such as the G1, S, and G2 phases and mitosis [35]. Some cytokines are essential to development, growth and cycling of hair follicles. Discussion Several plants have been widely used for treating or preventing diseases various countries. For instance, GSE has been used in folk remedies to treat diarrhea, bacterial infection, and anti-gastric ulcer agent [2]. Re- cently, scientific studies have shown that GSE holds pharmacological activity including anti-inflammatory, anti-bacterial function, anti-diarrhea effect and anti- gastric ulcer action as well as high ability of DPPH radical scavenging [11, 12, 14, 15]. However, the ef- fects of extracts from this plant on hair growth have The total polyphenol and flavonoid contents of GSE in this study were 305.9 mg TAE/g extract and 104 mg Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 7 of 9 Fig. 4 Hair growth-stimulating effect of GSE in an in vivo model. a Observation of C57BL/6 mice hair growth for 3 weeks. b Histological analysis of hair follicles, mast cells, and stem cell factor expression on the back skin of C57BL/6 mice. c The numbers of mast cells were counted by toluidine blue staining. d Expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1) in the back skin of C57BL/6 mice. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. NC: negative control of C57BL/6 mice skin treated with dimethyl sulfoxide; PC: positive control of C57BL/6 mice skin treated with 5% minoxidil; GSE: C57BL/6 mice skin treated with 1,000 ppm GSE Fig. 4 Hair growth-stimulating effect of GSE in an in vivo model. a Observation of C57BL/6 mice hair growth for 3 weeks. b Histological analysis of hair follicles, mast cells, and stem cell factor expression on the back skin of C57BL/6 mice. c The numbers of mast cells were counted by toluidine blue staining. d Expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1) in the back skin of C57BL/6 mice. The fold changes were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase; the values are expressed as the mean ± SEM. Values sharing the same superscript letters differ significantly at p < 0.05 by Duncan’s multiple range test. Discussion The results revealed that HGF and VEGF mRNA expression level of hDPCs following treatment with GSE was signifi- cantly upregulated compared with NC or PC (p < 0.05). Although GSE treatment did not reduce TGF- β1 mRNA expression significantly compared with the NC group, the PC group showed significantly higher mRNA expression of TGF-β1 than the GSE group (1.21 vs. 0.87). Since HGF and VEGF are known growth modulators of the follicular papilla in the anagen stage [34], it stands to reason that the induc- tion of these growth factors by GSE treatment would promote the anagen stage or active hair growth. The downregulation of TGF-β1 may contribute partially to the prevention of hair loss, because this is involved in the catagen stage of hair development or the apop- tosis of hair follicle cells [33]. Considering these find- ings in the in vitro model, GSE treatment may promote the anagen stage of hair growth by modulat- ing growth regulators. Cell proliferation and migration are the essential fac- tors for hair growth [32–34]. 19.5 ppm of GSE showed cell proliferation and migration of hDPCs significantly higher than those of NC and PC (Fig. 2). Since GSE in- duced the proliferation and migration of cultured hDPCs possibly, GSE could enhance hair growth positively. However, GSE more than 156.3 ppm caused toxic effect on hDPCs. The induction of Ki-67 in vitro model Page 8 of 9 Page 8 of 9 Boisvert et al. BMC Complementary and Alternative Medicine (2017) 17:109 Page 8 of 9 We had also conducted in vivo study to determine hair growth effect of GSE in induced telogenic C57BL/6 mice. From the histological analysis, the depth and de- velopment of dermal papillar and the long hair shaft of hair follicles in the GSE group indicated active hair growth rather than in the NC and PC groups. As mast cells play a key role in stress reaction and inflammation, the degranulation of inflammatory mediators by mast cells triggers the catagen stage of hair follicles, intrafolli- cular apoptosis, and hair loss [2, 36]. In this study, mast cells are observed in dermal connective tissues rather than near hair follicles. Considering a few number of mast cells in GSE group, GSE treatment contribute anti- inflammatory action on dermal tissues rather than on hair follicular epithelium. channel pore coupled with sulfonylurea receptor on the mitochondrial inner membrane. Funding h This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (2011–0013949) and by the Chung-Ang University Research Grants in 2015. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Discussion The downregulation of TGF-β1 in both in vitro and in vivo models indicated that GSE may have the same mechanism with minoxidil in hair growth activity. Although corilagin and gallic acid were identified as active components in GSE, there was a limitation that hair growth effect of each chemical was not measured. Therefore, their hair growth effect and mech- anism should be investigated in further study. All data of this study is included in this article. All data of this study is included in this article. Conclusions This study confirmed that GSE treatment significantly promoted hair growth, both in vitro and in vivo, by the proliferation and enhanced migration of hDPCs. GSE treatment also modulated the expression of HGF, VEGF, and TGF-β1, which are involved in the growth and in- hibition of hair follicular cells. The reduced number of mast cells and the induction of SCF also contributed to hair regeneration events. Therefore, we concluded that GSE could enhance hair growth and prevent hair loss. Interestingly, this study confirmed that GSE induced SCF in vivo model by immunohistochemistry. SCF was strongly expressed in mouse epidermal keratinocytes and epithelium of hair follicular bulbs. Since the SCF plays a role in preserving stem cells and participating in the growth of hair follicles, it is detectable in the anagen stage [37]. The induction of endothelin-1 and SCF was correlated with the regeneration of hair follicles [38]. In concurrence with the previous studies, the strong induc- tion of SCF in the GSE-treated mice skin suggested that hDPCs underwent the regeneration process of hair follicles. Acknowledgement h h This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (2011–0013949) and by the Chung-Ang University Research Grants in 2015. Abbreviations ACN: Acetonitrile; CCK-8: Cell Counting Kit-8; DEX: Dexamethasone; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: Dimethylsulfoxide; EDA: Electron donating ability; GAPDH: Glyceraldehyde 3-phosphate dehydrogen- ase; GSE: Geranium sibiricum L. extract; hDPCs: Human dermal papilla cells; HGF: Hepatocyte growth factor; HPLC: High pressure liquid chromatography; MeOH: Methanol; MXD: Minoxidil; NC: Negative control, NE, naringin equivalent; PC: Positive control; qPCR: Quantitative real-time polymerase chain reaction; SCF: Stem cell factor; TAE: Tannic acid equivalent; TGF-β1: Transforming growth factor-beta 1; VEGF: Vascular endothelial growth factor Interestingly, the treatment of 1,000 ppm GSE signifi- cantly downregulated mRNA expression of VEGF, HGF, and TGF-β1 in the mouse skin (Fig. 4d). While GSE upregulated expression of HGF and VEGF on hDPCs, the expression of HGF and VEGF was downregulated in mouse skin tissues. Since induction of VEGF and HGF were required in active anagen phase, they are expressed in actively growing hair follicles. As 24 h of GSE treatment induced anagen phase of hDPCs, ex- pression of VEGF and HGF was upregulated in GSE- treated hDPCs. However, hair follicles in mouse skin tissues after 3 weeks of depilation were in catagen phase rather than anagen when it is considered that GSE-treated group finished the hair growth faster than PC or NC. Therefore, catagen phase could explain the reduced expression of VEGF and HGF in GSE-treated mouse hair follicles for 3 weeks. Authors’ contributions BHL was responsible for this research project, experimental design, and preparation of this manuscript. WAB, MY, YC, GHJ, Y-LZ, and CC were in charge of animal experiment, histopathology, data production, and draft preparation. CS and LS were in charge of literature study and figure produc- tion from HPLC analysis. All authors read and approved the final manuscript. It was surprising finding that TGF-β1 was downregu- lated in both models. As the function of TGF-β1 is associated with hair loss, upregulation of TGF-β1 in hair growth inhibits the growth of hair follicular cells by accelerating the transition of catagen and telogen. Thus, downregulation of TGF-β1 could prevent hair loss in GSE-treated skin by maintaining catagen phase of hair follicles and viability of follicular cells. Otomo [39] reported that minoxidil inhibits of TGF-β induced apoptosis of hair matrix cells by opening the Kir 6.0 Competing interests Consent for publication All authors consent to publish this study in BMC Complementary and Alternative Medicine. All authors consent to publish this study in BMC Complementary and Alternative Medicine. References 27. Jia L, Jin H, Zhou J, Chen L, Lu Y, Ming Y, et al. A potential anti-tumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through blocking the TGF-β signaling pathways. 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Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • 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 y p and we will help you at every step: 19. Boyera N, Galey I, Bernard BA. Biphasic effects of minoxidil on the proliferation and differentiation of normal human keratinocytes. Skin Pharmacol Physiol. 1997;10:206–20. • We accept pre-submission inquiries 20. Evans CP, Elfman F, Cunha G, Shuman MA. Decreased prostate cancer cell migration by inhibition of the insulin-like growth factor II/Mannose-6- Phosphate receptor. Urol Oncol. 1997;3:166–70. 21. 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COPYRIGHT COPYRIGHT © 2023 Dong, Xu, Zhang, Shi, Du, Jia, Wang and Wang. 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. TYPE Correction PUBLISHED 07 February 2023 DOI 10.3389/fped.2023.1142196 KEYWORDS KEYWORDS EEG analysis, infantile spasm, functional brain networks, local network, global network APPROVED BY Frontiers in Pediatrics, Frontiers Media SA, Switzerland *CORRESPONDENCE Yan Dong yjs6690@126.com SPECIALTY SECTION This article was submitted to Pediatric Neurology, a section of the journal Frontiers in Pediatrics RECEIVED 11 January 2023 ACCEPTED 18 January 2023 PUBLISHED 07 February 2023 CITATION Dong Y, Xu R, Zhang Y, Shi Y, Du K, Jia T, Wang J and Wang F (2023) Corrigendum: Different frequency bands in various regions of the brain play different roles in the onset and wake-sleep stages of infantile spasms. Front. Pediatr. 11:1142196. doi: 10.3389/fped.2023.1142196 APPROVED BY Frontiers in Pediatrics, Frontiers Media SA, Switzerland *CORRESPONDENCE Yan Dong yjs6690@126.com SPECIALTY SECTION This article was submitted to Pediatric Neurology, a section of the journal Frontiers in Pediatrics RECEIVED 11 January 2023 ACCEPTED 18 January 2023 PUBLISHED 07 February 2023 CITATION Dong Y, Xu R, Zhang Y, Shi Y, Du K, Jia T, Wang J and Wang F (2023) Corrigendum: Different frequency bands in various regions of the brain play different roles in the onset and wake-sleep stages of infantile spasms. Front. Pediatr. 11:1142196. doi: 10.3389/fped.2023.1142196 Yan Dong 1*, Ruijuan Xu 1, Yaodong Zhang 2, Yali Shi 1, Kaixian Du 1, Tianming Jia 1, Jun Wang 3 and Fang Wang 4 Yan Dong 1*, Ruijuan Xu 1, Yaodong Zhang 2, Yali Shi 1, Kaixian Du 1, Tianming Jia 1, Jun Wang 3 and Fang Wang 4 1Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou, China, 2 Henan Key Laboratory of Children’s Genetics and Metabolic Diseases, Henan Neurodevelopment Engineering Research Center for Children, Children’s Hospital Affiliated to Zhengzhou University, Zhengzhou, China, 3Department of Children’s Rehabilitation, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China, 4Department of Medical Record Management, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China PUBLISHED 07 February 2023 Dong Y, Xu R, Zhang Y, Shi Y, Du K, Jia T, Wang J and Wang F (2023) Corrigendum: Different frequency bands in various regions of the brain play different roles in the onset and wake-sleep stages of infantile spasms. Front. Pediatr. 11:1142196. doi: 10 3389/fped 2023 1142196 Attribution License (CC BY). The use, Dong Y, Xu R, Zhang Y, Shi Y, Du K, Jia T, et al. (2022) Front. Pediatr. 10:878099. doi: 10.3389/fped. 2022.878099 In the published article, there was an error in institution namesof affiliations 1, 3 and 4. The original affiliations “1Henan Provincial Key Laboratory of Child Brain Injury, Department of Pediatrics, Third Associated Hospital of ZhengZhou University, Zhengzhou, China”, “3Department of Children’s Rehabilitation, Third Associated Hospital of ZhengZhou University, Zhengzhou, China” and “4Department of Medical Record Management, Third Associated Hospital of ZhengZhou University, Zhengzhou, China”, have been changed to “1Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, the Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou, China”, “3Department of Children’s Rehabilitation, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China” and “4Department of Medical Record Management, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China”. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. Frontiers in Pediatrics 01 frontiersin.org
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Synthesis of Schiff’s Bases from 2-Amino-4H-1, 3-Oxazine / Thiazine and Substituted Aldehydes and their Transition Metal Complexes
International Journal of Current Science Research and Review
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ABSTRACT Objective: Many methods can be found in literature for the synthesis a Oxazines and Thiazines. Few are reported for one-pot multi component cyclo-condensation of an alkayane urea / Thiourea and Aldehyde to 2- amino-4H-1, 3-Oxazines or Thiazines. Different catalysts are reported like Trifluoro-acetic acid, glacial acetic acid under reflux condition Mandal and Co-workers have utilized perchloric acid (HClO4) supported on silica gel as catalyst under solvent free condition ytterbium trifluorate [Yb(OTF)3] plays role of Lewis acid. It is also used in several Diel’s-Alder cyclo-addition reactions. Inspired these observations, development of simple effective synthetic strategy for the synthesis of 2-amino-4H-1, 3- Oxazium & Thiazines was attempted. Material and Methods: One pot multi-component synthesis of Schiff’s base as new ligand an their complexes with Ni, Fe, Co and Cu and developed using 2-amino-4H-1,3 Oxazines /Thiazines and substituted Aldehydes, different catalyst are used to synthesize of 2-amino 4H-1,3 Oxazines. Its characterization is confirmed by taking IR, NMR spectra. The Schiff’s base act as ligand is bidentate, when it is treated to metal complexes in 1:1 ratio (metal: ligand) complexes are formed. Result: By using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by using different aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. And these Schiff’s bases treated with bioactive metals like Co, Fe, Cu, Zn which enhances their bio-activity. y Conclusion: All these synthesized Schiff’ Bases and their metal complexes having bioactive properties. KEYWORDS: Coordination complexes and Characterization, Oxazines and substituted aldehydes, Synthesis of Schiff’s bases, 2- amine-4H-1, 3. Synthesis of Schiff’s Bases from 2-Amino-4H-1, 3-Oxazine / Thiazine and Substituted Aldehydes and their Transition Metal Complexes D.T. Biradar1, Dr. M. B. Swami2 1Department of Chemistry, Research Centre, Shivaji Mahavidyalaya, Udgir, Dist.Latur 2Dept. of Chemistry, Bahirji Smarak Mahavidyalaya, Basmath, dist. Hingoli-431512[M.S] www.ijcsrr.org www.ijcsrr.org Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 677 *Corresponding Author: Dr. M. B. Swami International Journal of Current Science Research and Review Preparation of 2-Amino-4H-1, 3-Oxazine / Thiazine: Preparation of 2-Amino-4H-1, 3-Oxazine / Thiazine: reparation of 2-Amino-4H-1, 3-Oxazine / Thiazine A solution of benzaldehyde (1 m mole), (2- bromovinyl) 1 m mole and Urea 3 m mole in water (3 m mole) was taken in 20 ml vial and subject to M.W. irradiated at (1200-2500C) reaction completed monitored by TLC, reaction mixture was cooled, filter wash with acetone and re-crystallize by ethanol to give pure product. Table. No.1 Table. No.1 Sr. NO. R1 R2 X Yield in % 1 H C O 89 2 P-CH3 C6H5 O 72 3 O-OCH3 C6H5 O 84 4 M-NO2 C6H5 O 85 5 H C6H5 S 76 6 P-CH3 C6H5 S 72 7 P-Cl C6H5 S 77 8 P-NO2 C6H5 S 66 Preparation of Schiff’s bases : Take m mol of 2- amino 4H-1, 3 Oxazine or Thiazine compound in mortar and add benzaldehyd add 2-3 drops of ethanol Preparation of Schiff’s bases : Preparation of Schiff’s bases : INTRODUCTION In chemical research and industry has increased efficient, economical clean short time procedure increases attention in recent years1.. Development of such methods is great demand in coordination chemistry. In chemical research and industry has increased efficient, economical clean short time procedure increases a Development of such methods is great demand in coordination chemistry. Development of such methods is great demand in coordination chemistry. Compounds containing an Oxazine or Thaizine moiety have been found to passes a wide range of bioactivities like antifungal antibiotic2-3. A derivative of oxazine has also exhibited anti- HIV activity Compounds containing an Oxazine or Thaizine moiety have been found to passes a wide range of bioactivities like antifungal antibiotic2-3. A derivative of oxazine has also exhibited anti- HIV activity Many methods can be found in literature for the synthesis a oxazines and thiazines. Few are reported for one-pot multicomponent cyclocondensation of an alkayane urea/thiourea and aldehyde to 2- amino-4H-1, 3-oxazine or thiazine3. Different catalysts are reported like trifluoraoacetic acid glacial acetic acid under reflux condition Mandal and co-workers have utilized perchloric acid (HClO4) supported on silica gel as catalyst under solvent free condition ytterbium trifluorate [yb(OTF)3] plays role of Lewis acid It is also used in several Diel’s-Alder cyclo-addition reactions4. Inspired these observations, development of simple effective synthetic strategy for the synthesis of 2-amino-4H-1, 3- Oxazine & Thiazine was attempted5-7. Schiff’s bases also have applications of ant-inflammatory, antitumor, antibiotic, antifungal activities. We want to enhance the bioactivity by containing these two moiety using different methods are used8-9. To synthesize the Schiff’s bases. Different methods are reported like constant stirring at moderate temperature use of catalysts like H2SO4, AlCl3 etc. p If such bases are treated with biologically active metals like Zn, CO, Cu, Fe it enhance the biological ac p If such bases are treated with biologically active metals like Zn, CO, Cu, Fe it enhance the biological activity of compounds. If such bases are treated with biologically active metals like Zn, CO, Cu, Fe it enhance the biological activity of compounds. 677 *Corresponding Author: Dr. M. B. INTRODUCTION Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org Synthesis of Metal complexes: Synthesis of Metal complexes: Solution of ligand in methanol & metal salt were mixed thoroughly in 1:1 and 2:1 metal legend ratio and 1% mixed KOH solution in methanol was added to adjust PH between 7-8 and reflux for 1-2 hr. We get complex which monitored on TLC. y p Solution of ligand in methanol & metal salt were mixed thoroughly in 1:1 and 2:1 metal legend ratio and 1% mixed KOH solution in methanol was added to adjust PH between 7-8 and reflux for 1-2 hr. We get complex which monitored on TLC. M (CH3COO)2 (H2O)4 +1 or 2 ln or M ln2 M= Cu, Fe, Co, Zn j g p CH3COO)2 (H2O)4 +1 or 2 ln or M ln2 Cu, Fe, Co, Zn ct spectroscopic data Band at 525-425,450-350 NMR- H Ar. at7.60 SULT AND DISCUSSION using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by usin stituted aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. these Schiff’s bases treated with bioactive metals like Co, Fe, Cu, and Zn which enhances their bio-activity10-11. t spectroscopic data Band at 525-425,450-350 NMR- H Ar. at7.60 Select spectroscopic data Band at 525-425,450-350 NMR- H Ar. at7.60 Select spectroscopic data Band at 525-425,450-350 NMR- H Ar. at7.60 Preparation of Schiff’s bases : Preparation of Schiff s bases : Take m mol of 2- amino 4H-1, 3 Oxazine or Thiazine compound in mortar and add benzaldehyd add 2-3 drops of ethanol grind for 10-15 min or it stir for 1-2 Hrs. We get the products. Pour this product on crushed ice, filter and re-crystallize it using ethanol. Strong singlet at 9.41 in NMR and 3400-3500 and in IR does no observed indicates NH2 convert in R2C = NR (imines) Take m mol of 2- amino 4H-1, 3 Oxazine or Thiazine compound in mortar and add benzaldehyd add 2-3 drops of ethanol grind for 10-15 min or it stir for 1-2 Hrs. We get the products. Pour this product on crushed ice, filter and re-crystallize it using ethanol. Strong singlet at 9.41 in NMR and 3400-3500 and in IR does no observed indicates NH2 convert in R2C = NR (imines) Band at 3650-3584 due to free -OH band at 1689-1471 due to imines stretching 678 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 678 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org Synthesis of Metal complexes: Solution of ligand in methanol & metal salt were mixed thoroughly in 1:1 and 2:1 metal legend ratio and 1% mixed KOH solution in methanol was added to adjust PH between 7-8 and reflux for 1-2 hr. We get complex which monitored on TLC. M (CH COO) (H2O) +1 or 2 ln or M ln International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org 679 *Corresponding Author: Dr. M. B. Swami RESULT AND DISCUSSION SU N SCUSS ON By using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by using different substituted aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. By using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by using different substituted aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. By using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by using different substituted aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. By using the bioactive molecules like 2-amino-4H-1, 3 Oxazine / Thiazines, Schiff’s Bases can be synthesized by using different substituted aldehydes. These Schiff’s Bases has antitumor, antipyretic, anti-inflammatory property. And these Schiff’s bases treated with bioactive metals like Co, Fe, Cu, and Zn which enhances their bio-activity10-11. ses treated with bioactive metals like Co, Fe, Cu, and Zn which enhances their bio-activity10-11. And these Schiff’s bases treated with bioactive metals like Co, Fe, Cu, and Zn which enhances their bio-a Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 679 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 679 *Corresponding Author: Dr. M. B. Swami International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org International Journal of Current Science Research and Review ISSN: 2581-8341 rnational Journal of Current Science Research and Review www.ijcsrr.org ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org 680 *Corresponding Author: Dr. M. B. RESULT AND DISCUSSION Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 Spectral data of 2-Amino-4H-1, 3-oxazine / thiazine: Sr.No Name of Compound Nature M.P Result in % H NMR IR 1 4,6-Dipheny1-4H-1,3- Oxzine-2-amine pale yellow crystals 219- 2210c 89% 9.45 (S,2H.NH2), 7.76 (m, J=2Hz.4Hz,2H,Ar), 7.46 (M,3H,HAr), 7.52 (m, J = 2Hz, 4Hz, 8H, HAr) 6.62 ( d J = 4Hz, 1H, CH) , 5.58 ( d J= 4Hz, 1H, CH) 3400-3500, 3078, 1710, 1664, 1371, 1330, 1263, 1096 2 6-pheny-4 (P-toly) -4H- 1-3-oxazine-2-amine pale yellow crystal 176- 1780c 84% 9.43 (S,2H,NH2), 7.72 (m J =2 Hz, 4Hz ,2H,HAr), 7.46 (m,3H, HAr), 7.28 (d J= 4Hz, 2H, Ar) 7.16 (d,J=4Hz, 2H, HAr), 6.60 (d, J=4Hz, 1H CH) 5.56 (d, J=4Hz, 1H; CH), 2.43 (S, 3H,CH3) 3400-3500, 3078, 3100, 1710, 1664, 1371, 1330, 1263, 1096 3 4-(O-Methoxyphenyl-6 -phyenyl - 4H-1, 3- Oxazine – 2-amine) Pale yellow crystal 186- 1880c 72% 9.42 (Cs 2H, NH2), 7.72 (m,J= 2Hz, 4Hz, 2H, HAr) 7.43 (m, J = 2Hz 4Hz, 5H, HAr), 7.22 (d, J = 4Hz) 7.43 (m,J= 2Hz 4Hz, 5H, HAr), 7.22 (d,J=4Hz, 1H, HAr), 7.09 (d J= 4Hz, 1H HAr), 6.61 (d,J= 4Hz.1H CH), 5.54 (d,J=4Hz, 1H CH) 3.87 (S,3H, OCH3) 3400-3500, 3078, 3120, 1710, 1650, 1471, 1330, 1247, 1040 4 (m-Nitrophenyl) 6- Pheuyl- 4H-13oxazine- 2-amine Pale yellow crystal 168- 1700c 86% 9.41 (s, 2H NH2), 8.21 (m, J= 2Hz, 4Hz, 2H, HAr) 7.99 (m,J =2Hz, 4Hz, 2H, HAr) 7.72 (m.2H, HAr), 6.42 (d,J = 4Hz 1H, CH) 5.55 (d, J= 4H2, 1H, CH) 3400-3500, 3078, 1710, 1664, 1371, 1330, 1263, 1096 5 4,6-diphenyl-4H-1, 3- Thiazine-2-amine Pale yellow crystals 178- 1800c 76% 9.88 (S 2H, NH2), 7.42 (M,7H, HAR) 7.38 (M,2H,HAr) , 7.18 (d J=4Hz, 1H, HAr) 6.62 (d,J=2Hz 1H CH) 6.22 ( d, J= 2Hz 1 H CH) 3400-3500, 3080, 1523, 1710, 1660, 1371 1330, 1263, 1096 680 *Corresponding Author: Dr. M. B. RESULT AND DISCUSSION Swami International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org 6 6 (phenyl-4(P-Tolyl)- 4H-1, 3 thiazin- 2- amine Pale yellow crystal 180- 1820c 72% 9.86 (S, 2H, NH2) 7.41 (d, J= 8Hz, 1H, HAR) 7.34 (d,J= 8Hz, 1H, HAR) , 7.22 (m,7H HAr), 6.71 (d, J= 2Hz, 1H, CH), 6.24 (d,J=2Hz, 1H, CH) 2.42 (S,3H, CH3) 3400-3500, 3078, 3100, 1710, 1664, 1371, 1330, 1263, 1096 7 4 (P-Chlorophenyl) -6 phenyl-4 H-1, 3- thiazine-2amine Pale yellow crystals 170- 1720c 77% 9.88 (S,2H, NH2) 7.42 (m, 4Hz, HAr), 7.32 (m, 2H, HAr) , 6.26 (M, 3H, HAr), 6.72 (d, J = 2Hz 1H CH), 6.40 (d,I= 2Hz, 1H, CH) 3400-3500, 3078, 1720, 1660, 1371, 1330, 1263, 1096 Spectral data of Schiff’s bases Sr. No Name of Compound H NMR IR 1 4,6 – Dipheny-4H-1, 3 Oxazine-2, imine (2- hydroxyl) benzene 7.76 (m, J = 2Hz, 4Hz, 2H, Ar) , 7.52 (m, J= 2Hz, 4Hz, 8H, HAr) 6.62 ( t, J = 4Hz, 1 H CH) 5.58 (d, J= 4Hz, 1H CH), 5.48 (d J = 2Hz 1 H CH), 5.20 ( S 1H, OH) 3650-3584, 3080, 1710, 1664, 1470, 1263, 1096 2 6- Phenyl -4 ( P-tolyl)- 4H-1-3 Oxazine -2 imine (2-hydroxy) benzene 7.72 (m, J = 2Hz 4Hz, 2H, Ar) 7.46 (m, 3H Har), 7.28 ( d J = 4Hz 2H, HAr) 7.16 (d,J = 4Hz, 2H, HAr), 6.62 (d J = 4Hz, 1H CH) , 5.56 (t ,J=4Hz 1H, CH) 5.48 (d , 2Hz, 1H, CH) 5.20 (S, 1H, OH), 2.43 (S,3H, CH3) 3650-3584, 3080, 1710, 1689, 1650, 1470, 1263, 1096 3 4- (O- methoxy phenyl -6 phenyl – 4H,1 3-Oxazine 2- Imine (2-hydroxy) benzene 7.72 (m,J-2Hz, 4Hz, 2H HAr), 7.43 (m,J=2Hz 4Hz, 5H HAr), 7.22 (d, J= 4Hz, 1H, HAr), 7.09 (d J=4Hz, 1H, HAr) 6.61 (d J = 4Hz, 1H CH) 5.54 (d J= 4Hz 1 H CH) 5.48 ( d 2H 1H CH) 5.20 (S, 1H, OH), 3 87 (S 3H OCH3) 3650-3584, 3080, 3120, 1710, 1630, 1471, 1330, 1247, 1040 International Journal of Current Science Research and Review ISSN: 2581-8341 rnational Journal of Current Science Research and Review www.ijcsrr.org Spectral data of Schiff’s bases 681 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 681 *Corresponding Author: Dr. M. B. 681 *Corresponding Author: Dr. M. B. Swami RESULT AND DISCUSSION Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 International Journal of Current Science Research and Review ISSN: 2581-8341 4 (M-Nitrophyenyl) 1- 6 Phyenyl-4H-1, 3- Pxazome-2 imine (2-hydroxy)benzene 8.21 (m, J = 2Hz, 4Hz, 2H, HAr), 7.99 ( m, J = 2z, 4z, 2H, HAr), 7.72 (M, 2H, HAr) , 6.42 (D, J=4z, 1H, CH), 5.55 (d , J – 4Hz, 1H, CH) 5.48 ( d, J = 2Hz, 1H CH) , 5.20 (S, 1H, OH) 3650-3584, 3080, 1710, 1630, 1471, 1330, 1247, 1040 5 4, 6 diphenyl-4H-1, 3- thiazine-2 imine (2-Hydroxy) benzene 7.42 (m,7H, HAr), 7.38 (m, 2H, HAr), 7.18 (d, J = 4Hz, 1H, HAr), 6.62 (d, J = 2Hz, 1H, CH) , 6.22 (d, J=2z, 1H, CH), 5.58 (d, J = 2Hz, 1H, CH) 5.20 (S,, 1H, OH) 3650-3584, 3080, 1710, 1660, 1371, 1330, 1263, 1096 6 6-Phenyal-4 (P-tolyl)-4H-1, 3 –Thizine-2-imine (2- hydroxy)benzene 7.41 (d, J = 8Hz, 1H, HAr), 7.34 (d, J=8Hz, 1H HAr) 7.22 (m, 7H,HAr) 6.71 (d , J = 2Hz, CH) , 6.24 ( d, J= 2Hz, 1H, CH), 5.58 (d J = 2Hz, 1H,CH) 5.20 (S, 1H, OH) 2.42 (S, 3H, CH3) 3650-3584, 3078, 3100, 1710, 1664, 1371, 1330, 1263, 1096 7 4 (P.Chlorophenyl) -6 phenyl- 4 H-1, 3-thiazine- 2-imine (2- hydroxy) benzene 7.42 (m, 4H, HAr) 7.32 (m, 2H, HAr), 7.26 (m, 3H, HAr) , 6.72 (d,J= 2Hz 1H CH), 6.60 (d, J = 2H2 1H CH), 5.50 (d,I= 2Hz, 1H, CH) 5.20 (S,J 1H OH) 3650-3584, 3078, 1710, 1660, 1371, 1330, 1263, 1096 8 4 (P-Nitrophyanyl ) -6 phenyl- 4 H-1, 3-thiazine- 2-imine (2- hydroxy)benzene 8.32 (m, 2H, HAr) 7.82 (m, 2H, HAr) 7.45 (m, 5H, HAr) 6.67 (d,J= 2Hz, 1H CH) 6.26 (d,J=2H2, 1H CH), 5.58 (d,J=2H2 1H CH), 5.20(S, 1H CH) 3650-3584, 3078, 1710, 1660, 1523, 1371, 1330, 1263, 1096 CONCLUSION CONCLUSION 682 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 CONCLUSION All the synthesized Schiff’ Bases and their metal complexes having bioactive properties like anti-inflammatory, antitumor, antibiotic, antifungal activities. REFERENCES 1. Mathew, B.P. Kumar, A.Sharma, S. Shukla, P.K. Nath, M.Eur, J. Med. Chem. 45, 1502 (2010) 2. Haneisi J. : Okazaki, T. Hata , T, Jamura L, Nom. Arura M.J. Antibiot,24, 797 (1971) 3. Sasaki K. Kusalabe, Y. Esumi, S.J. Aautibiot 25,151(1972) 4. Pictsh M : Custchow M.J. Med. Chem. 48,8270 (2005) All the synthesized Schiff’ Bases and their metal complexes having bioactive properties like anti-inflammatory, antitumor, antibiotic, antifungal activities. 682 *Corresponding Author: Dr. M. B. Swami 5. Hung S. : Pan Y. : Zhu Y : Wu, A. Org, Lett. 7,3797 (2005) 6. Cecchetti, V : Cruciani, G : Filliponi , E : Fravolini A; Tabarrini O, T.Bioorg. Med.Chem. 5, 1339(1997) 7. Remillard, S. Rebhu, L.I Howie, G.A. Kupchan, S.M .Science 189 ,1002 (1975) 8. Sokolova, A,S ; Rayabokon’, N.A; Erashova, Yu.A; Andreeva, N.A. ; Nemeryuk, M.P; Kermov, A.F. Traven, N.I; Yadrovskaya, V.A; Chernov, V.A ; Safonova, T.S.Pharm. Chem.J 11 (9) ,49 (1977) 9. Pietsch, M, Gutschow M, M.J.Med.Chem, 48,8270 (2005) 10. Diwagh, S.S; Piste, P.B. Int. J. Pharm. Sci, Res. 4,2045 (2013) 11. Mandal, P.K.Misra, A.K. Lett. Org. Chem. 3, 848 (2006) Cite this Article: D.T. Biradar, Dr. M. B. Swami (2021). Synthesis of Schiff’s Bases from 2-Amino-4H-1, 3-Oxazine /Thiazine and Substituted Aldehydes and their Transition Metal Complexes. International Journal of Current Science Research and Review, 4(7), 677-683 683 *Corresponding Author: Dr. M. B. Swami REFERENCES Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 International Journal of Current Science Research and Review ISSN: 2581-8341 Volume 04 Issue 07 July 2021 DOI: 10.47191/ijcsrr/V4-i7-09, Impact Factor: 5.825 IJCSRR @ 2021 www.ijcsrr.org International Journal of Current Science Research and Review 683 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683 Cite this Article: D.T. Biradar, Dr. M. B. Swami (2021). Synthesis of Schiff’s Bases from 2-Amino-4H-1, 3-Oxazine /Thiazine and Substituted Aldehydes and their Transition Metal Complexes. International Journal of Current Science Research and Review, 4(7), 677-683 Cite this Article: D.T. Biradar, Dr. M. B. Swami (2021). Synthesis of Schiff’s Bases from 2-Amino-4H-1, 3-Oxazine /Thiazine and Substituted Aldehydes and their Transition Metal Complexes. International Journal of Current Science Research and Review, 4(7), 677-683 683 *Corresponding Author: Dr. M. B. Swami Volume 04 Issue 07 July 2021 Available at: ijcsrr.org Page No.-677-683
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Investigation of Plant Surfaces with Environmental Scanning Electron Microscopy (ESEM®) - A Comparison with Conventional SEM
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Associations between Neurocardiovascular Signal Entropy and Physical Frailty
Entropy
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Associations between neurocardiovascular signal entropy and physical frailty Knight, S. P., Newman, L., O’Connor, J. D., Davis, J., Kenny, R. A., & Romero-Ortuno, R. (2020). Associations between neurocardiovascular signal entropy and physical frailty. Entropy, 23(1), Article 4. https://doi.org/10.3390/e23010004 Queen's University Belfast - Research Portal: Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University Publisher rights © 2020 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Publisher rights © 2020 The Authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. 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Published in: Entropy Document Version: Publisher's PDF, also known as Version of record Article Associations between Neurocardiovascular Signal Entropy and Physical Frailty Silvin P. Knight 1,2,* , Louise Newman 1,2 , John D. O’Connor 1,2,3 , James Davis 1,2 , Rose Anne Kenny 1,2,4 and Roman Romero-Ortuno 1,2,4,5 * , Louise Newman 1,2 , John D. O’Connor 1,2,3 , James Davis 1,2 , Rose Anne Kenny 1,2,4 o-Ortuno 1,2,4,5 1 The Irish Longitudinal Study on Ageing (TILDA), School of Medicine, Trinity College Dublin, D02 R590 Dublin, Ireland; louise.newman@tcd.ie (L.N.); john.oconnor@qub.ac.uk (J.D.O.); davisj5@tcd.ie (J.D.); rkenny@tcd.ie (R.A.K.); romeroor@tcd.ie (R.R.-O.) 1 The Irish Longitudinal Study on Ageing (TILDA), School of Medicine, Trinity College Dublin, D02 R590 Dublin, Ireland; louise.newman@tcd.ie (L.N.); john.oconnor@qub.ac.uk (J.D.O.); davisj5@tcd.ie (J.D.); rkenny@tcd.ie (R.A.K.); romeroor@tcd.ie (R.R.-O.) j y 2 Discipline of Medical Gerontology, School of Medicine, Trinity College Dublin, D02 R590 Dublin, Ireland 3 School of Medicine, Dentistry and Biomedical Sciences, The Patrick G Johnston Centre for Cancer Research, Queen’s University, Belfast BT9 7BL, UK 2 Discipline of Medical Gerontology, School of Medicine, Trinity College Dublin, D02 R590 Dublin, Ireland 3 School of Medicine, Dentistry and Biomedical Sciences, The Patrick G Johnston Centre for Cancer Research Queen’s University, Belfast BT9 7BL, UK 4 Mercer’s Institute for Successful Ageing (MISA), St. James’s Hospital, D08 NHY1 Dublin, Ireland 5 Global Brain Health Institute, Trinity College Dublin, D02 DK07 Dublin, Ireland 4 Mercer’s Institute for Successful Ageing (MISA), St. James’s Hospital, D08 NHY1 Dublin, Ireland 5 Global Brain Health Institute, Trinity College Dublin, D02 DK07 Dublin, Ireland g g p 5 Global Brain Health Institute, Trinity College Dublin, D02 DK07 Dublin, Ireland * Correspondence: silvin.knight@tcd.ie Abstract: In this cross-sectional study, the relationship between noninvasively measured neurocar- diovascular signal entropy and physical frailty was explored in a sample of community-dwelling older adults from The Irish Longitudinal Study on Ageing (TILDA). The hypothesis under inves- tigation was that dysfunction in the neurovascular and cardiovascular systems, as quantified by short-length signal complexity during a lying-to-stand test (active stand), could provide a marker for frailty. Frailty status (i.e., “non-frail”, “pre-frail”, and “frail”) was based on Fried’s criteria (i.e., exhaustion, unexplained weight loss, weakness, slowness, and low physical activity). Approximate entropy (ApEn) and sample entropy (SampEn) were calculated during resting (lying down), active standing, and recovery phases. There was continuously measured blood pressure/heart rate data from 2645 individuals (53.0% female) and frontal lobe tissue oxygenation data from 2225 participants (52.3% female); both samples had a mean (SD) age of 64.3 (7.7) years.   Citation: Knight, S.P.; Newman, L.; O’Connor, J.D.; Davis, J.; Kenny, R.A.; Romero-Ortuno, R. Associations between Neurocardiovascular Signal Entropy and Physical Frailty. Entropy 2021, 23, 4. https://dx.doi.org/10.3390/e23010004 Keywords: approximate entropy; sample entropy; physical frailty; cardiovascular; neurovascular; blood pressure; heart rate; frontal lobe oxygenation; near infrared spectroscopy; NIRS; TILDA Received: 30 November 2020 Accepted: 19 December 2020 Published: 22 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional affiliations. Article Associations between Neurocardiovascular Signal Entropy and Physical Frailty Results revealed statistically significant associations between neurocardiovascular signal entropy and frailty status. Entropy differ- ences between non-frail and pre-frail/frail were greater during resting state compared with standing and recovery phases. Compared with ApEn, SampEn seemed to have better discriminating power between non-frail and pre-frail/frail individuals. The quantification of entropy in short length neurocardiovascular signals could provide a clinically useful marker of the multiple physiological dysregulations that underlie physical frailty. Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 entropy entropy 1. Introduction Frailty can be defined as a biologically driven decrease in reserve and resistance to stressors, resulting from collective declines across multiple physiological systems, which causes increased vulnerability to adverse outcomes such as mortality, institutionalization, falls, and hospitalization [1–4]. In this study, we used the frailty phenotype as proposed by Fried et al. [1] to define non-frail, pre-frail, and frail groups. This model has been extensively used in clinical practice and research [5]. The phenotype is based on five components, namely, unintentional weight loss, self-reported exhaustion, weakness, slow walking speed, and low physical activity. By this operationalization, pre-frailty is defined (independently of age and sex) as the presence of one or two criteria, and frailty as having three or more [1]. Despite its long conceptual and operational history, the intrinsic dynamic physiological mechanisms of frailty are not well understood [6]. Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). https://www.mdpi.com/journal/entropy Entropy 2021, 23, 4. https://dx.doi.org/10.3390/e23010004 Entropy 2021, 23, 4 2 of 19 Dysregulation of the neurovascular and cardiovascular systems under conditions of stress have been shown to be associated with risk of frailty [7–9]. A simple method to stress the neurocardiovascular system in clinical practice is by asking a person to remain lying supine for a few minutes, and then asking him/her to stand quickly. This is generally referred to as the orthostatic “active stand” test. The active stand challenges the body’s ability to compensate for the natural drop in blood pressure that occurs after standing due to gravity, and humans may perform this up to 50 times per day [10]. Continuously monitoring cardiovascular and neurovascular activity (such as blood pressure, heart rate, and frontal lobe brain oxygenation levels) during this challenge can provide clinically useful information with regards a person’s ability to compensate and recuperate from the stressor of standing. Currently, there is no consensus as to the most appropriate way to analyze and interpret continuously-measured active stand data; however, the Irish Longitudinal Study on Ageing (TILDA) has pioneered research in this area, using both standard statistical methods [11,12], as well as advanced data-driven approaches [7,13]. standard statistical methods [11,12], as well as advanced data-driven approaches [7,13]. Disorder in physiological signals can be assessed by means of entropy [14–17]. 1. Introduction En- tropy is essentially a measure of irregularity/unpredictability, assigning lower entropy values to periodic, predictable data, and higher entropy values to irregular, unpredictable data. Multiple different implementations of entropy have been proposed for the analysis of time-varying physiological signals, including approximate entropy (ApEn), sample entropy (SampEn), multi-scale entropy, and cross entropy [17–20]. In the present work, we investigated two of the most widely used entropy measures for investigating physio- logical time series data, namely, ApEn and SampEn. Having been initially developed for physiological applications, both ApEn and SampEn have been demonstrated to provide reliable estimates of signal complexity in cardiovascular data [14,17–19,21–24]. ApEn was first proposed in 1991 by Pincus et al. [25]. Briefly, given a time-series of length N, ApEn approximates the negative average logarithm of the conditional probability that two trajec- tories of length m remain similar in the next timestep, within a tolerance specified as ±r * standard deviation (SD) of the time-series. ApEn provides a unit-less number from 0 to 2. Notably, ApEn counts each subset as matching itself, and therefore, the ApEn algorithm is inherently biased towards regularity. In 2000, Richman and Moorman [17] introduced SampEn. Similar to ApEn, SampEn is defined as the negative natural logarithm of the conditional probability that two trajectories of length m remain similar for m + 1; however, self-matches are not considered in the probability calculation in this instance. Additionally, it has been demonstrated that SampEn is largely independent of the data length and can potentially provide more consistent results than ApEn [17]. In the present study, we utilized both ApEn and SampEn for the analysis of neurocardiovascular signal complexity. p p y g p y To date, a handful of studies have used both ApEn and SampEn specifically for the analysis of cardiovascular signal complexity in the context of frailty [21–23,26]. However, previously reported results have been contradictory, with some studies reporting higher complexity for frail individuals [23,26], while others reporting a reduction in complexity for frail individuals [21,22]. This may be due to differences in type of physiological signals analyzed, frailty operationalization methodologies employed, and/or small sample sizes. Because frailty is associated with adverse health outcomes in older people, it is im- portant to detect it as soon as possible before it manifests as a visible disability. 2.2. Neurocardiovascular Measurements Participants began the assessment with the affixing of a digital photoplethysmograph to the middle finger of the left hand (Finometer MIDI device, Finapres Medical Systems BV, Amsterdam, the Netherlands). This arm was then placed in a sling (to discourage its use during transition from supine to stand), resulting in the measurement site on the finger being roughly at the level of the heart throughout (further height changes adjusted for using the built-in height sensor on the Finometer device). Beat-to-beat blood pressure and heart rate were measured at 200 Hz using the Finometer device. Cerebral oxygenation was also measured simultaneously at 50 Hz using a near-infrared spectroscopy (NIRS) device (Portalite; Artinis Medical Systems, Zetten, the Netherlands) that was fixed to the forehead in approximately the FP1 (left frontal) position of the 10 to 20 electrode system (3 cm lateral and 3.5 cm superior to the nasion) [32]. This NIRS device uses 3 transmitters and 1 receiver, with each transmitter emitting 2 different wavelengths of light (760 nm and 850 nm) that propagate through the skull to a depth of approximately 2–3 cm and are absorbed at different rates by oxygenated haemoglobin (O2Hb) and deoxygenated haemoglobin (HHb). Hence, serum concentration levels of these molecules can be measured on the basis of the principle of absorption of electromagnetic radiation as described by the modified Beer–Lambert law. Multiple transmitters enable absolute concentration values to be determined via spatial resolved spectroscopy [33]. O2Hb and HHb concentrations were recorded, and tissue saturation index (TSI) was calculated as the O2Hb value expressed as a percentage of the sum of O2Hb and HHb values. The influence of environmental light was minimized via a black headband covering the sensor. All measurements were carried out in a comfortably lit room at an ambient temperature between 21 and 23 ◦C. Participants laid supine for ≈10 min before transitioning to a standing position and remained standing still for 3 min while data were continuously recorded. 2.1. Study Population This cross-sectional study utilized data from TILDA, an ongoing nationally representa- tive prospective cohort study of community-dwelling adults (representing approximately 1 in 150 individuals in Ireland aged ≥50 years) established in 2009 (N = 8507). TILDA’s study design and sampling methods are detailed elsewhere [29–31]. Briefly, sampling was based on geographic clustering. Ongoing health, social, and economic data collection involves a computer-assisted personal interview (CAPI) and a self-completed questionnaire (SCQ) approximately every 2 years. Every second wave, participants take part in a comprehensive health assessment at a dedicated health center. The primary exposure variables for this study were measured at Wave 3 of TILDA, which took place between March 2014 and December 2015. Ethical approval was granted from the Health Sciences Research Ethics Committee at Trinity College Dublin (granted 9 June 2014 for Wave 3; approval reference “Main Wave 3 Tilda Study”) and all participants provided written informed consent. All research was performed in accordance with the Declaration of Helsinki. 1. Introduction Indeed, medical research has shown that interventions can delay and even reverse frailty, especially when it presents in the early stages [27,28]. For that purpose, it is important to consider a frailty measure that does not include disability in its definition, and in that regard, Fried’s physical frailty phenotype is not only suitable, but also one of the most widely used in clinical practice [5]. We hypothesized that a simple automated measure of neurocardiovas- cular signal complexity (entropy) could provide a clinically useful marker of the multiple physiological dysregulations that underlie physical frailty. Given the critical importance of the performance of cardiovascular and neurovascular systems in helping us deal with stressors, our aim was to explore the relationship between entropy in these physiological Entropy 2021, 23, 4 3 of 19 3 of 19 signals and physical frailty. In other words, we undertook to classify frailty groups on the basis of entropy measures (ApEn, SampEn) and other demographic and health variables. signals and physical frailty. In other words, we undertook to classify frailty groups on the basis of entropy measures (ApEn, SampEn) and other demographic and health variables. 2.3. Signal Processing Signals for systolic blood pressure (sBP), diastolic blood pressure (dBP), mean arterial pressure (MAP), heart rate (HR), O2Hb, HHb, and TSI were extracted using MATLAB (R2019b, TheMathWorks, Inc., Natick, MA, USA). For the resting state, data from the last minute of supine rest were utilized in this study (prior to 60 s active stand “baseline” recording). For the active stand data, we analyzed a 1-min section, starting from when the participant began their stand (“challenge”), and another 1-min section taken from 120 to 180 s post-stand was analyzed as “recovery” from the active stand challenge. Time-to-stand was determined using the Finometer device’s built-in height sensor [34]. All data were Entropy 2021, 23, 4 4 of 19 down sampled to 5 Hz prior to analysis, providing N = 300 data points for analysis, and no filtering was applied. These values were chosen to provide a data collection timeframe that is easily transferable for clinical use (60 s), has an appropriate number of data points for analysis (time series with <200 data points are not recommended for either ApEn or SampEn due to inadequate vector matching [24,35]), while still capturing physiologically relevant signal components. Stationarity of the data was assessed via augmented Dicky– Fuller tests on both the original raw data and transformed data (data were transformed by subtracting the mean and dividing it by the standard deviation to increase the stationarity of the data series [36]). 2.4.2. Sample Entropy (SampEn) 2.4.2. Sample Entropy (SampEn) SampEn [17] was calculated as SampEn(m, r, N):= log   N−m ∑ i=1 Cm i (r)  −log   N−m−1 ∑ i=1 Cm+1 i (r)  , (2) (2) however, in this instance, Cm i (r) does not count self-matches. For SampEn, an r of 0.15 was selected, in line with previous recommendations for similar physiological data [37,38]. To assess the effects of data stationarity on entropy measures, we calculated ApEn and SampEn for both the original raw and transformed data. 2.4. Entropy Analysis Entropy analysis was performed using MATLAB. Previously developed MATLAB scripts were used to calculate ApEn and SampEn [15,16]. A detailed description of the algorithms used to compute both ApEn and SampEn has been previously reported in detail [17,25]; however, below we provide a brief overview. 2.4.1. Approximate Entropy (ApEn) ApEn [25] was calculated as 2.4.1. Approximate Entropy (ApEn) ApEn [25] was calculated as ApEn [25] was calculated as ApEn(m, r, N):= 1 (N −m + 1) N−m+1 ∑ i=1 log Cm i (r) Cm+1 i (r) , (1) (1) where Cm i (r) is the number of points found within the distance r for any point x(i) within the points xm i : = [x(i),..., x(i + m −1)], divided by N −m + 1. In this study, m (embedding dimension; the length of the data segment being compared) was set to 2 for both ApEn and SampEn, as this has been shown to show good statistical validity for ApEn and SampEn, especially for biological data [14,35]. The effects of increasing m were also investigated, with results from m = 2, 3, and 4 presented comparatively in Appendix B. An optimal r (similarity criterion) was computed to give the maximum ApEn using r from 0 to 0.6 in increments of 0.02, as per the method proposed by Chon et al. [14]. 2.6. Other Measures As part of the TILDA assessment, the following self-reported measures were also recorded at Wave 3 of the study: educational attainment, cardiovascular conditions (angina, high blood pressure, heart failure, heart murmur, abnormal heart rhythm, heart attack (ever), stroke (ever), or transient ischemic attack (TIA, ever)), diabetes, alcohol consumption habits (CAGE) [44], smoking history, and anti-hypertensive medication use (coded using the Anatomical Therapeutic Chemical Classification (ATC): antihypertensive medications (ATC C02), diuretics (ATC C03), β-blockers (ATC C07), calcium channel blockers (ATC C08), and renin-angiotensin system agents (ATC C09)). Depressive symptoms were assessed using the CESD scale [45]. Time taken to stand was calculated using data from the Finometer device’s built-in height sensor, as previously described [34]. To describe the general level of disability, we recorded the number of difficulties in performing activities of daily living (ADL). The original ADL scale, developed by Katz et al., encompasses “activities which people perform habitually and universally”, such as dressing (including putting on shoes and socks), walking across a room, bathing or showering, eating (such as cutting up food), getting in or out of bed, and using the toilet (including getting up and down) [46]. 2.5. Frailty Phenotype The calculation of the frailty phenotype was conducted following the methodology proposed by Fried et al. [1]. Full details have been described previously [39–41]; briefly, the frailty phenotype was operationalized using population-specific cut-off points re- lated to differences in the assessments of weakness (sex- and body mass index-adjusted grip strength measured with dynamometer on the dominant hand), physical activity (sex-adjusted kilocalories from the International Physical Activity Questionnaire—Short Form [42]), and walking speed (sex- and height-adjusted cm per second using the GAITRite portable walkway (CIR Systems, Inc., Sparta, NJ, USA)). Weight loss was determined by the question “In the past year have you lost 10 pounds (4.5 kg) or more in weight when you were not trying to?” Exhaustion was captured using 2 items from the Centre for Epi- demiological Studies Depression (CESD) scale [43]. Participants were asked how often they Entropy 2021, 23, 4 5 of 19 felt that “I could not get going” and “I felt that everything I did was an effort”. A response of “moderate amount/all of the time” to either question was considered as “exhaustion”. felt that “I could not get going” and “I felt that everything I did was an effort”. A response of “moderate amount/all of the time” to either question was considered as “exhaustion”. 2.7. Statistical Analysis Statistical analysis was performed using STATA 14.1 (StataCorp, College Station, TX, USA). The data were visually assessed for normality via Q-Q plots and histograms. All multivariate analysis was completed using robust linear regression with residual analysis completed to assess model assumptions. Statistical significance was set at P < 0.05. Multi- ple models were utilized to examine the relationships between the neurocardiovascular entropy measures and frailty phenotype status. Additional potential correlates controlled for in all models were age, sex, education, number of cardiovascular conditions (0, 1, 2+), diabetes, antihypertensive medication, alcohol consumption habits, smoking, and depres- sion. The models for active stand data additionally controlled for stand time. Results from absolute coefficients were reported as point estimates in appropriate units, presented with 95% confidence intervals (CI). 2.8. Data Availability Statement The datasets generated during and/or analyzed during the current study are not publicly available due to data protection regulations but are accessible at TILDA on reasonable request. The procedures to gain access to TILDA data are specified at https: //tilda.tcd.ie/data/accessing-data/. q p //tilda.tcd.ie/data/accessing-data/. 3.1. Participant Characteristics In total 2645 participants had complete blood pressure (BP) resting state and active stand data and 2225 had full NIRS data; full exclusions leading to these cohorts are provided in Figure 1. In total 2172 individuals had both BP and NIRS data. Participants’ mean (SD) age was 64.3 (7.7) years in both samples. In the BP cohort, 53.0% were female and in the NIRS cohort 52.3% were female. Similar distributions of frailty phenotype status were seen across both cohorts (non-frail: 59.1% (BP), 59.5% (NIRS); pre-frail: 37.2% (BP), 36.7% (NIRS); frail 3.7% (BP), 3.8% (NIRS)). Full demographic characteristics for both cohorts are presented in Table 1. Entropy 2021, 23, 4 6 of 19 notype 37.2% b h Figure 1. Flow chart describing sample selection and exclusions. Figure 1. Flow chart describing sample selection and exclusions. Figure 1. Flow chart describing sample selection and exclusions. Figure 1. Flow chart describing sample selection and exclusions. Entropy 2021, 23, 4 7 of 19 Table 1. Demographic characteristics of the study samples. BP Cohort (n = 2645) NIRS Cohort (n = 2225) Age [years] 64.3 (SD: 7.7, range: [50–93]) 64.3 (SD: 7.7, range: [50–93]) Sex [% (n)] Female: 53.0% (1401) Female: 52.3% (1163) Education [% (n)] Primary/none 16.5% (436) 16.5% (368) Secondary 39.9% (1055) 40.1% (891) Third/higher 43.6% (1154) 43.4% (966) Frailty Phenotype [% (n)] Non-frail 59.1% (1564) 59.5% (1325) Pre-frail 37.2% (984) 36.7% (816) Frail 3.7% (97) 3.8% (84) Disability [% (n)] Number of ADL disabilities Non-frail 0 98.9% (1547) 98.9% (1310) 1 1.0% (15) 1.0% (13) +2 0.1% (2) 0.1% (2) Pre-frail 0 96.1% (946) 96.3% (786) 1 2.3% (23) 2.1% (17) +2 1.6% (15) 1.6% (13) Frail 0 79.4% (77) 78.6% (66) 1 13.4% (13) 15.5% (13) +2 7.2% (7) 5.9% (5) No. 3.1. Participant Characteristics of Cardiovascular Conditions a [% (n)] 0 40.4% (1069) 40.6% (904) 1 35.6% (942) 35.9% (768) 2+ 24.0% (634) 23.5% (523) Self-reported diabetic [%] 6.8% (179) 6.5% (144) Antihypertensive medications b [% (n)] 37.7% (997) 37.2% (827) CAGE alcohol scale CAGE < 2 76.7% (2029) 77.2% (1718) CAGE ≥2 12.4% (328) 12.3% (274) No response 10.9% (288) 10.5% (233) Smoker [% (n)] Never 47.9% (1268) 48.0% (1067) Past 42.6% (1127) 42.5% (945) Current 9.5% (250) 9.5% (213) CESD [% (n)] Non-depressed (CESD < 9) 89.1% (2358) 89.3% (1986) Depressed (CESD ≥9) 10.9% (287) 10.7% (239) Time to stand [seconds] 7.2 (SD: 2.8, range: [2–27]) 7.2 (SD: 2.8, range: [2–26]) a Cardiovascular conditions: angina, high blood pressure, heart failure, heart murmur, abnormal heart rhythm, heart attack (ever), stroke (ever), or transient ischemic attack (TIA, ever). b Coded using the Anatomical Therapeutic Chemical Classification (ATC): antihypertensive medications (ATC C02), diuretics (ATC C03), β-blockers (ATC C07), calcium channel blockers (ATC C08), and renin–angiotensin system agents (ATC C09). Abbreviations: ADL, activities of daily living; CESD, Center for Epidemiologic Studies Depression scale. Table 1. Demographic characteristics of the study samples. Entropy 2021, 23, 4 8 of 19 3.2. Associations of Entropy with Frailty Phenotype 3.2. Associations of Entropy with Frailty Phenotype Figure 2 visually illustrates data from three example participants with “low” (0.20), “medium” (0.45), and “high” (0.70) levels of ApEn in their resting state sBP data. As was generally the case, individuals with higher entropy in one measure (in the case of Figure 2 sBP) generally had higher entropy in the other physiological measures investigated. Figure 2 illustrates this well, with the stratification of signal disorder still visually apparent in the frontal lobe NIRS measures (O2Hb, HHb, and TSI). REVIEW 9 of 20 Figure 2 visually illustrates data from three example participants with “low” (0.20), “medium” (0.45), and “high” (0.70) levels of ApEn in their resting state sBP data. As was generally the case, individuals with higher entropy in one measure (in the case of Figure 2 sBP) generally had higher entropy in the other physiological measures investigated. Figure 2 illustrates this well, with the stratification of signal disorder still visually apparent in the frontal lobe NIRS measures (O2Hb, HHb, and TSI). REVIEW 9 of 20 ( , , ) Figure 2. 3.1. Participant Characteristics Example plots of data from three different participants with “low” (0.20), “medium” (0.45), and “high” (0.7 levels of approximate entropy (ApEn) measured in the resting state systolic blood pressure (sBP) data. Individuals wi higher entropy in one measure investigated (in this case, resting state sBP) also generally had higher entropy in the oth physiological measures investigated, as visually illustrated above. re 2. Example plots of data from three different participants with “low” (0.20), “medium” (0.45), and “high s of approximate entropy (ApEn) measured in the resting state systolic blood pressure (sBP) data. Individua er entropy in one measure investigated (in this case, resting state sBP) also generally had higher entropy in th iological measures investigated, as visually illustrated above. Figure 2. Example plots of data from three different participants with “low” (0.20), “medium” (0.45), and “high” (0.70) levels of approximate entropy (ApEn) measured in the resting state systolic blood pressure (sBP) data. Individuals with higher entropy in one measure investigated (in this case, resting state sBP) also generally had higher entropy in the other physiological measures investigated, as visually illustrated above. Figure 2. Example plots of data from three different participants with “low” (0.20), “medium” (0.45), and “high” (0.70) levels of approximate entropy (ApEn) measured in the resting state systolic blood pressure (sBP) data. Individuals with higher entropy in one measure investigated (in this case, resting state sBP) also generally had higher entropy in the other physiological measures investigated, as visually illustrated above. Entropy 2021, 23, 4 9 of 19 9 of 19 Results from augmented Dicky–Fuller tests revealed low proportions of stationarity (0.2% to 23.1%) for the raw data; after transforming the data, 18.1% to 92.6% of cases were stationary. Despite the increase in stationarity, which in some instances was large (e.g., resting state O2Hb stationarity increased from 0.2% to 92.6%), no differences in ApEn and SampEn measures were found between the original and transformed data (see Appendix A, Table A1). Thus, the results from the original time series were used for statistical analysis. Tables 2 and 3 provide the mean, SD, and range for ApEn and SampEn calculated from resting state, active stand (stand 0–60s), and recovery (120–180s post-stand), stratified by physical frailty status. Across all measures, absolute mean entropy was higher in pre-frail and frail groups compared with the non-frail group. Mean sBP, dBP, MAP, and HR entropy measures were highest during recovery and lowest during stand. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. 3.1. Participant Characteristics For O2Hb, HHb, and TSI mean values increased from resting state to stand to recovery. Overall, absolute BP and HR SampEn measures were 3% to 50% lower than ApEn measures. Similarly, absolute SampEn was 6% to 36% lower than ApEn measures for NIRS resting state and active stand measures; however, absolute SampEn values in NIRS recovery were higher than ApEn measures. Of note, SD values reported in Tables 2 and 3 are absolute and are not statistically adjusted for any confidence level. Table 2. Approximate entropy (ApEn) results by frailty phenotype grouping. ApEn Non-Frail (N = 1325–1564) Pre-Frail (N = 816–984) Frail (N = 84–97) Resting State Mean (SD, [Range]) Mean (SD, [Range]) Mean (SD, [Range]) sBP 0.52 (0.07, [0.26–0.98]) 0.54 (0.08, [0.22–0.77]) 0.56 (0.08, [0.31–0.75]) dBP 0.45 (0.08, [0.20–0.96]) 0.47 (0.09, [0.22–0.78]) 0.50 (0.11, [0.29–0.83]) MAP 0.46 (0.08, [0.17–1.01]) 0.48 (0.08, [0.22–0.79]) 0.52 (0.09, [0.30–0.80]) HR 0.49 (0.09, [0.02–0.81]) 0.49 (0.09, [0.05–0.77]) 0.51 (0.10, [0.29–0.77]) O2Hb 0.44 (0.07, [0.16–0.68]) 0.45 (0.07, [0.18–0.65]) 0.46 (0.07, [0.23–0.60]) HHb 0.43 (0.07, [0.09–0.65]) 0.43 (0.07, [0.16–0.64]) 0.45 (0.07, [0.27–0.60]) TSI 0.39 (0.06, [0.12–0.62]) 0.40 (0.07, [0.11–0.63]) 0.41 (0.06, [0.28–0.54]) Stand (0–60s) sBP 0.44 (0.07, [0.20–0.97]) 0.46 (0.08, [0.21–0.80]) 0.48 (0.08, [0.33–0.70]) dBP 0.40 (0.07, [0.17–0.90]) 0.42 (0.09, [0.19–0.83]) 0.45 (0.10, [0.25–0.76]) MAP 0.41 (0.08, [0.17–0.93]) 0.43 (0.08, [0.18–0.82]) 0.45 (0.09, [0.30–0.80]) HR 0.45 (0.08, [0.07–0.80]) 0.46 (0.09, [0.18–0.95]) 0.49 (0.11, [0.23–0.81]) O2Hb 0.79 (0.12, [0.36–1.37]) 0.80 (0.12, [0.32–1.34]) 0.79 (0.14 [0.38–1.08]) HHb 0.69 (0.14, [0.21–1.37]) 0.70 (0.15, [0.30–1.36]) 0.69 (0.14, [0.40–0.99]) TSI 0.77 (0.12, [0.40–1.39]) 0.79 (0.12, [0.41–1.33]) 0.79 (0.12, [0.53–1.08]) Stand (120–180s) sBP 0.55 (0.07, [0.29–1.04]) 0.56 (0.07, [0.33–0.84]) 0.58 (0.07, [0.39–0.78]) dBP 0.49 (0.08, [0.27–1.02]) 0.50 (0.09, [0.26–0.85]) 0.53 (0.10, [0.32–0.84]) MAP 0.51 (0.07, [0.22–1.01]) 0.52 (0.08, [0.26–0.81]) 0.54 (0.09, [0.28–0.84]) HR 0.49 (0.08, [0.02–0.88]) 0.50 (0.09, [0.23–0.85]) 0.52 (0.11, [0.20–0.79]) O2Hb 0.91 (0.12, [0.43–1.40]) 0.91 (0.12, [0.55–1.37]) 0.92 (0.13 [0.50–1.12]) HHb 0.87 (0.14, [0.40–1.42]) 0.88 (0.14, [0.42–1.36]) 0.90 (0.11, [0.62–1.18]) TSI 0.92 (0.11, [0.50–1.34]) 0.92 (0.11, [0.59–1.34]) 0.95 (0.11, [0.60–1.15]) Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated Table 2. Approximate entropy (ApEn) results by frailty phenotype grouping. Entropy 2021, 23, 4 10 of 19 Table 3. Sample entropy (SampEn) results by frailty phenotype grouping. 3.1. Participant Characteristics SampEn Non-Frail (N = 1325–1564) Pre-Frail (N = 816–984) Frail (N = 84–97) Resting State Mean (SD, [Range]) Mean (SD, [Range]) Mean (SD, [Range]) sBP 0.49 (0.13, [0.07–1.41]) 0.51 (0.14, [0.08–0.93]) 0.55 (0.14, [0.15–0.87]) dBP 0.39 (0.13, [0.06–1.34]) 0.41 (0.13, [0.06–0.95]) 0.45 (0.16, [0.18–0.99]) MAP 0.40 (0.13, [0.05–1.27]) 0.43 (0.14, [0.08–0.84]) 0.48 (0.15, [0.15–0.89]) HR 0.44 (0.15, [0.01–1.00]) 0.44 (0.16, [0.02–1.03]) 0.46 (0.19, [0.08–0.99]) O2Hb 0.34 (0.14, [0.01–0.71]) 0.35 (0.14, [0.05–0.78]) 0.38 (0.14, [0.07–0.67]) HHb 0.31 (0.13, [0.03–0.80]) 0.32 (0.14, [0.04–0.72]) 0.36 (0.14, [0.12–0.62]) TSI 0.26 (0.10, [0.03–0.72]) 0.27 (0.11, [0.03–0.74]) 0.28 (0.11, [0.06–0.52]) Stand (0–60 s) sBP 0.30 (0.12, [0.04–1.26]) 0.32 (0.13, [0.04–0.98]) 0.35 (0.14, [0.12–0.82]) dBP 0.20 (0.11, [0.03–1.03]) 0.22 (0.14, [0.04–1.03]) 0.28 (0.16, [0.03–0.87]) MAP 0.24 (0.11, [0.04–1.11]) 0.26 (0.13, [0.04–0.92]) 0.30 (0.15, [0.05–0.99]) HR 0.31 (0.14, [0.02–1.08]) 0.33 (0.17, [0.03–1.21]) 0.40 (0.21, [0.03–1.05]) O2Hb 0.73 (0.37, [0.04–2.69]) 0.74 (0.39, [0.01–2.70]) 0.68 (0.38, [0.03–1.54]) HHb 0.44 (0.33, [0.02–2.88]) 0.47 (0.35, [0.01–2.80]) 0.44 (0.29, [0.03–1.30]) TSI 0.61 (0.36, [0.07–2.73]) 0.64 (0.36, [0.06–2.63]) 0.67 (0.36, [0.12–1.67]) Stand (120–180 s) sBP 0.52 (0.13, [0.14–1.28]) 0.53 (0.13, [0.10–1.13]) 0.57 (0.14, [0.27–0.96]) dBP 0.43 (0.13, [0.10–1.57]) 0.44 (0.14, [0.10–1.10]) 0.50 (0.19, [0.13–1.17]) MAP 0.47 (0.13, [0.06–1.50]) 0.48 (0.14, [0.09–0.99]) 0.51 (0.16, [0.11–1.07]) HR 0.42 (0.14, [0.01–1.06]) 0.44 (0.16, [0.03–1.12]) 0.47 (0.19, [0.08–0.98]) O2Hb 1.16 (0.38, [0.06–2.77]) 1.16 (0.39, [0.09–2.57]) 1.15 (0.39, [0.09–1.91]) HHb 0.89 (0.42, [0.07–2.80]) 0.93 (0.45, [0.06–2.60]) 0.97 (0.42, [0.12–2.05]) TSI 1.08 (0.36, [0.15–2.58]) 1.08 (0.37, [0.15–2.78]) 1.18 (0.36, [0.28–2.18]) Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Table 3. Sample entropy (SampEn) results by frailty phenotype grouping. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. P: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated ntration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Figure 3 reports multivariate-adjusted point estimates from the regression models, with error bars showing the 95% CIs corresponding to the 95% confidence level (i.e., P ≤0.05 considered significant). Overall, models controlled only for age and sex provided similar results to the fully controlled models (controlling for age, sex, education, number of cardiovascular conditions (0, 1, 2+), diabetes, antihypertensive medication, alcohol consumption habits, smoking, and depression). 3.1. Participant Characteristics Models for active stand data also controlled for stand time). Beta coefficients were slightly lower for some of the measures in the fully controlled versus age and sex-controlled models, however, significance of the results was generally consistent, with the exception of the O2Hb and HHb resting state data, which were no longer significant in the fully controlled models. The magnitude of the beta coefficients from the regression analysis were in line with the absolute differences reported in Tables 2 and 3. In fully controlled models of resting state data, all four Finometer measures were significantly higher in pre-frail (ApEn—sBP: β = 0.01, P = 0.004; dBP: β = 0.01, P ≤0.001; MAP: β = 0.01, P ≤0.001; HR: β = 0.01, P = 0.049. SampEn—sBP: β = 0.01, P = 0.050; dBP: β = 0.02, P = 0.001; MAP: β = 0.02, P = 0.001; HR: β = 0.01, P = 0.038) and frail groups Entropy 2021, 23, 4 MAP HR 11 of 19 7]) 8]) 11 of 19 7]) 8]) (ApEn—sBP: β = 0.02, P = 0.006; dBP: β = 0.04, P = 0.001; MAP: β = 0.04, P ≤0.001; HR: β = 0.03, P = 0.007. SampEn—sBP: β = 0.05, P = 0.004; dBP: β = 0.05, P = 0.003; MAP: β = 0.06, P ≤0.001; HR: β = 0.04, P = 0.048). 0.89 (0.42, [0.07 2.80]) 0.93 (0.45, [0.06 2.60]) 0.97 (0.42, [0.12 2.05]) 1.08 (0.36, [0.15–2.58]) 1.08 (0.37, [0.15–2.78]) 1.18 (0.36, [0.28–2.18]) lic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; lobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. (ApEn—sBP: β = 0.02, P = 0.006; dBP: β = 0.04, P = 0.001; MAP: β = 0.04, P ≤0.001; HR: β = 0.03, P = 0.007. SampEn—sBP: β = 0.05, P = 0.004; dBP: β = 0.05, P = 0.003; MAP: β = 0.06, P ≤0.001; HR: β = 0.04, P = 0.048). 0.89 (0.42, [0.07 2.80]) 0.93 (0.45, [0.06 2.60]) 0.97 (0.42, [0.12 2.05]) 1.08 (0.36, [0.15–2.58]) 1.08 (0.37, [0.15–2.78]) 1.18 (0.36, [0.28–2.18]) lic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; lobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. (ApEn—sBP: β = 0.02, P = 0.006; dBP: β = 0.04, P = 0.001; MAP: β = 0.04, P ≤0.001; HR: β = 0.03, P = 0.007. 3.1. Participant Characteristics SampEn—sBP: β = 0.05, P = 0.004; dBP: β = 0.05, P = 0.003; MAP: β = 0.06, P ≤0.001; HR: β = 0.04, P = 0.048). ( , [ ]) ( , [ ]) ( , [ ]) 1.08 (0.36, [0.15–2.58]) 1.08 (0.37, [0.15–2.78]) 1.18 (0.36, [0.28–2.18]) lic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; lobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Figure 3. Results from robust multivariate linear regression showing beta coefficients (β) for both approximate entropy (ApEn) and sample entropy (SampEn). All models adjusted for age, sex, education, diabetes, number of cardiovascular conditions, antihypertensive medication use, alcohol consumption habits, smoking, and depression. Model for active stand data additionally controlled for stand time. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb: oxygenated hemoglobin concentration; HHb: deoxygenated hemoglobin concentration; TSI: tissue saturation index; β: beta coefficient. Figure 3. Results from robust multivariate linear regression showing beta coefficients (β) for both approximate entropy (ApEn) and sample entropy (SampEn). All models adjusted for age, sex, education, diabetes, number of cardiovascular conditions, antihypertensive medication use, alcohol consumption habits, smoking, and depression. Model for active stand data additionally controlled for stand time. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb: oxygenated hemoglobin concentration; HHb: deoxygenated hemoglobin concentration; TSI: tissue saturation index; β: beta coefficient. Figure 3. Results from robust multivariate linear regression showing beta coefficients (β) for both approximate entropy (ApEn) and sample entropy (SampEn). All models adjusted for age, sex, education, diabetes, number of cardiovascular conditions, antihypertensive medication use, alcohol consumption habits, smoking, and depression. Model for active stand data additionally controlled for stand time. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb: oxygenated hemoglobin concentration; HHb: deoxygenated hemoglobin concentration; TSI: tissue saturation index; β: beta coefficient. Figure 3. Results from robust multivariate linear regression showing beta coefficients (β) for both approximate entropy (ApEn) and sample entropy (SampEn). All models adjusted for age, sex, education, diabetes, number of cardiovascular conditions, antihypertensive medication use, alcohol consumption habits, smoking, and depression. Model for active stand data additionally controlled for stand time. 4. Discussion Results from this study demonstrate significant associations between peripherally measured neurocardiovascular signal entropy and physical frailty status. Even though the magnitude of these associations was shown to be similar for resting state, active stand, and recovery data, the differences between non-frail and pre-frail/frail BP and HR entropy measures did increase during the stand and recovery phases, most notably for HR. For frail individuals, TSI was significantly higher during the recovery from stand, compared with non-frail. Even though, overall, absolute SampEn values were 2 to 50% lower than ApEn values, while β coefficients from statistically significant models were up to 198% higher when using SampEn, which suggests potentially better discriminating power between non-frail and pre-frail/frail individuals for SampEn. These results support the hypothesis that a simple automated measure of neurocardiovascular signal entropy could provide a clinically useful marker of the multiple physiological dysregulations that underlie physical frailty. p y y Only a handful of smaller scale studies have investigated the associations of cardio- vascular signal entropy with frailty. Results reported to date are contradictory, with some reporting higher levels of entropy and disorder in cardiovascular data for pre-frail and frail individuals versus non-frail [23,26], while others contrariwise report lower entropy values in these groups [21,22]. However, there are several important methodological differ- ences between these studies, as well as between those and the present work, which most likely account for this. Most similar to the present work in terms of methodology, and reporting similar results, Takahashi et al. [23], in a study on 80 individuals, found higher ApEn and conditional entropy for both pre-frail and frail groups, compared with non-frail, with frail participants having the highest entropy overall; in addition, the authors also reported lower absolute entropy in their stand data versus rest, which is also in line with our results, as well as other previous work [47]. Analogously, some studies have reported a reduction in entropy after a head-up tilt test [48,49], which is another type of orthostatic challenge, though less representative of daily living. Exploring the physiological origins of these signal complexity differences between resting state and orthostatic challenge would be of interest, particularly in relation to baroreflex control, which has been shown to be associated with frailty status [50]. Conversely, Chaves et al. indicated that lower values of ApEn were associated with a higher probability of an individual being frail [21]. Chaves et al. 3.1. Participant Characteristics Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb: oxygenated hemoglobin concentration; HHb: deoxygenated hemoglobin concentration; TSI: tissue saturation index; β: beta coefficient. For the fully controlled stand 0–60s models, all BP/HR entropy measures were significantly associated with frailty status (pre-frail: ApEn—sBP: β = 0.01, P = 0.001; dBP: β = 0.01, P = 0.001; MAP: β = 0.01, P = 0.001; HR: β = 0.01, P ≤ 0.001. SampEn—sBP: β = 0.02, For the fully controlled stand 0–60s models, all BP/HR entropy measures were signifi- cantly associated with frailty status (pre-frail: ApEn—sBP: β = 0.01, P = 0.001; dBP: β = 0.01, P = 0.001; MAP: β = 0.01, P = 0.001; HR: β = 0.01, P ≤0.001. SampEn—sBP: β = 0.02, P = 0.004; dBP: β = 0.01, P = 0.020; MAP: β = 0.01, P = 0.018; HR: β = 0.02, P = 0.012. frail: ApEn—sBP: β = 0.03, P = 0.003; dBP: β = 0.03, P = 0.001; MAP: β = 0.03, P = 0.003; HR: β = 0.04, P ≤0.001. SampEn—sBP: β = 0.04, P = 0.011; dBP: β = 0.06, P = 0.001; MAP: β = 0.04, P = 0.010; HR: β = 0.09, P ≤0.001). Likewise, for the fully controlled stand 120–180s models, again all BP/HR entropy measures were significantly associated with frailty status (pre-frail: ApEn—sBP: β = 0.01, P = 0.004; dBP: β = 0.01, P = 0.008; MAP: β = 0.01, P = 0.006; HR: β = 0.01, P ≤0.001. Entropy 2021, 23, 4 12 of 19 12 of 19 SampEn—sBP: β = 0.01, P = 0.010; dBP: β = 0.01, P = 0.028; MAP: β = 0.02, P = 0.007; HR: β = 0.02, P = 0.001. Frail: ApEn—sBP: β = 0.03, P = 0.001; dBP: β = 0.04, P ≤0.001; MAP: β = 0.03, P = 0.001; HR: β = 0.04, P = 0.003. SampEn—sBP: β = 0.05, P = 0.003; dBP: β = 0.07, P ≤0.001; MAP: β = 0.06, P = 0.001; HR: β = 0.06, P = 0.004). 3.1. Participant Characteristics β β For the fully controlled models, NIRS (O2Hb, HHb, and TSI) entropy measurements were not significantly associated with frailty status, with the exception of TSI at resting state, which was higher in the pre-frail group (ApEn: β = 0.01, P = 0.011; SampEn: β = 0.01, P = 0.007); and in the recovery data, TSI entropy was significantly higher for frail partici- pants (ApEn: β = 0.03, P = 0.010; SampEn: β = 0.10, P = 0.022). For all models investigated, the magnitude of statistically significant differences between frailty groups were larger for SampEn compared with ApEn, with β coefficients up to 102% higher in BP and 198% higher in NIRS SampEn results compared with ApEn. The effect of increasing m was also investigated, with multivariate-adjusted point estimates from the regression models for m = 2, 3, and 4 presented in Appendix B, Figure A1. For BP and HR, all significant associations described above remained significant as m was increased. However, the associations of frail status with recovery TSI lost significance as m was increased. 4. Discussion This suggests that the patterns of increased disorder associated with frailty status in BP and HR data occur within the scale of 0.4 to 0.8 s. The present study has several strengths. To date, ours is the largest study to investigate the associations between entropy measures in neurocardiovascular signals and physical frailty status (N = 2225/2645). Additionally, to the best of our knowledge, this is the first study to examine frontal lobe oxygenation entropy (as measured using NIRS) with the physical frailty phenotype. The rich data available as part of TILDA meant that models could be comprehensively controlled for a number of covariates known to affect physical and neurocardiovascular function. Additionally, the richness of the continuously, simulta- neously measured neurovascular and cardiovascular data allowed for the assessment of several physiological measurements, recorded within the same experimental paradigm. p y g p p g From a clinical relevance point of view, it is important to notice the generally low levels of disability in this sample, with almost 80% of frail participants not having any ADL impairments (Table 1). Indeed, Fried’s physical frailty phenotype intends to capture a pre-disability state [53]. That, coupled with the fact that less than 4% of the sample were classified as frail, highlights the remarkable sensitivity of entropy measures in automatically identifying frailty status at resting state, when often it is very difficult for clinicians to identify frailty with the naked eye. y y y The methodologies presented herein were specifically designed to be highly trans- ferable for use in a clinical setting. All measures were non-invasive and non-ionizing. The short data length required (60 s) would be feasible and practical for use in a busy clinic. Entropy provides a single-number measure, which could theoretically be calculated at and displayed on the measurement device itself, allowing for easy use by clinicians. Additionally, since the associations of entropy levels in resting state, active stand, and recovery data with frailty phenotype were all similar, this suggests that resting state en- tropy might be sufficient as a clinical marker for frailty, further increasing the ease by which this measure could be recorded in the clinic. 4. Discussion operationalized the original Fried’s criteria; however, direct comparison between that study and the present is not possible due to large difference in the methods used; for example, only females were investigated (N = 389), and a dichotomous classification of frailty was adopted (frail or non-frail). Most notably, their study utilized a much longer Entropy 2021, 23, 4 13 of 19 13 of 19 dataset (2–3 h), recorded while participants underwent transitions through a number of diverse postural positions (e.g., lying, sitting, standing). As such, their study is likely to be reporting on the flexibility of the cardiovascular system, i.e., the ability of the system to adapt to multiple challenges over a longer time period, a measure that is known to be indicative of a more positive health status. Another recent study by Rangasamy et al. likewise reported lower entropy in frail versus non-frail (N = 364) [22]. However, again methodological differences do not allow for direct comparison with the present study, including in this case the vastly different process used to quantify the dichotomous frailty status used (which was based on demographics, anthropometrics, and blood biomarkers). ( g p p ) Heart rate variability complexity is generally expected to decrease with pathology [51]; however, in the present work, higher entropy in both BP and HR were found to be as- sociated with increased frailty. We postulate that entropy calculated in short length neu- rocardiovascular data, as reported herein, is not measuring system flexibility, but rather systemic disorder, or “jitter”, resulting from dysregulation of the neurocardiovascular system. It would therefore be expected that this negative state of higher disorder on a short time frame would be associated with physical frailty or systemic dysregulation. One pos- sible cause for this dysregulation could be an increase of sympathetic activity and/or modulation directed to the heart and/or blood vessels with increased frailty status, as previously described in abnormal ageing states [52]. Other potential influencing factors could be modified cardiac reserve, changes in arterial structure (e.g., increased stiffness, decreased compliance, and endothelial dysfunction), as well as changes of diastolic fill- ing and increased collagen in the left ventricle [47]. Increasing the embedded dimension (m = 2, 3, and 4) did not have a major effect on the main significant results of this study (see Appendix B, Figure A1). 4. Discussion Moreover, the similarity between BP and HR entropy measures, in relation to frailty status, suggests that these measures may provide complementary information, as has been previously reported [54], and as such a univariant approach (i.e., the assessment of one of these measures) may be sufficient for clinical use. Input parameters and implementation of ApEn and SampEn calculations Entropy 2021, 23, 4 14 of 19 14 of 19 were based on recommendations for similar physiological data from previous studies (m = 2 (also reported in Appendix B m = 3, 4) [14,35]; r = 0.15 (SampEn) [37,38], optimum calculated [0 to 0.6] (ApEn) [14]; N > 200 [24,35]); however, a consensus with regards the optimal methodologies to use, as well as normative age- and sex-adjusted reference values, would be required for widespread clinical adoption. Further work is necessary to establish the prognostic implications of entropy measures vis-à-vis other clinical markers (e.g., for the prediction of mortality and other adverse health events). Future longitudinal work investigating how these measures vary over time would also be of interest, since this may provide an “early warning” measure for potential transitions from less to more adverse frailty statuses, for use in a clinical setting. There are several further limitations to this study that should be kept in mind when interpreting the results. Analyses were cross-sectional and, as such, causality or even tem- porality of the observed relationships could not be inferred. There was a small number of frail individuals compared with the other groups; however, the proportion of frail individ- uals was in line with previously reported TILDA studies [55,56]. This slight discrepancy in the proportion of frail persons may have been due to the rigor of the data quality exclusion criteria used to ensure high internal validity of the study. Since a convenience sample was used in the current work (i.e., participants without full neurovascular or cardiovascular data and physical frailty data could not be included), we do not propose these results are population-representative, despite the large cohort sizes; it is reasonable to assume that a higher proportion of participants unable to provide these data (particularly active stand data) may have been from the frail group. 4. Discussion Data utilized in this study had relatively high proportions of non-stationarity, as is commonly the case with physiological data [57], which may have potentially biased the estimates of complexity since non-stationarities have been shown to diminish the abso- lute level of complexity as assessed by conditional entropy [58]. However, there was no difference between entropy measures from the original raw data and data transformed to increase stationarity, even though stationarity increased by up to 92% in some instances, which is in line with previous work [35]. Further work exploring different methods to increase stationarity of these types of data, while still retaining the physiologically relevant signal complexity information, would be of interest. Another important caveat to keep in mind when interpreting the results of this study is that the ApEn results presented may be biased towards regularity, since ApEn counts self matches. Methods have been proposed to correct this bias in ApEn measures [48]; however, this was not done in the present study as we also present SampEn results in parallel, which are not subject to the same potential self-matching bias. Additionally, due to the fact that SampEn displays relative consistency under conditions where ApEn does not (e.g., data length) [17], it would be recommended to use the SampEn results from this study for future cross-study comparisons. In the present study, ApEn and SampEn were used to investigate each neurocardiovascular measure individually and at a single scale; future work using other entropy methods, such as multi-scale and cross entropy, would be of interest. Finally, this study does support the need for future longitudinal work to determine the clinical significance of these findings, and as such, this study should be considered preliminary and exploratory. 5. Conclusions Results from this study demonstrated significant associations between peripher- ally measured neurovascular/cardiovascular signal entropy and physical frailty status. These results support the hypothesis that a simple automated measure of neurocardio- vascular signal entropy at rest could provide a clinically useful marker of physical frailty. Our future work will focus on the study of physiological signal entropy as an early marker of the physiological dysregulation seen in frailty, which may open the possibility to detect early physiological dysregulation before the onset of obvious physical disability, and the opportunity to translate this work into opportunities to improve physiological resilience in older adults. Entropy 2021, 23, 4 15 of 19 15 of 19 Author Contributions: Conceptualization, S.P.K. and R.R.-O.; data curation, S.P.K., L.N. and J.D.O.; formal analysis, S.P.K.; funding acquisition, R.A.K. and R.R.-O.; investigation, S.P.K., J.D.O., J.D. and R.R.-O.; methodology, S.P.K. and R.R.-O.; project administration, R.A.K. and R.R.-O.; resources, R.A.K. and R.R.-O.; software, S.P.K., L.N. and J.D.O.; supervision, R.R.-O.; validation, S.P.K., L.N. and J.D.O.; visualization, S.P.K.; writing—original draft, S.P.K. and R.R.-O.; writing—review and editing, S.P.K., L.N., J.D., R.A.K. and R.R.-O. All authors have read and agreed to the published version of the manuscript. gy p j R.A.K. and R.R.-O.; software, S.P.K., L.N. and J.D.O.; supervision, R.R.-O.; validation, S.P.K., L.N. and J.D.O.; visualization, S.P.K.; writing—original draft, S.P.K. and R.R.-O.; writing—review and editing, S.P.K., L.N., J.D., R.A.K. and R.R.-O. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Science Foundation Ireland (SFI), grant number 18/FRL/6188. The Irish Longitudinal Study on Ageing (TILDA) is funded by Atlantic Philanthropies, the Irish Department of Health and Irish Life. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Health Sciences Research Ethics Committee of Trinity College Dublin (protocol reference: “Main Wave 3 Tilda Study”, approval granted 9 June 2014). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The datasets generated during and/or analyzed during the current study are not publicly available due to data protection regulations but are accessible at TILDA on reasonable request. The procedures to gain access to TILDA data are specified at https://tilda.tcd.ie/ data/accessing-data/. Acknowledgments: The authors would like to acknowledge the continued commitment and cooper- ation of the TILDA participants and research team. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Appendix A Table A1. Pre-processing results from augmented Dicky–Fuller test for stationarity, as well as differences between raw and standardized data for approximate entropy (∆ApEn) and sample entropy (∆SampEn) measures. X X− ¯ X σX ∆ApEn ∆SampEn Resting State N = 3273–3817 N [%] Stationary N (%) Stationary Mean [Range] Mean [Range] sBP 59 (1.6%) 921 (24.1%) −0.1 [−2.3 to 2.6] × 10−14 <1 × 10−32 dBP 57 (1.5%) 879 (23.0%) 0.1 [−1.5 to 2.0] × 10−14 <1 × 10−32 MAP 103 (2.7%) 690 (18.1%) −0.1 [−2.4 to 2.8] × 10−14 <1 × 10−32 HR 18 (0.5%) 1958 (51.3%) −0.1 [−2.4 to 2.3] × 10−14 <1 × 10−32 O2Hb 8 (0.2%) 3032 (92.6%) 0.8 [−1.0 to 2.9] × 10−14 <1 × 10−32 HHb 28 (0.9%) 1835 (56.1%) 1.1 [−0.9 to 3.2] × 10−14 <1 × 10−32 TSI 15 (0.5%) 2280 (69.7%) 1.0 [−1.0 to 2.8] × 10−14 <1 × 10−32 Stand (0–60s) N = 2583–3538 sBP 267 (7.6%) 1532 (43.3%) <1 × 10−32 <1 × 10−32 dBP 478 (13.5%) 1283 (36.3%) <1 × 10−32 <1 × 10−32 MAP 817 (23.1%) 1871 (52.9%) <1 × 10−32 <1 × 10−32 HR 85 (2.5%) 2051 (58.0%) <1 × 10−32 <1 × 10−32 O2Hb 94 (3.6%) 1913 (74.1%) <1 × 10−32 <1 × 10−32 HHb 82 (3.2%) 1264 (48.9%) <1 × 10−32 <1 × 10−32 TSI 79 (3.1%) 885 (34.3%) <1 × 10−32 <1 × 10−32 Entropy 2021, 23, 4 16 of 19 Table A1. Cont. X X− ¯ X σX ∆ApEn ∆SampEn Stand (120–180s) N = 2583–3365 sBP 23 (0.7%) 927 (27.6%) <1 × 10−32 <1 × 10−32 dBP 26 (0.8%) 850 (25.3%) <1 × 10−32 <1 × 10−32 MAP 34 (1.0%) 694 (20.6%) <1 × 10−32 <1 × 10−32 HR 14 (0.4%) 1585 (47.1%) <1 × 10−32 <1 × 10−32 O2Hb 3 (0.1%) 2266 (87.7%) <1 × 10−32 <1 × 10−32 HHb 22 (0.9%) 1493 (57.8%) <1 × 10−32 <1 × 10−32 TSI 4 (0.2%) 1766 (68.4%) <1 × 10−32 <1 × 10−32 Abbreviations: X: raw data; X: mean of raw data; σX: standard deviation of raw data; ∆ApEn: difference in approximate entropy (raw vs. standardized); ∆SampEn: difference in sample entropy (raw vs. standardized); sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Appendix A Entropy 2020 22 x FOR PEER REVIEW 17 of 20 Abbreviations: X: raw data; X: mean of raw data; σX: standard deviation of raw data; ∆ApEn: difference in approximate entropy (raw vs. standardized); ∆SampEn: difference in sample entropy (raw vs. standardized); sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Entropy 2020, 22, x FOR PEER REVIEW 17 of 20 Abbreviations: X: raw data; X: mean of raw data; σX: standard deviation of raw data; ∆ApEn: difference in approximate entropy (raw vs. standardized); ∆SampEn: difference in sample entropy (raw vs. standardized); sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb oxygenated hemoglobin concentration; HHb deoxygenated hemoglobin concentration; TSI: tissue saturation index. Entropy 2020, 22, x FOR PEER REVIEW 17 of 20 Appendix B py , , Appendix B Figure A1. Results from robust multivariate linear regression showing beta coefficients (β) and 95% confidence intervals (CI) for both approximate entropy (ApEn) and sample entropy (SampEn) with the embedded dimension (m) set to 2 (as reported in the main manuscript), 3, and 4. All models adjusted for age, sex, education, diabetes, number of cardiovascular conditions, antihypertensive medication use, alcohol consumption habits, smoking, and depression. Model for active stand data additionally controlled for stand time. Abbreviations: sBP: systolic blood pressure; dBP: diastolic blood pressure; MAP: mean arterial pressure; HR: heart rate; O2Hb: oxygenated hemoglobin concentration; HHb: deoxygenated hemoglobin concentration; TSI: tissue saturation index. References Figure A1. 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Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage
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RESEARCH ARTICLE Tibor T. Glant1, Timea Ocsko2, Adrienn Markovics2, Zoltan Szekanecz3, Robert S. Katz4, Tibor A. Rauch2, Katalin Mikecz1* 1 Section of Molecular Medicine, Departments of Orthopedic Surgery, Biochemistry and Internal Medicine, Rush University Medical Center, Chicago, Illinois, 60612, United States of America, 2 Department of Orthopedic Surgery, Rush University Medical Center, Chicago, Illinois, 60612, United States of America, 3 Department of Rheumatology, Institute of Medicine, University of Debrecen, Faculty of Medicine, Debrecen, H-4012, Hungary, 4 Rheumatology Associates, Rush University Medical Center, Chicago, Illinois, 60612, United States of America * Katalin_Mikecz@rush.edu * Katalin_Mikecz@rush.edu OPEN ACCESS Citation: Glant TT, Ocsko T, Markovics A, Szekanecz Z, Katz RS, Rauch TA, et al. (2016) Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage. PLoS ONE 11(3): e0150784. doi:10.1371/journal. pone.0150784 Citation: Glant TT, Ocsko T, Markovics A, Szekanecz Z, Katz RS, Rauch TA, et al. (2016) Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage. PLoS ONE 11(3): e0150784. doi:10.1371/journal. pone.0150784 Citation: Glant TT, Ocsko T, Markovics A, Szekanecz Z, Katz RS, Rauch TA, et al. (2016) Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage. PLoS ONE 11(3): e0150784. doi:10.1371/journal. pone.0150784 Methods CitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and West- ern blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoanti- bodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by National Institutes of Health/National Institute of Arthritis, Musculoskeletal and Skin Diseases. R01 AR064206 for KM and R01 AR059356 for TTG. The study was supported in part by the J.O. Galante MD, DMSc Endowment Chair of Orthopedic Surgery (Rush University Medical Center, Chicago, IL, USA) for TTG, and by The Grainger Foundation (Lake Forest, IL, USA) for TTG and KM. The funders had no role in Background Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endoge- nous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromole- cules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA. Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Received: October 22, 2015 Accepted: February 17, 2016 Published: March 4, 2016 Copyright: © 2016 Glant et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Editor: Peter Szodoray, Institute of Immunology, Rikshospitalet, NORWAY Received: October 22, 2015 Accepted: February 17, 2016 Published: March 4, 2016 Copyright: © 2016 Glant 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: © 2016 Glant 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. Characterization and Localization of Citrullinated Proteoglycan Aggrecan in Human Articular Cartilage Tibor T. Glant1, Timea Ocsko2, Adrienn Markovics2, Zoltan Szekanecz3, Robert S. Katz4, Tibor A. Rauch2, Katalin Mikecz1* Introduction Rheumatoid arthritis (RA) is an autoimmune disease of the synovial joints causing chronic inflammation and profound tissue destruction in affected patients. The pathological features of RA include infiltration of the joints by inflammatory cells and formation of invasive synovial pannus, ultimately resulting in cartilage and bone erosion and loss of joint function [1][2]. The autoimmune character of RA is underscored by prominent production of autoantibodies (autoAbs) such as those against IgG (rheumatoid factor, RF), and a broad array of joint tissue- specific and other endogenous citrullinated proteins [3][4][5]. Citrullination is a post-translational protein modification catalyzed by peptidyl arginine dei- minase (PAD) enzymes, resulting in the conversion of protein-bound arginine to citrulline. Among PAD enzymes, PAD4 has been implicated in physiological processes such as the nor- mal regulation of gene expression via citrullination of histones as well as in autoimmunity by generating autoantigens (neoepitopes) through citrullination of self-proteins in RA [6][7]. Anti-citrullinated protein Abs (ACPA) can be detected in the serum of an even higher propor- tion of RA patients than RF [3][4][8], and ACPA positivity is employed as a diagnostic and prognostic tool for this disease [4][8][9][10]. The serum ACPA-reactive proteins identified thus far include citrullinated filaggrin, fibrin- ogen, vimentin, type II collagen (CII), α-enolase, and a few viral antigens (reviewed in [5] [6] [7][8][10]). Previous studies have described T-cell reactivity with citrullinated proteoglycan (PG) aggrecan peptides in RA patients [11][12][13] and one group reported the presence of PG G1 domain-specific autoAbs in RA synovial fluid (SF) [14]. However, PG-specific ACPA have not been described, and it is not known if cartilage PG undergoes citrullination in vivo. Citrullinated proteins and PAD4 enzyme have been identified in rheumatoid synovial tissue [15][16]. In addition, elevated concentrations of ACPA in the SF relative to the serum level in the same RA patients suggest that SF ACPA (reactive with multiple citrullinated proteins) might be preferentially retained or locally produced in the joint [17][18][19]. The citrullinated proteins within joint tissues provide obvious targets for ACPA, leading to immune complex formation [20]. As complement-fixing Abs/immune complexes can trigger inflammatory cell recruitment [6][21], ACPA have a significant potential to initiate inflammation or amplify the inflammatory cascade in the RA joint. We found high ACPA levels in the sera of mice immunized with cartilage PG (PG-induced arthritis, PGIA) [22], but not in non-immunized BALB/c mice or in those immunized with human CII. Conclusions CitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trig- ger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: Ab, antibody; ACPA, anti-citrullinated protein Ab; CCP, citrullinated cyclic peptide; CII, type II collagen; CitCII, citrullinated CII; CitPG, citrullinated PG; hG1, G1 domain of human proteoglycan (aggrecan); mAb, monoclonal Ab; MCV, mutated citrullinated vimentin; PBS, phosphate-buffered saline; PG, proteoglycan (aggrecan); OA, osteoarthritis; RA, rheumatoid arthritis; rhG1, recombinant G1 domain of human cartilage PG aggrecan; RF, rheumatoid factor; SF, synovial fluid. Findings Sera from ACPA+ RA patients reacted with PG purified from normal human cartilage speci- mens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ 1 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage. study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Human cartilage and serum samples PG aggrecan molecules (PG monomers) were isolated from normal human cartilage and puri- fied by repeated cesium chloride (CsCl) gradient ultracentrifugation as previously described [23][24]. We used “archived” samples of PG monomers purified from knee cartilage of adult organ donors (with no history of joint disease and having macroscopically normal-looking car- tilage). CII was also isolated from normal human cartilage by limited pepsin digestion and NaCl precipitation as described [25]. Purified PG and CII were stored at -80°C under nitrogen. PG aggrecan molecules (PG monomers) were isolated from normal human cartilage and puri- fied by repeated cesium chloride (CsCl) gradient ultracentrifugation as previously described [23][24]. We used “archived” samples of PG monomers purified from knee cartilage of adult organ donors (with no history of joint disease and having macroscopically normal-looking car- tilage). CII was also isolated from normal human cartilage by limited pepsin digestion and NaCl precipitation as described [25]. Purified PG and CII were stored at -80°C under nitrogen. Cartilage tissue was obtained from patients undergoing knee joint replacement surgery. Written informed consent was obtained from each patient before their participation, and carti- lage specimens were provided through the Orthopedic Information, Tissue and Implant Repository Study approved by the Institutional Review Board (IRB) of Rush University Medi- cal Center Chicago, IL (IRB approval number L00011021). These knee cartilage samples were pulverized in liquid nitrogen and crude extracts were prepared with 4.0 M guanidine hydro- chloride in the presence of protease inhibitors [26][27]. The samples were dialyzed, analyzed for PG content [26][27] and stored at -80°C. Cartilage tissue was obtained from patients undergoing knee joint replacement surgery. Written informed consent was obtained from each patient before their participation, and carti- lage specimens were provided through the Orthopedic Information, Tissue and Implant Repository Study approved by the Institutional Review Board (IRB) of Rush University Medi- cal Center Chicago, IL (IRB approval number L00011021). These knee cartilage samples were pulverized in liquid nitrogen and crude extracts were prepared with 4.0 M guanidine hydro- chloride in the presence of protease inhibitors [26][27]. The samples were dialyzed, analyzed for PG content [26][27] and stored at -80°C. Peripheral blood was obtained from RA patients (84 ACPA-positive and 20 ACPA-nega- tive) and 16 healthy volunteers at Rheumatology Associates, Rush University Medical Center, and at the Department of Rheumatology, Faculty of Medicine, University of Debrecen, Hun- gary. Human cartilage and serum samples All subjects provided their written informed consent to participate in this study. Collec- tion of blood from the study participants at these two rheumatology clinics was approved by the IRB of Rush University Medical Center (approval number L89050101) and the Ethics Committee of the Faculty of Medicine, University of Debrecen (approval number 29643/2012/ EKU), respectively. All RA patients fulfilled the ACR 1987 RA classification criteria [28], 84 of them were positive for serum ACPA (>25 U/ml) and had a disease duration of 10.56 ± 0.88 years (mean ± SEM). None of the RA patients received B-cell depletion therapy such as Rituximab. Introduction As these mice were injected with human cartilage PG aggrecan (henceforth PG) emulsified in a synthetic (protein-free) adjuvant, we suspected that the PG used for 2 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage immunization had already contained citrullinated molecules. Thus, we sought to detect citrul- line in PG isolated and purified from normal articular cartilage specimens. Indeed, we found that some samples of PG purified from normal adult cartilage contained citrulline residues, as the sera from ACPA+ RA patients reacted with these PG samples. Moreover, we could identify citrullinated (ACPA-reactive) PG epitopes in crude extracts and tissue sections of cartilage obtained from osteoarthritic (OA) and RA joints and most of the citrulline residues were located in the G1 domain of the core protein of cartilage PG aggrecan. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Dot blot, gel electrophoresis and Western blot Dot blots were performed using a 96-well transfer system with nitrocellulose membranes (Bio- Rad, Hercules, CA). Dots of purified PG, CII or crude cartilage extracts as well as serum IgG controls were applied to the membranes. Dotted membrane strips were probed with ACPA + or ACPA- human serum (1:2,000 dilution), followed by horseradish peroxidase (HRP)-con- jugated anti-human IgG (1:5,000 dilution). Control probes included anti-IgG Abs, or PG- or CII-specific monoclonal Abs (mAbs) (1:5,000–1:10,000 dilutions). For electrophoresis, samples (30 μg protein/lane) were loaded onto 8% sodium dodecyl-sulfate polyacrylamide gels (SDS-PAGE), and stained with 0.1% toluidine blue (for glycosaminoglycan, GAG) or Coomas- sie blue (for proteins). To facilitate the entry of macromolecules into the gels and subsequent resolution of protein bands, the chondroitin sulfate (CS) GAG side chains of PG were removed (truncated) by digestion with chondroitinase ABC (Seikagaku America, East Falmouth, MA) as previously described [23]. Equal amounts of protein (5 μg/lane) of the deglycosylated OA cartilage extract were loaded onto 8% SDS-PAGE and transferred onto nitrocellulose mem- brane. Vertical strips of the membrane were probed with ACPA+ or ACPA- human serum, or with mAbs to hPG G1- (G18) [22] or G3- (LEC-7, Acris Antibodies, San Diego, CA) domain, mAbs to hCII and C4S (BE123, Chemicon International Temecula, CA), or with rabbit Abs to enzyme-generated -VDIPEN and -NITEGE neoepitopes [29]. The Ab-stained dots or bands were visualized with HRP-labeled second-step antibodies and enhanced chemiluminescence (Luminol enhancer solution, Amersham/GE Healthcare Life Sciences, Piscataway, NJ). Assaying ACPA levels in human serum The ACPA levels in serum samples of the 84 ACPA+ and 20 ACPA- RA patients (and healthy controls) were re-assessed simultaneously using enzyme-linked immunosorbent assay (ELISA) kits for anti-mutated citrullinated vimentin (MCV, Antibodies-online, Inc., Atlanta, GA) and anti-citrullinated cyclic peptide (CCP3 kit, Inova Diagnostics, San Diego, CA). The sera were assayed at 1:100 and 1:500 dilutions, and the assays were performed according to the manufac- turers’ instructions. We selected 2 RA serum samples (ACPA+#9 and ACPA+#20) with high anti-MCV and anti-CCP3 titers (both ~1100 U/ml) to detect citrullinated proteins by dot blot, Western blot, and immunohistochemistry. As an additional positive control for dot blot, we also used the “Calibrator A” component, a serum standard (pre-diluted to 250 U/ml) supplied with the anti-CCP3 kit. ACPA-negative (<20 U/ml) normal human serum samples served as negative controls in different immunoassays. 3 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Removal of contaminating serum IgG from the cartilage extracts and immunodepletion of the G1 domain While the highly purified PG samples were free of serum proteins, the crude extracts of human cartilage specimens were contaminated with human serum components. Therefore, it was nec- essary to remove serum IgG contamination from the cartilage extracts prior to using them for dot blot or Western blot. The crude extracts were first incubated with purified goat anti- human IgG (Invitrogen). The IgG-anti-IgG immune complexes were then removed by repeated incubation with Protein G-Sepharose CL4B until the HRP-conjugated goat anti- human IgG antibody no longer stained the cartilage extract on the membrane. A similar method was employed to deplete the G1 domain-containing PG fragments in the crude extracts by immune absorption using the anti-G1 mAb (G18) followed by incubation with Pro- tein G-Sepharose CL4B. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Immunohistochemistry Cryostat sections of knee cartilage (tibial plateaus of OA and RA patients) were fixed in cold methanol for 5 min, blocked with 1% normal goat serum at room temperature for 1 hour, and incubated with ACPA+ or ACPA- human sera at 1:50–1:100 dilutions. After extensive washing in phosphate-buffered saline (PBS; pH 7.4), sections were incubated with Texas Red (TR)-con- jugated goat anti-human IgG or biotinylated anti-human G1 mAb (G18) followed by Alexa Fluor (AF)488-labeled streptavidin, and mounted in DAPI-containing VectashieldR Hard SetTM mounting medium (Vector Laboratories, Burlingame, CA). ACPA- sera or non-biotiny- lated G18 mAb and fluorochrome-labeled reagents (without primary Abs) were used as con- trols. Fluorescence staining was examined and images were created using a Nikon FXA epifluorescence microscope (Nikon, Melville, NY) equipped with a digital color camera and MetaMorph image processing program (Meta Imaging Series, Universal Imaging Corporation, Downingtown, PA). 4 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Three-dimensional (3-D) molecular modeling of the G1 domain of human PG The theoretical structure of the G1 domain was assembled via protein homology modeling using the SWISS-MODEL Workspace program (http://www.swissmodel.expasy.org). The molecular coordinate files of the Aloop, Bloop and B’loop of the G1 domain were created based on the SWISS-MODEL repository templates 1qz1A, 1o7bT, and 1pozA, respectively [33][34]. The 3D structures of the protein were visualized and the arginine residues were highlighted using RasMol software (http://www.RasMol.org) [35]. Statistical analysis The statistical analysis of data was performed using GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Pearson’s test was employed to determine the correlation coefficients (r), and the best-fit curves were created using linear regression. P values of <0.05 were consid- ered significant. In vitro citrullination of the rhG1 domain and human CII, and detection of anti-CitPG and anti-CitCII Abs by ELISA Purified rhG1 protein (2.5 mg) was dissolved in citrullination buffer (0.1 M Tris-HCl [pH 7.6] containing 10 mM CaCl2 and 5 mM dithiothreitol) [30]. Lyophilized human CII [25] was first dissolved in 0.1 M acetic acid (5 mg/ml) and dialyzed exhaustively against the citrullination buffer. Proteins were incubated with rabbit skeletal muscle PAD4 (Sigma-Aldrich), at a con- centration of 4 U/mg protein for 4 hours at 37°C. Citrullination was terminated by addition of 20 mM EDTA and the material was dialyzed successively against 10 mM Tris-HCl buffer con- taining 5 mM EDTA (pH 7.6) at 4°C. Control (non-citrullinated) rhG1 and CII proteins were treated identically, except that no PAD4 was added. The effect of PAD4 treatment (citrullination status) on rhG1 and CII was detected using ACPA+ serum or ACPA-independent methods. In brief, untreated and PAD4-treated proteins were labeled with rhodamine-conjugated phenylglyoxal, a citrulline-specific probe (Cayman Chemical, Ann Arbor, Michigan), according to a protocol described in [31] before SDS-PAGE, and fluorescent bands were detected with a Bio-Rad ChemiDoc imaging system (Bio-Rad, Her- cules, CA). Another method of citrullinated protein detection involved the use of an anti-modi- fied citrulline detection kit, following the manufacturer’s instructions (EMD Millipore, Billerica, MA) [32]. The optimal coating concentrations for ELISA were 0.2 μg CitPG or rhG1 (non-CitPG), and 0.5 μg CitCII or non-citrullinated CII per well. Sera from RA patients were diluted to 1:100 and 1:500, and 100 μl of the serum samples were incubated with the immobilized antigens at room temperature for 1 hour, followed by washing and incubation with HRP-conjugated goat anti- human IgG for 1 hour. The color reaction was developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BD Biosciences, San Diego, CA). Optical density (OD) values (at 450 nm) of non-citrullinated proteins (PG and CII) were subtracted from those of CitPG or CitCII, respec- tively. The specific reactivity of the sera with CitPG or CitCII was expressed as ΔOD. Citrullinated Proteoglycan in Human Cartilage Fig 1. Recognition of proteoglycan (PG) aggrecan purified from normal human cartilage by ACPA-positive human sera. (A-C) Dots of human PG aggrecan that was purified from 6 normal cartilage samples (1–6) were applied to nitrocellulose membranes (upper dots: 2 μg, lower dots: 0.2 μg PG) along with various control IgGs purified from human (Hu), rabbit (Rb) or mouse (Mo) serum (upper dots: 20 ng, lower dots: 2 ng IgG) and human type II collagen (hCII) purified from normal cartilage (upper dot: 10 μg, lower dot: 1 μg hCII). The membranes were subjected to immunostaining with ACPA-positive sera including the (A) “Calibrator” serum from the anti-CCP3 assay kit and (B, C) sera from two ACPA+ RA patients (#20 and #9), followed by horseradish peroxidase (HRP)-labeled anti-human IgG. (D-G) Specificity controls included blotting with (D) a biotinylated monoclonal antibody (mAb) specific to human PG G1 domain (anti-hG1-biot) followed by streptavidin (SA)-HRP, (E) ACPA- serum followed by anti-human IgG-HRP, (F) anti-rabbit IgG-HRP, and (G) mouse antibody to hCII (anti-hCII) followed by anti-mouse IgG-HRP. Fig 1. Recognition of proteoglycan (PG) aggrecan purified from normal human cartilage by ACPA-positive human sera. (A-C) Dots of human PG aggrecan that was purified from 6 normal cartilage samples (1–6) were applied to nitrocellulose membranes (upper dots: 2 μg, lower dots: 0.2 μg PG) along with various control IgGs purified from human (Hu), rabbit (Rb) or mouse (Mo) serum (upper dots: 20 ng, lower dots: 2 ng IgG) and human type II collagen (hCII) purified from normal cartilage (upper dot: 10 μg, lower dot: 1 μg hCII). The membranes were subjected to immunostaining with ACPA-positive sera including the (A) “Calibrator” serum from the anti-CCP3 assay kit and (B, C) sera from two ACPA+ RA patients (#20 and #9), followed by horseradish peroxidase (HRP)-labeled anti-human IgG. (D-G) Specificity controls included blotting with (D) a biotinylated monoclonal antibody (mAb) specific to human PG G1 domain (anti-hG1-biot) followed by streptavidin (SA)-HRP, (E) ACPA- serum followed by anti-human IgG-HRP, (F) anti-rabbit IgG-HRP, and (G) mouse antibody to hCII (anti-hCII) followed by anti-mouse IgG-HRP. doi:10.1371/journal.pone.0150784.g001 (collected from 6 normal adult cartilage samples, age range 26-76-years) [23][24] along with control samples including purified human, rabbit, and mouse IgGs as well as hCII to a nitrocel- lulose membrane (Fig 1). The calibrator serum from CCP3 ELISA kit reacted with 2 of the 6 PG dots (Fig 1A), whereas both ACPA+#20 and ACPA+#9 sera recognized 4 of the 6 PG sam- ples (Fig 1B and 1C). None of the ACPA+ sera reacted with hCII, and the secondary Ab was positive only with human IgG (Figs 1A–1C). When the membrane was probed with the hG1-specific mAb G18 [22], all of the PG samples, but not the control IgGs or hCII, were rec- ognized by this mAb (Fig 1D). Importantly, an ACPA- serum did not react with any of the PG or other control dots. Specificity controls (human, mouse, rabbit IgGs) used in subsequent experiments reacted only with the specific antibodies (Fig 1D–1G). CitPG (Cit-Aggrecan) is detected in normal adult human cartilage by ACPA+ sera The intact PG monomers in normal cartilage are very large molecules (~ 2x106 Da) and cannot be resolved in SDS-PAGE. Therefore, we applied dots of highly purified PG aggrecan molecules 5 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Fig 2. Recognition of PG in crude extracts of osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage specimens by ACPA and PG-specific antibodies. Dots of crude extracts of cartilage from OA donors (row A, samples 1–5) and RA donors (samples 6–10) were applied to a nitrocellulose membrane strip. Purified human PG and CII (far left and far right dots, respectively) served as controls. Blotting with the secondary antibody (anti-human IgG-HRP) revealed positive reactions (row A) with all crude extracts, but not with purified hPG or hCII. The “positive” reaction disappeared after the contaminating IgG was removed from the crude extracts (row B). These IgG-free cartilage extracts were used for subsequent dot blots. The ACPA+#20 serum (row A) reacted with all cartilage extracts and purified PG (row C). Similarly, the G1 domain-specific mAb recognized purified PG and PG in the crude extracts (row D), but no reaction was detected when the G1 domain was removed by immune absorption (row E). The reactivity of the cartilage extracts with ACPA+#20 serum (row F) was nearly completely lost after G1 domain immunodepletion (row G). As demonstrated by the anti-chondroitin 4-sulfate (C4S)- specific antibody, PG in all extracts and in the purified PG sample were glycosylated (row H). Some extracts and the purified PG contained small amounts of the PG G3 domain (row I), and all crude extracts contained cartilage-specific CII (row J). doi:10 1371/journal pone 0150784 g002 Fig 2. Recognition of PG in crude extracts of osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage specimens by ACPA and PG-specific antibodies. Dots of crude extracts of cartilage from OA donors (row A, samples 1–5) and RA donors (samples 6–10) were applied to a nitrocellulose membrane strip. Purified human PG and CII (far left and far right dots, respectively) served as controls. Blotting with the secondary antibody (anti-human IgG-HRP) revealed positive reactions (row A) with all crude extracts, but not with purified hPG or hCII. The “positive” reaction disappeared after the contaminating IgG was removed from the crude extracts (row B). These IgG-free cartilage extracts were used for subsequent dot blots. The ACPA+#20 serum (row A) reacted with all cartilage extracts and purified PG (row C). Similarly, the G1 domain-specific mAb recognized purified PG and PG in the crude extracts (row D), but no reaction was detected when the G1 domain was removed by immune absorption (row E). The reactivity of the cartilage extracts with ACPA+#20 serum (row F) was nearly completely lost after G1 domain immunodepletion (row G). As demonstrated by the anti-chondroitin 4-sulfate (C4S)- specific antibody, PG in all extracts and in the purified PG sample were glycosylated (row H). Some extracts and the purified PG contained small amounts of the PG G3 domain (row I), and all crude extracts contained cartilage-specific CII (row J). doi:10.1371/journal.pone.0150784.g002 including IgG, as the dots of all cartilage extracts gave positive reaction with anti-human IgG-HRP Ab (Fig 2, top row A). The crude cartilage extracts of OA and RA patients were sub- jected to immune absorption with goat anti-human IgG-Protein G-Sepharose, which resulted in the removal of serum IgG contamination (Fig 2, row B). All 10 pre-absorbed extracts (and purified human PG, but not purified human CII) reacted with the ACPA+ serum (Fig 2. row C). Importantly, all samples (and purified PG) also reacted with the G18 anti-hG1 mAb (Fig 2, row D). The reaction was specific, as the G18 mAb did not react after G1 depletion (Fig 2, row E). The ACPA+ RA serum stained the purified human PG dots as well as all of the OA and RA cartilage extracts, suggesting that all these samples contained citrullinated PG molecules (Fig 2, row F). This positive staining with the ACPA+ serum disappeared after removal of the G1 domain by immune absorption (Fig 2, row G) indicating that most, if not all, of the citrulline residues were in the N-terminal G1 domain of PG or G1 domain-containing PG fragments. The PG molecules in all tested samples had chondroitin sulfate side chains or stubs as indicated by positive reactions with an anti-C4S mAb (Fig 2, row H). A few samples also contained the C-terminal G3 domain of PG (Fig 2, row I). Finally, all crude cartilage extracts, but not purified PG, contained CII, as they gave positive reaction with the anti-hCII mAb (Fig 2, row J). Collec- tively, these data suggested that crude extracts of OA and RA cartilage specimens contained including IgG, as the dots of all cartilage extracts gave positive reaction with anti-human IgG-HRP Ab (Fig 2, top row A). The crude cartilage extracts of OA and RA patients were sub- jected to immune absorption with goat anti-human IgG-Protein G-Sepharose, which resulted in the removal of serum IgG contamination (Fig 2, row B). CitPG is also present in cartilage extracts from OA and RA patients We hypothesized that if PG citrullination occurs in macroscopically intact (normal) human cartilage (Fig 1), it should also occur in joint diseases such, as OA or RA. As noted above, due to its large size, the intact cartilage PG aggrecan molecule cannot be resolved by SDS-PAGE, unless the GAG side chains are removed or the core protein is degraded, or both. Although limited core protein degradation occurs during the normal turnover of PG, the core protein is degraded extensively in diseased OA or even more in RA cartilage [36]. Unlike intact PG from normal cartilage, PG fragments present in OA or RA cartilage cannot be separated or purified from other proteins by CsCl gradient ultracentrifugation. Therefore, we used crude extracts of RA and OA knee cartilage specimens (without any purification) to identify citrullinated PG by dot blots. However, these crude extracts were contaminated with human serum proteins PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 6 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 To identify these bands, replicate strips of the membrane were probed with antibodies against the G1 or G3 domain of PG and a pair of antibodies recognizing protease-generated PG neoepitopes. The respective antibodies showed reactions with the G1 (lane 4), G3 (lane 5) as well as with the neoepitopes -NITEGE and -VDIPEN (lane 6). There were additional bands above 55 kDa (depicted with asterisks in lane 3) that could not be identified as PG fragments. One representative sample of over 10 Western blots (using different crude extracts and ACPA+ sera) is shown. Fig 3. Western blots to identify PG domains and fragments of OA cartilage extract recognized by ACPA-positive serum. The crude extract of OA cartilage (shown as sample 4 in Fig 2) was loaded onto 8% SGS-PAGE gel and stained with toluidine blue (TB) to visualize PG GAG chains. Essentially all of the TB- positive PG material remained in the stacking gel. To facilitate resolution, chondroitin sulfate chains were removed by digestion with chondroitinase ABC, and aliquots of the deglycosylated extract was loaded onto 6 lanes of a SDS-PAGE gel. Coomassie blue (CB) staining of the gel (lane 1) showed good resolution of the proteins of the OA cartilage extract after deglycosylation. Following transfer onto a nitrocellulose membrane, vertical strips of the membrane were probed with human sera or PG-specific antibodies (lanes 2–6). Immunostaining with ACPA- (normal) serum followed by anti-human IgG-HRP revealed a single protein band most likely corresponding to the heavy chain of contaminating IgG (lane 2). The ACPA+ serum detected several additional bands (lane 3). To identify these bands, replicate strips of the membrane were probed with antibodies against the G1 or G3 domain of PG and a pair of antibodies recognizing protease-generated PG neoepitopes. The respective antibodies showed reactions with the G1 (lane 4), G3 (lane 5) as well as with the neoepitopes -NITEGE and -VDIPEN (lane 6). There were additional bands above 55 kDa (depicted with asterisks in lane 3) that could not be identified as PG fragments. One representative sample of over 10 Western blots (using different crude extracts and ACPA+ sera) is shown. doi:10.1371/journal.pone.0150784.g003 doi:10.1371/journal.pone.0150784.g003 citrullinated PG, and that the majority of the citrulline residues were located within the G1 domain of the PG molecule. To corroborate these results, we sought to identify citrullinated G1 domain (and perhaps other citrullinated fragments) of the PG molecule by SDS-PAGE and Western blotting. All 10 pre-absorbed extracts (and purified human PG, but not purified human CII) reacted with the ACPA+ serum (Fig 2. row C). Importantly, all samples (and purified PG) also reacted with the G18 anti-hG1 mAb (Fig 2, row D). The reaction was specific, as the G18 mAb did not react after G1 depletion (Fig 2, row E). The ACPA+ RA serum stained the purified human PG dots as well as all of the OA and RA cartilage extracts, suggesting that all these samples contained citrullinated PG molecules (Fig 2, row F). This positive staining with the ACPA+ serum disappeared after removal of the G1 domain by immune absorption (Fig 2, row G) indicating that most, if not all, of the citrulline residues were in the N-terminal G1 domain of PG or G1 domain-containing PG fragments. The PG molecules in all tested samples had chondroitin sulfate side chains or stubs as indicated by positive reactions with an anti-C4S mAb (Fig 2, row H). A few samples also contained the C-terminal G3 domain of PG (Fig 2, row I). Finally, all crude cartilage extracts, but not purified PG, contained CII, as they gave positive reaction with the anti-hCII mAb (Fig 2, row J). Collec- tively, these data suggested that crude extracts of OA and RA cartilage specimens contained 7 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Fig 3. Western blots to identify PG domains and fragments of OA cartilage extract recognized by ACPA-positive serum. The crude extract of OA cartilage (shown as sample 4 in Fig 2) was loaded onto 8% SGS-PAGE gel and stained with toluidine blue (TB) to visualize PG GAG chains. Essentially all of the TB- positive PG material remained in the stacking gel. To facilitate resolution, chondroitin sulfate chains were removed by digestion with chondroitinase ABC, and aliquots of the deglycosylated extract was loaded onto 6 lanes of a SDS-PAGE gel. Coomassie blue (CB) staining of the gel (lane 1) showed good resolution of the proteins of the OA cartilage extract after deglycosylation. Following transfer onto a nitrocellulose membrane, vertical strips of the membrane were probed with human sera or PG-specific antibodies (lanes 2–6). Immunostaining with ACPA- (normal) serum followed by anti-human IgG-HRP revealed a single protein band most likely corresponding to the heavy chain of contaminating IgG (lane 2). The ACPA+ serum detected several additional bands (lane 3). PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 However, PG fragments in crude extracts of OA cartilage were still too large to enter the running gel (Fig 3, lane “TB”: toluidine blue-stained for GAG content). Deglycosylation of the PG (removal of GAG chains) of the OA cartilage extract provided good resolution on SDS-PAGE (Fig 3, lane “CB”: Coomassie blue-stained for proteins). Aliquots of this OA extract were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. As shown in the other lanes of Fig 3, the normal (ACPA-) human serum did not stain the OA extract, and only some residual contaminating IgG was recognized by the secondary anti- human IgG Ab (lane 2). In contrast, several protein bands were observed after blotting with the ACPA+ RA serum (lane 3). Using PG domain- and neoepitope-specific antibodies, the bands recognized by the ACPA+ serum (lane 3) could be identified as citrullinated PG frag- ments containing the G1 domain (lane 4), the G3 domain (lane 5) and G1-associated frag- ments with protease cleavage sites (lane 6). Four of the bands stained with the ACPA+ serum (lane 3, depicted by ) could not be identified as either G1-, interglobular- or G3 domain-containing fragments. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 8 / 16 Citrullinated Proteoglycan in Human Cartilage Fig 4. Immunohistochemical localization of ACPA-reactive (citrullinated) epitopes in OA and RA cartilage sections. (A-D) Frozen sections of OA knee (tibial plateau) cartilage were immunostained with (A) Texas red-labeled anti-human IgG (anti-hIgG-TR), (B) normal (ACPA-negative) human serum followed by anti-hIgG-TR, (C) ACPA+ serum (RA#9) followed by anti-hIgG-TR, or (D) a biotinylated anti-human G1 antibody followed by Alexa Fluor 488-labeled streptavidin (SA-AF488). (E-H) Frozen sections prepared from the RA tibial plateau cartilage were immunostained with the same sera and antibodies as listed for A-D. Cell nuclei in all sections were visualized by DAPI staining. (A and E) Anti-hIgG-TR alone did not stain the sections, and (B and F) negligible reaction (red fluorescence) was observed when the tissues were first stained with ACPA- serum. (C) The ACPA+ serum primarily stained the chondrocyte pericellular matrix in the OA cartilage, but (G) it diffusely stained the entire matrix of the RA cartilage. Similar staining patters to those with ACPA + serum were observed when (D) the OA and (H) RA cartilage sections were incubated with biotinylated anti-hG1 mAb (green fluorescence), suggesting at least partial co-localization of PG G1 and citrullinated epitopes in both OA and RA cartilage. Fig 4. Immunohistochemical localization of ACPA-reactive (citrullinated) epitopes in OA and RA cartilage sections. (A-D) Frozen sections of OA knee (tibial plateau) cartilage were immunostained with (A) Texas red-labeled anti-human IgG (anti-hIgG-TR), (B) normal (ACPA-negative) human serum followed by anti-hIgG-TR, (C) ACPA+ serum (RA#9) followed by anti-hIgG-TR, or (D) a biotinylated anti-human G1 antibody followed by Alexa Fluor 488-labeled streptavidin (SA-AF488). (E-H) Frozen sections prepared from the RA tibial plateau cartilage were immunostained with the same sera and antibodies as listed for A-D. Cell nuclei in all sections were visualized by DAPI staining. (A and E) Anti-hIgG-TR alone did not stain the sections, and (B and F) negligible reaction (red fluorescence) was observed when the tissues were first stained with ACPA- serum. (C) The ACPA+ serum primarily stained the chondrocyte pericellular matrix in the OA cartilage, but (G) it diffusely stained the entire matrix of the RA cartilage. Similar staining patters to those with ACPA + serum were observed when (D) the OA and (H) RA cartilage sections were incubated with biotinylated anti-hG1 mAb (green fluorescence), suggesting at least partial co-localization of PG G1 and citrullinated epitopes in both OA and RA cartilage. doi:10.1371/journal.pone.0150784.g004 Immunolocalization of CitPG epitopes in tissue sections from OA and RA cartilage specimens Incubation of OA and RA cartilage sections with anti-human IgG-Texas Red (anti-hIgG-TR, Fig 4A and 4E) or with normal (ACPA-) human serum, followed by anti-hIgG-TR (Fig 4B and 4F) resulted in some background staining, likely due to the presence of trace amounts of human IgG in these cartilage specimens. When ACPA+ human serum was used as a source of primary Ab, strong positive staining was observed in both the OA and RA cartilage sections (Fig 4C and 4G). ACPA positivity was primarily observed in the intra- and pericellular areas of the OA cartilage (Fig 4C), whereas it was detected throughout the entire matrix in the RA carti- lage (Fig 4G). Immunohistochemistry on neighboring sections of the same OA and RA speci- mens with G18 (anti-hG1) mAb resulted in staining patterns (Fig 4D and 4H) similar to those obtained with the ACPA+ serum (Fig 4C and 4G). Potential sites of citrullination within the G1 domain of human PG and detection of citrullinated rhG1 by ACPA and other probes Potential sites of citrullination within the G1 domain of human PG and detection of citrullinated rhG1 by ACPA and other probes The G1 domain of human PG contains 23 arginine residues (S1 Fig) that can be potentially converted to citrulline by PAD enzymes. Molecular modeling revealed that most arginine resi- dues were displayed on the surfaces of the three subdomains/loops (A, B, and B’) of the G1 9 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Fig 5. Positions of arginine residues within the three-dimensional structure of the human G1 domain and detection of citrullinated rhG1. (A) 3-D images of the A, B, and B’ loops of the G1 domain illustrate the location of arginine (R) residues (red-yellow balls with numbers) and the two previously reported immunodominant epitopes (sequences highlighted), both of which contain arginine residues that may become citrulline (R/Cit) [11][12][13]. The three loops of G1 were rotated relative to each other using RasMol software in order to expose the R-rich surfaces. The amino acid sequence is shown in S1 Fig. (B) In vitro citrullination of rhG1 was performed using PAD4 enzyme. The native (lanes 1) and PAD4-treated (lanes 2) rhG1 proteins were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. Two of the membranes were probed with either anti-hG1 mAb: (first panel) or with ACPA+ RA serum (second panel). The citrulline residues present in the same proteins on the third membrane were subjected to chemical modification and then probed with an Ab specific for chemically-modified citrulline (anti-modif. Cit Ab, third panel). Native and PAD4-treated rhG1 proteins were also reacted with citrulline-specific phenylglyoxal conjugated with rhodamine (Rhod-Phe-Gly) and subjected to SDS-PAGE (fourth panel). While the anti-hG1 mAb reacted with both the native and citrullinated forms of the protein, only the citrullinated hG1 was detected by ACPA, the anti-modified citrulline Ab, and the phenylglyoxal probe. Fig 5. Positions of arginine residues within the three-dimensional structure of the human G1 domain and detection of citrullinated rhG1. (A) 3-D images of the A, B, and B’ loops of the G1 domain illustrate the location of arginine (R) residues (red-yellow balls with numbers) and the two previously reported immunodominant epitopes (sequences highlighted), both of which contain arginine residues that may become citrulline (R/Cit) [11][12][13]. The three loops of G1 were rotated relative to each other using RasMol software in order to expose the R-rich surfaces. The amino acid sequence is shown in S1 Fig. (B) In vitro citrullination of rhG1 was performed using PAD4 enzyme. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Potential sites of citrullination within the G1 domain of human PG and detection of citrullinated rhG1 by ACPA and other probes The native (lanes 1) and PAD4-treated (lanes 2) rhG1 proteins were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. Two of the membranes were probed with either anti-hG1 mAb: (first panel) or with ACPA+ RA serum (second panel). The citrulline residues present in the same proteins on the third membrane were subjected to chemical modification and then probed with an Ab specific for chemically-modified citrulline (anti-modif. Cit Ab, third panel). Native and PAD4-treated rhG1 proteins were also reacted with citrulline-specific phenylglyoxal conjugated with rhodamine (Rhod-Phe-Gly) and subjected to SDS-PAGE (fourth panel). While the anti-hG1 mAb reacted with both the native and citrullinated forms of the protein, only the citrullinated hG1 was detected by ACPA, the anti-modified citrulline Ab, and the phenylglyoxal probe. doi:10.1371/journal.pone.0150784.g005 doi:10.1371/journal.pone.0150784.g005 doi:10.1371/journal.pone.0150784.g005 domain, theoretically accessible to PADs (Fig 5A). The localization of recently identified T-cell epitopes within the A and B’ loops of the G1 domain is highlighted in Fig 5A. To test the specificity of the PG/G1-reactive ACPA+ RA serum (ACPA+#9) against the citrullinated form of PG, we treated the rhG1 domain of PG [22] with PAD4 enzyme, and then subjected these native and citrullinated rhG1 proteins to Western blotting with anti-hG1 mAb, ACPA+#9 serum, and an Ab to chemically modified citrulline. As an Ab-independent method, we also used a rhodamine-labeled phelylglyoxal probe that specifically reacts with citrulline [31]. While the anti-hG1 mAb reacted with both the native and citrullinated form of rhG1 (Fig 5B, first panel), only the citrullinated protein was recognized by the ACPA+ serum (Fig 5B, sec- ond panel). Citrullination of rhG1 (and the specificity of ACPA) was further confirmed by an ACPA-independent method using an Ab against modified citrulline (Fig 5B, third panel) and by a chemical method using the citrulline-specific phenylglyoxal probe (Fig 5B, fourth panel). 10 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Using CitrhG1 to detect PG-specific ACPA in the sera of RA patients We sought to determine the relative proportions of citrullinated PG (CitrhG1/CitPG)-specific and citrullinated CII (CitCII)-specific ACPA in our collection of 84 RA serum samples (all of which were previously found to be ACPA+). We assayed these samples simultaneously using commercially available ELISA kits for anti-MCV and anti-CCP3, and in-house ELISAs employing PAD4-treated rhG1 (CitPG) and PAD4-treated human CII (CitCII). As seen in Fig 6A, all 84 of the previously tested ACPA+ sera had comparable reactivity with MCV (range: 12–1285 U/ml) and CCP3 (range: 10–1160 U/ml), showing a strong positive correlation (r = 0.95, p < 0.0001). Of note, a few RA samples contained less than 20 U/ml ACPA (the cut- off value for ACPA positivity) in one or both of the ACPA assays. Of the same 84 RA serum samples, 61 (72.6%) reacted with CitPG, 51 (60.7%) reacted with both CitPG and CitCII, and only 23 (27.4%) of the CCP3+ sera were negative for CitPG and/or CitCII (Fig 6B and 6C). There was relatively poor, but significant, correlation (r = 0.54, p < 0.0001) between the anti- CitPG and anti-CCP3 levels (Fig 6B), whereas the anti-CitPG and anti-CitCII ΔOD values showed better correlation (r = 0.64, p < 0.0001) (Fig 6C). Citrullinated Proteoglycan in Human Cartilage Fig 6. Correlations between ACPA of different specificities including citrullinated PG in the sera of RA patients. (A) Concentrations of antibodies to mutated citrullinated vimentin (MCV) and cyclic citrullinated peptides (CCP) were measured in serum samples from 84 RA patients using commercial ELISA kits. Results were expressed as units/ml (U/ml). Correlation analysis revealed strong positive correlation between anti-MCV and anti-CCP3 levels (r = 0.95, R square = 0.9, p<0.0001). (B) Concentrations of anti-CitPG antibodies were measured by in-house ELISA using in vitro citrullinated rhG1 (shown in Fig 5B) as antigen (CitPG). The results were expressed as delta optical density (ΔOD, the OD values of anti-CitPG minus the OD values of anti-PG as described in the Methods). The ΔOD values were correlated with the concentrations (U/ml) of anti-CCP in the same 84 RA serum samples. (C) Purified hCII was also citrullinated by PAD4, and CitCII was used to measure anti-CitCII levels in the 84 RA serum samples by ELISA. The dotted lines indicate the 95% confidence intervals. Fig 6. Correlations between ACPA of different specificities including citrullinated PG in the sera of RA patie s between ACPA of different specificities including citrullinated PG in the sera of RA patients. (A) Concentrat of different specificities including citrullinated PG in the sera of RA patients. (A) Concentrations of antibodies to Fig 6. Correlations between ACPA of different specificities including citrullinated PG in the sera of RA patients. (A) Concentrations of antibodies to mutated citrullinated vimentin (MCV) and cyclic citrullinated peptides (CCP) were measured in serum samples from 84 RA patients using commercial ELISA kits. Results were expressed as units/ml (U/ml). Correlation analysis revealed strong positive correlation between anti-MCV and anti-CCP3 levels (r = 0.95, R square = 0.9, p<0.0001). (B) Concentrations of anti-CitPG antibodies were measured by in-house ELISA using in vitro citrullinated rhG1 (shown in Fig 5B) as antigen (CitPG). The results were expressed as delta optical density (ΔOD, the OD values of anti-CitPG minus the OD values of anti-PG as described in the Methods). The ΔOD values were correlated with the concentrations (U/ml) of anti-CCP in the same 84 RA serum samples. (C) Purified hCII was also citrullinated by PAD4, and CitCII was used to measure anti-CitCII levels in the 84 RA serum samples by ELISA. The dotted lines indicate the 95% confidence intervals doi:10.1371/journal.pone.0150784.g006 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Discussion In this study we demonstrate that ACPA+ sera from RA patients react with human cartilage PG aggrecan. Our investigations were prompted by the observation that immunization of BALB/c mice with purified PG [37] or crude extracts of OA cartilage [26] induced both arthri- tis and abundant production of ACPA [22]. Indeed, we found that ACPA of RA patients reacted with PG fragments of OA and RA cartilage, suggesting that cartilage from diseased joints contained in vivo citrullinated PG molecules. Unexpectedly, ACPA of RA patients also reacted with highly purified PG monomers isolated from visually normal cartilage specimens of adult human subjects. The significance of our observations is three-fold. First, PG can be citrullinated in vivo in the joints of adult human subjects; second, CitPG-specific Abs are pres- ent in the serum of ACPA+ RA patients; and third, CitPG epitopes may also induce ACPA pro- duction in patients with RA. 11 / 16 PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Citrullinated Proteoglycan in Human Cartilage Removal of the G1 domain of human PG by immunodepletion resulted in substantial weak- ening of reactivity indicating a relatively high concentration of citrullinated epitopes located within the G1 region. The 325 amino acid-long G1 domain contains 23 arginine residues, and the G2 and G3 domains contain 13 and 20 arginines, respectively. The linear GAG-attachment domains have only 11 arginine residues. While all 67 arginine residues of PG (56 of which are found in the globular domains) are potential sites of in vivo citrullination by PAD enzymes, steric hindrance by GAG side chains and PG-bound hyaluronan may limit the access of PAD to these sites either within the GAG-attachment domains or in the B loop of the G1 domain [38][39]. Here, we show that ACPA from RA serum preferentially react with G1- and G3-con- taining PG fragments. Identification of citrulline residues within the G1 and G3 domains of human PG and detection of ACPA that preferentially bind to citrulline-containing epitopes (peptides) within these regions, are the subjects of our ongoing investigations. We were able to detect ACPA-reactive epitopes within OA and RA cartilage specimens by immunohistochemistry. While ACPA-reactive regions were restricted to the intra- and pericel- lular compartments of OA cartilage, ACPA reactivity was distributed throughout the entire matrix of RA cartilage. Discussion The similar immunolocalization of PG (G1)-specific and ACPA-reac- tive regions suggested that at least some of the citrullinated epitopes recognized by ACPA are CitPG epitopes in both OA and RA cartilage sections. The wide distribution of citrullinated epitopes within RA cartilage suggests that PADs released from inflammatory cells in synovial fluid or from synovial tissue may gain access to the extensively damaged cartilage matrix in RA joints. In contrast, the intra- and pericellular localization of the citrullinated epitopes in OA cartilage suggests that chondrocyte-derived PADs may contribute to the citrullination of matrix molecules, including PG. Further studies are warranted to determine if PADs produced by chondrocytes are involved in the citrullination of human cartilage matrix molecules. A human PG peptide representing the so-called 5/4E8 epitope (ATEGRVRVNSAYQDK) was found by our group to be a dominant T-cell epitope in PG-immunized arthritic BALB/c mice [38][40][41]. This epitope was also the target of in vitro studies in two independent labo- ratories [11][12] showing that a citrullinated and extended version of the 5/4E8 epitope-con- taining synthetic peptide (VVLLVATEGCitVRVNSAYQDK) elicited strong T-cell responses in RA patients. This citrullinated peptide induced substantial cytokine (IL-17, IL-22, IL-6, TNFα, and IFNγ) production by peripheral blood T cells from the majority of RA patients [11] [12]. T cells from RA patients responded poorly to the native (non-citrullinated) peptide in both studies, and T cells from healthy subjects did not respond at all [11] or responded only to the citrullinated peptide by producing IL-6 [12]. More recently, Aggarwal et al. [13] reported that another citrullinated peptide (AGWLADCitSVRYPI) from human PG induced as much T-cell proliferation as citrullinated vimentin or fibrinogen peptides in a significant number of RA patients. Interestingly, this peptide sequence (in a non-citrullinated form) is included in de Jong’s predicted and tested PG T-cell epitopes in RA patients [42] and was also found to be immunogenic in HLA-DR4 transgenic mice [43]. Regarding the humoral responses, an earlier report [14] identified Abs in RA SF that reacted with human PG. The same study suggested that these Abs in RA SF were specific to the G1 domain of PG [14], but it was not known whether such Abs recognized native or citrullinated epitope(s) within the G1 domain. Using the in vitro citrullinated rhG1 domain of PG and hCII, we were able to detect Abs specific to CitPG and CitCII in the majority of ACPA+ sera from RA patients. PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 Acknowledgments The authors thank Beata Tryniszewska and András Vida for expert technical assistance and the tissue repository research coordinators and nurses for providing the OA and RA cartilage spec- imens and peripheral blood samples from the consenting patients for this study. Supporting Information S1 Fig. Amino acid sequence with the arginine residues and immunodominant epitopes depicted in the recombinant G1 domain of human PG aggrecan. Amino acid sequence of the recombinant hG1 protein containing 329 amino acids of the G1 domain and 44 amino acids of the neighboring interglobular domain of human PG aggrecan. Numbering of amino acids starts after the signal peptide. Most or all of the 24 arginine (R, red font) residues (23 in the G1 domain and 1 in the interglobular region) may be converted to citrulline by peptidyl arginine deiminase (PAD) enzymes. The sequences of two confirmed T-cell epitopes [11][12] [13] within the G1 domain are highlighted in boldface. The 3-dimensional structure of the G1 domain is shown in Fig 5A. Blue fonts depict the neoepitopes generated by stromelysin (VDI- PEN) and aggrecanase (NITEGE) cleavage. The complete amino acid sequence of human PG aggrecan (including the signal peptide and the whole core protein) is found at http://www. uniprot.org/uniprot/P16112#sequences. (TIF) Discussion We found mod- erate but significant correlations of the serum levels of anti-CitPG Abs with the levels of Abs to CCP. The correlation between anti-CitPG and anti-CitCII was higher, suggesting that in vivo citrullination of cartilage matrix molecules may contribute to ACPA production in RA patients. However, both anti-CitPG- and anti-CitCII-specific Abs were present at much lower levels in the patients’ sera than those against MCV or CCP3, indicating that autoAbs directed PLOS ONE | DOI:10.1371/journal.pone.0150784 March 4, 2016 12 / 16 Citrullinated Proteoglycan in Human Cartilage to CitPG and/or CitCII represent a relatively small proportion of the ACPA pool, and may arise as a result of epitope spreading to citrullinated self-proteins during the course of RA [8] [44][45]. To our knowledge, our study is the first to show the presence of ACPA-reactive citrullinated human PG epitopes and to demonstrate humoral immunity against CitPG in RA patients. CitPG (and other citrullinated matrix molecules) in cartilage are likely accessible by ACPA present in the serum and/or SF of RA patients with joint inflammation. Because ACPA is not typically produced by healthy individuals or patients with OA, the presence of CitPG in normal or OA articular cartilage may remain unnoticed by the immune system. As reported by Law et al [12], some healthy individuals carrying the “shared epitope” in the HLA-DR0401 allele exhibit T-cell reaction to an epitope in CitPG, but it is much lower in magnitude than the T- cell response of RA patients to the same epitope. It is likely that, even if T-cell reactivity to CitPG is present in healthy or OA subjects, it does not reach the threshold required for B-cell help and autoAb production, and does not result in pathologic reactions against cartilage in the joints. In contrast, CitPG can become a target of ACPA in RA joints, as CitPG can provoke robust T-cell responses and autoAb production in the case of an insufficiently controlled adap- tive immune system. Although the involvement of ACPA in RA joint pathology is unclear, the arthritogenic potential of ACPA and some citrullinated proteins has been demonstrated in ani- mal models of RA [46][47]. Therefore, it is conceivable that immune complexes formed between CitPG and anti-CitPG Abs are able to trigger local inflammatory reactions, thus con- tributing to the initiation or perpetuation of joint inflammation in ACPA+ RA patients. References 1. Fox DA. Etiology and Pathogenesis of Rheumatoid Arthritis. In: Koopman WJ, Moreland LW, editors. Arthritis and Allied Conditions: A Textbook of Rheumatology. Philadelphia: Lippincott Williams & Wil- kins; 2005. pp. 1085–1102. 2. Firestein GS. Rheumatoid arthritis: Etiology and pathogeneis of rheumatoid arthritis. In: Ruddy S, Harris ED, Sledge CB, Kelley WN, editors. Kelley's Textbook of Rheumatology. Philadelphia, PA: W.B.Saun- ders Co.; 2005. pp. 996–1045. 3. Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibod- ies. J Clin Invest 1998; 101: 273–281 PMID: 9421490 4. Bas S, Perneger TV, Seitz M, Tiercy JM, Roux-Lombard P, Guerne PA. Diagnostic tests for rheumatoid arthritis: comparison of anti-cyclic citrullinated peptide antibodies, anti-keratin antibodies and IgM rheu- matoid factors. Rheumatology (Oxford) 2002; 41: 809–814 5. Trouw LA, Huizinga TW, Toes RE. Autoimmunity in rheumatoid arthritis: different antigens—common principles. Ann Rheum Dis 2013; 72 Suppl 2: ii132–ii136 doi: 10.1136/annrheumdis-2012-202349 PMID: 23253931 6. Wegner N, Lundberg K, Kinloch A, Fisher B, Malmstrom V, Feldmann M, et al. Autoimmunity to specific citrullinated proteins gives the first clues to the etiology of rheumatoid arthritis. Immunol Rev 2010; 233: 34–54 doi: 10.1111/j.0105-2896.2009.00850.x PMID: 20192991 7. Klareskog L, Ronnelid J, Lundberg K, Padyukov L, Alfredsson L. Immunity to citrullinated proteins in rheumatoid arthritis. Annu Rev Immunol 2008; 26: 651–675 doi: 10.1146/annurev.immunol.26. 021607.090244 PMID: 18173373 8. Rantapaa-Dahlqvist S, de Jong BA, Berglin E, Hallmans G, Wadell G, Stenlund H, et al. Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 2003; 48: 2741–2749 PMID: 14558078 9. Szodoray P, Szabo Z, Kapitany A, Gyetvai A, Lakos G, Szanto S, et al. Anti-citrullinated protein/peptide autoantibodies in association with genetic and environmental factors as indicators of disease outcome in rheumatoid arthritis. Autoimmun Rev 2010; 9: 140–143 doi: 10.1016/j.autrev.2009.04.006 PMID: 19427413 10. van Venrooij WJ, van Beers JJ, Pruijn GJ. Anti-CCP antibodies: the past, the present and the future. Nat Rev Rheumatol 2011; 7: 391–398 doi: 10.1038/nrrheum.2011.76 PMID: 21647203 11. von Delwig A, Locke J, Robinson JH, Ng WF. 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Support for a point-of-sale cigarette display ban among smokers: findings from the international tobacco control (ITC) Netherlands survey
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Support for a point-of-sale cigarette display ban among smokers: Findings from the International Tobacco Control (ITC) Netherlands Survey Citation for published version (APA): van Mourik, D.-JA., Candel, M. J. J. M., Nagelhout, G. E., Willemsen, M. C., Fong, G. T., Hummel, K., van den Putte, B., & de Vries, H. (2018). Support for a point-of-sale cigarette display ban among smokers: Findings from the International Tobacco Control (ITC) Netherlands Survey. BMC Public Health, 18, Article 740. https://doi.org/10.1186/s12889-018- 5666-4 Citation for published version (APA): van Mourik, D.-JA., Candel, M. J. J. M., Nagelhout, G. E., Willemsen, M. C., Fong, G. T., Hummel, K., van den Putte, B., & de Vries, H. (2018). Support for a point-of-sale cigarette display ban among smokers: Findings from the International Tobacco Control (ITC) Netherlands Survey. 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BMC Public Health (2018) 18:740 https://doi.org/10.1186/s12889-018-5666-4 Open Access Support for a point-of-sale cigarette display ban among smokers: findings from the international tobacco control (ITC) Netherlands survey Dirk-Jan A. van Mourik1* , Math J. J. M. Candel2, Gera E. Nagelhout1,3,4, Marc C. Willemsen1,5, Geoffrey T. Fong6,7,8, Karin Hummel1, Bas van den Putte9 and Hein de Vries1 Abstract Background: Displaying tobacco products at point-of-sale (PoS) has become an important marketing strategy for the tobacco industry. This study was designed to (1) examine how support for a PoS cigarette display ban changed among Dutch smokers between 2010 and 2015 and (2) identify the variables that predict support among smokers for a PoS cigarette display ban. Methods: Longitudinal data from six annual survey waves (2010-2015) from the International Tobacco Control (ITC) Netherlands Survey were analyzed. The sample consisted of between 1279 and 1800 smokers per year. Smokers were asked whether they supported a complete ban on displays of cigarettes inside shops and stores. Results: Support for a PoS cigarette display ban increased from 28.9% in 2010 to 42.5% in 2015 (OR = 1.40, p < 0.001). A multiple logistic regression analysis revealed that support for a PoS display ban of cigarettes was more likely among smokers who had more knowledge about the health risks of smoking (OR = 3.97, p < 0.001), believed smoking-related health risks to be severe (OR = 1.39, p < 0.001), had a more positive attitude towards quitting smoking (OR = 1.44, p = 0. 006), reported stronger social norms to quit smoking (OR = 1.29, p = 0.035), had a higher self-efficacy for quitting smoking (OR = 1.31, p = 0.001), and had stronger intentions to quit smoking (OR = 1.23, p = 0.006). Conclusions: This paper showed that support for a PoS display ban of cigarettes increased among smokers in the Netherlands over the years. To further increase support, educational campaigns about the dangers of smoking, and campaigns that encourage quitting may be needed. Keywords: Support, Tobacco display ban, Point of sale, The Netherlands, Smokers Background Internal documents of the tobacco industry suggested that tobacco displays are used to shape positive attitudes and beliefs about tobacco brands and products [6]. Dis- playing tobacco products at PoS can act as a cue to smoke [7–11], even among people who try to avoid smoking [12]. Research has also shown that exposure to PoS tobacco displays increases susceptibility for smoking uptake among youth [4, 13, 14]. Restrictions on PoS to- bacco displays can lead to fewer display recalls [15] and may help to denormalise smoking [16]. As countries take action to reduce tobacco advertising, promotion, and sponsorship (TAPS), the tobacco indus- try has fewer opportunities to promote their products. Displaying tobacco products at point-of-sale (PoS) has become one of the most important remaining tools for the tobacco industry to communicate with current and potential customers [1–5], increasing the importance of PoS tobacco display bans to reduce TAPS. The World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) calls on the 180 parties (179 countries and the European Union) to * Correspondence: d.vanmourik@maastrichtuniversity.nl 1Department of Health Promotion, Maastricht University (CAPHRI), PO Box 616, 6200, MD, Maastricht, the Netherlands Full list of author information is available at the end of the article * Correspondence: d.vanmourik@maastrichtuniversity.nl 1Department of Health Promotion, Maastricht University (CAPHRI), PO Box 616, 6200, MD, Maastricht, the Netherlands Full list of author information is available at the end of the article * Correspondence: d.vanmourik@maastrichtuniversity.nl 1Department of Health Promotion, Maastricht University (CAPHRI), PO Box 616, 6200, MD, Maastricht, the Netherlands Full list of author information is available at the end of the article van Mourik et al. BMC Public Health (2018) 18:740 Page 2 of 9 Page 2 of 9 implement PoS tobacco display bans [17]. Several juris- dictions including Canada, Iceland, Norway, Finland, United Kingdom, and Ireland have introduced a PoS to- bacco display ban but global progress in this domain has been slow. In the Netherlands, a ban on PoS tobacco displays in supermarkets is planned for 2020. For other points of sale such as gas stations, convenience stores, drug stores, bars and cafes, evening shops, and kiosks, a ban on PoS tobacco displays is planned for 2022 [18]. A PoS tobacco display ban might be especially effective in the Netherlands where the number of inhabitants per PoS is low compared to other countries [19]. used to explain overt (directly observable), and covert (not immediately observable) health behaviors [27] such as supporting a PoS display ban. Based on these models we identified two groups of factors: (1) socio-demographic characteristics (age, gender, and educational level), and (2) smoking cessation related beliefs such as awareness (knowledge, cues such as noticing anti-tobacco informa- tion, and risk perception), motivation factors (attitude, so- cial norms, and self-efficacy for quitting smoking), and intention to quit smoking. Lowly educated smokers have lower intentions to quit smoking [28, 29]. Since intention to quit smoking pre- dicts support for a PoS tobacco display ban [25], the question arises whether lowly educated smokers are less often supportive. Insight into educational differences may enable policy makers to differentiate in the educa- tional approach on tobacco display bans. Therefore, this study aims to examine differences between low, moder- ate, and high educated smokers in predictors and trends of support for a PoS tobacco display ban. p High levels of public support for tobacco control mea- sures, particularly among smokers, may be an important condition for the adoption of these measures by the gov- ernment [20]. High levels of public support among smokers for a PoS tobacco display ban could prevent re- sistance that could endanger the implementation in 2020 and the continuation of this ban. This is of utmost importance in the Netherlands, where tobacco control policies have been reversed and delayed in the past [21]. In 2014, 60% of all Europeans and 56% of the Dutch population supported keeping tobacco products out of sight at PoS [22]. While non-smokers generally are more likely to support tobacco control measures than smoker [23, 24], many smokers are also supportive. In Ireland, 67% of the non-smokers and 63% of the smokers were supportive of a PoS display ban of cigarette and tobacco packs after the implementation of this tobacco control measure [15]. A study from Canada found that the levels of support for a ban on PoS displays of cigarettes ranged between 55 and 82% (in Canadian provinces) among adult smokers [25]. These studies show reasonable levels of support among smokers for a ban on PoS tobacco dis- plays but studies examining possible predictors of this support remain limited. This study aims to answer the following research ques- tions: (1) Did support among Dutch smokers for a PoS cigarette display ban change over time from 2010 to 2015? (2) Which factors predict support among smokers for a PoS cigarette display ban? (3) Are the findings from the first two research questions different for low, moder- ate, and high educated smokers? Methods Sample p Longitudinal data were obtained from the ITC Netherlands Survey. The surveys were administrated via the internet by the research firm Kantar Public (previ- ously TNS NIPO) which used a quota sample of respon- dents from a probability-based web database to retrieve a representative sample of Dutch smokers aged 15 years and older [30]. Respondents were categorized as smokers if they were currently smoking at least monthly and if they had smoked at least 100 cigarettes in their lifetime [31]. Sampling weights and tailored replenish- ment samples ensured representativeness by compensat- ing for attrition effects [32]. Respondents received incentives for participation in the form of points for each answered question, which could be exchanged for gift certificates. Identifying these predictors may help policy makers to increase support levels. Data from the International To- bacco Control (ITC) Canada Survey revealed that smokers with higher intention to quit were more likely to support a PoS cigarette display ban [25]. This study only focused on intention to quit smoking and socio-demographic characteristics but did not include smoking cessation related beliefs. The current study ex- amines which factors may predict support for a PoS to- bacco display among Dutch smokers. For our analyses, we used data from survey wave 4 (May to June 2010; N = 2060), wave 5 (May to June 2011; N = 2101), wave 6 (May to June 2012; N = 2022), wave 7 (May to June 2013; N = 1970), wave 8 (May to June 2014; N = 2008), and wave 9 (November to Decem- ber 2015; N = 1720). Attrition ranged from 17.1 to 23.9% between survey waves. We excluded quitters from our analyses. Exclusion due to smoking cessation ranged from 12.6 to 25.6% between survey waves. Figure 1 As a multitude of factors may be associated with sup- port for PoS display bans, we used two integrated behav- ior change models: the ITC Conceptual Model [26], and the Integrated Change Model (I-Change Model) [27]. The ITC Conceptual Model is used to explain how to- bacco control measures might work based on a combin- ation of health communication theories and existing psychological models [26]. The I-Change Model can be van Mourik et al. BMC Public Health (2018) 18:740 Page 3 of 9 Page 3 of 9 (3) high (Senior general secondary education, (pre-) uni- versity education and higher professional education). Outcome variable Support for a PoS cigarette display ban was measured by asking ‘Do you support complete bans on displays of cigarettes inside shops and stores?’ [25]. This measure used a three-point scale with the response options (0) not at all, (1) somewhat, and (2) a lot. This variable was dichotomized for the analyses by combining the last two response options. Risk perception was assessed by measuring per- ceived susceptibility and perceived severity. Perceived susceptibility was obtained by the question ‘If you continue to smoke the amount you do now, how likely do you think it is that you will develop lung cancer in the future?’ The scale ranged between (1) very low and (5) very high. Additionally, all smokers were asked about perceived severity via the question ‘If you develop lung problems due to smoking, how serious would you find this?’ The scale ranged be- tween (1) not at all serious and (5) extremely serious. Awareness variables Awareness variables Knowledge about the health risks of smoking was measured by asking eight questions. The following format was used: ‘The following are a few health ef- fects and diseases. Based on what you know or be- lieve, does smoking cause ...’ [33]. This question was asked for heart disease, impotence in male smokers, lung cancer, blindness, mouth and throat cancer, stroke, lung-cancer in non-smokers from secondhand smoke, and heart disease in non-smokers from sec- ondhand smoke (α in wave 8 = 0.84). Respondents could answer with (1) yes, (0) no, and (0) don’t know. Noticing anti-tobacco information was assessed by asking if respondents had noticed advertising or infor- mation about the dangers of smoking or advertising that encouraged quitting in the last 6 months [34]. There were five answering categories ranging between (1) never and (5) very often. Knowledge about the health risks of smoking was measured by asking eight questions. The following format was used: ‘The following are a few health ef- fects and diseases. Based on what you know or be- lieve, does smoking cause ...’ [33]. This question was asked for heart disease, impotence in male smokers, lung cancer, blindness, mouth and throat cancer, stroke, lung-cancer in non-smokers from secondhand smoke, and heart disease in non-smokers from sec- ondhand smoke (α in wave 8 = 0.84). Respondents could answer with (1) yes, (0) no, and (0) don’t know. In the analyses for research question 2 we used the two most recent survey waves (waves 8 and 9). Re- spondents were included in the analyses if they were categorized as a smoker in both waves. In wave 9, 1017 of 1565 smokers from wave 8 participated. Re- spondents were excluded if they had more than five missing values on the independent variables (out of 22 variables) or if they had not filled in the outcome variable on wave 9. This left 844 smokers eligible for the analyses of research question 2. Noticing anti-tobacco information was assessed by asking if respondents had noticed advertising or infor- mation about the dangers of smoking or advertising that encouraged quitting in the last 6 months [34]. There were five answering categories ranging between (1) never and (5) very often. Methods Sample shows the number of smokers of the initial cohort and of the replenishment samples that remained in the study for each wave, leading to a total number of smokers per survey wave. To answer research question 1, all smokers from survey waves 4-9 were included in the analyses. Motivational variables conditional specification method (with linear regression for scalar covariates) [40]. The number of imputations was set according to the percentage of cases that were incomplete [41]. Moreover, starting from this number of imputations, we systematically increased the number of imputations by 10 until results hardly differed. This re- sulted in 80 imputations. Also, we adjusted for the time a respondent participated in the cohort (time-in-sample) since this may influence responses [42]. The dependent variable was dichotomous and therefore the binomial distribution and the logit link was used [43]. Survey wave was the repeated measure variable. The interaction between educational level and survey wave was assessed in a separate analysis (research question 3). Attitude towards quitting smoking was asked via the questions ‘If you quit smoking within the next 6 months, this would be…’: (1) very foolish to (5) very wise and sensible, (1) very disagreeable to (5) very agreeable, and (1) very negative to (5) very positive. The three answers were summed into one attitude score (α in wave 8 = 0.83). Social norms for quitting smoking were assessed by asking ‘Thinking about the people who are important to you, how do you think most of them would feel about your quitting smoking within the next 6 months?’ [35]. Based on a five-point scale respondents could (1) strongly disapprove to (5) strongly approve. T-tests and Chi-square analyses were run to test differ- ences in independent variables (measured in 2014) be- tween supportive and non-supportive smokers of a cigarette display ban (measured in 2015). The associa- tions between independent variables on wave 8 with support for a PoS cigarette display ban on wave 9 (re- search question 2) were examined by performing mul- tiple logistic regression analyses, while controlling for gender, education and age. To examine total effects of predictor variables, we should not correct for possible mediators or descending proxies thereof [44]. For this reason, the variables were added in successive steps: first the awareness variables were entered (knowledge about the health risks of smoking, perceived severity, perceived susceptibility, and noticing anti-tobacco information), second the motivational variables (attitude, social norms and self-efficacy), and in the last step intention to quit smoking. The analysis results for the variables as re- ported, are those for the step in which they were first entered into the analysis model. Only 477 out of the eli- gible 844 respondents completed all independent vari- ables. Socio-demographic variables Respondents were asked whether they were (1) male or (2) female and were classified in one of the following age groups: (1) 15-24, (2) 25-39, (3) 40-45, or (4) 55 years and older. Education was divided into three categories: (1) low (Primary education and lower pre-vocational secondary education), (2) moderate (Middle pre-vocational second- ary education and secondary vocational education), and Fig. 1 Flowchart of the International Tobacco Control Netherlands Survey recruitment of smokers*. *R = Replenishment van Mourik et al. BMC Public Health (2018) 18:740 Page 4 of 9 Page 4 of 9 Intention to quit smoking variable Intention to quit smoking was measured by asking if the respondents were planning to quit smoking within the next 6 months [35, 36]. A five-point scale ranging be- tween (1) very unlikely and (5) very likely was used. Motivational variables For the remaining 367 respondents missing values were filled in by multiple imputation. The 884 subjects available for the analysis therefore contained no persons with missing values on the outcome, but with possibly missing values on predictor variables that were filled in by multiple imputation. This method improves the stat- istical power by increasing the sample size for the mul- tiple logistic regression [40]. Following the same procedure as for the trend analysis, the number of impu- tations was set at 100. The imputed values for the out- come variable were not employed in the analyses, since excluding cases with missing values on the outcome variable yields more stable estimates [45]. Because the variables education and age each were represented in the analyses through multiple dummy variables and SPSS provided only p-values for these separate dummy variables when analyzing multiple imputed datasets, a correction for multiple testing was applied. More specif- ically, for all tests involving the variables education and Self-efficacy to quit smoking was measured by asking how sure they were to succeed if they decided to give up smoking completely in the next 6 months [29]. This was measured on a five-point scale ranging from (1) not at all sure to (5) extremely sure. Smokers were also asked how easy or hard it would be to quit smoking if they wanted to. Answering categories ranged between (1) ex- tremely difficult and (5) not at all difficult. The two an- swers were summed into one self-efficacy score (α in wave 8 = 0.72). Smoking-related variables Smoking-related covariates were level of addiction to to- bacco, and ever having tried to quit smoking. The Heavi- ness of Smoking Index (HSI) was used to measure the level of addiction to tobacco. The HSI is based on the time before smoking the first cigarette of the day (61 + min, 31-60 min, 6-30 min, 5 min or less) and on the number of cigarettes smoked per day (0-10, 11-20, 21-30, 31+). This measure ranges between 0 and 6, with a higher score indicating a higher level of addiction to tobacco [37]. Also, the respondents were asked whether they had ever tried to quit smoking, (1) yes or (2) no. Support for a PoS cigarette display ban between 2010 and 2015 Table 2 shows that support for a PoS display ban of cigarettes increased from 28.9% in 2010 to 42.5% in 2015. A GEE analysis confirmed the overall linear trend (Odds Ratio (OR) = 1.40, 95% Confidence inter- val (CI): 1.25, 1.58). Predictors of support for a PoS cigarette display ban pp g p y The bivariate analyses from Table 3 show that smokers who support a PoS cigarette display had more know- ledge about the health risks of smoking, had a higher perceived severity of smoking-related lung problems, had a more positive attitude towards quitting smoking, perceived stronger social norms for quitting, had a higher self-efficacy for quitting smoking, and a higher intention to quit smoking. A separate GEE analysis including interaction terms of wave by educational level revealed that the support for a PoS cigarette display ban increased less among moderately educated smokers than among highly Table 1 Sample characteristics of smokers between 2010 and 2015a 2010 2011 2012 2013 2014 2015 Gender Male (%) 53.6 54.4 54.1 52.0 50.7 54.0 Female (%) 46.4 45.6 45.9 48.0 49.3 46.0 Age group 15-24 years (%) 12.7 22.8 13.7 11.5 11.1 15.5 25-39 years (%) 27.5 28.9 23.5 23.7 23.8 24.1 40-54 years (%) 32.3 25.1 32.1 32.2 29.8 27.9 55 years and older (%) 27.5 23.2 30.7 32.6 35.3 32.6 Educational level Low (%) 36.2 30.2 31.8 29.1 26.1 24.7 Moderate (%) 41.9 45.9 44.9 46.1 43.7 42.4 High (%) 21.9 24.0 23.3 24.8 30.2 33.0 Level of addiction to tobacco 0 to 1 (%) 29.2 30.8 28.0 28.7 29.4 31.7 2 to 4 (%) 64.4 58.1 64.5 64.4 64.5 62.1 5 to 6 (%) 6.4 11.2 7.5 6.9 6.0 6.2 Ever tried quitting smoking Yes (%) 65.1 60.7 60.9 60.2 57.8 64.1 No (%) 34.9 39.3 39.1 39.8 42.2 35.9 aEstimates were weighted for gender and age Table 1 Sample characteristics of smokers between 2010 and 2015a Table 1 Sample characteristics of smokers between 2010 and The results from the multiple logistic regression (Table 4) revealed that support for a PoS cigarette display ban was significantly associated with more knowledge about the health risks of smoking, a higher perceived severity, a more positive attitude towards quitting smoking, stronger social norms for quitting smoking, a higher self-efficacy for quitting smoking, and a higher intention to quit smoking. Statistical analysis Data were analyzed using the statistical software pro- gram SPSS 23. All statistical estimates and tests pre- sented were weighted for gender and age [38]. Trends in outcome measure (research question 1) were tested with Generalized Estimating Equations (GEE) analyses [39], while controlling for gender, age, ever having made a quit attempt, education, and level of addiction to tobacco. Missing data values on these vari- ables were imputed multiple times using the full van Mourik et al. BMC Public Health (2018) 18:740 Page 5 of 9 Page 5 of 9 Table 2 Percentages of smokers by educational level and wave who support a PoS cigarette display bana 2010 2011 2012 2013 2014 2015 Total group (%) 28.9 34.6 34.4 36.4 37.7 42.5 Low educational level (%) 24.6 34.8 33.7 38.4 37.6 38.6 Moderate education level (%) 30.5 33.1 31.0 34.5 36.7 38.2 High educational level (%) 30.8 37.4 41.2 38.5 39.1 50.9 aEstimates were weighted for gender and age Table 2 Percentages of smokers by educational level and wave who support a PoS cigarette display bana age, the Holm correction was applied to the significance level α = 0.05 [46]. Interactions between educational level and independent variables were added in a separate re- gression analysis to determine whether there were differ- ences between educational levels in the predictors for support for a PoS cigarette display ban (research ques- tion 3). This analysis was also done and reported with the same steps as delineated above, but then also adding each time the interaction terms with education. aEstimates were weighted for gender and age educated smokers (OR of interaction = 0.87, 95% CI: 0.78, 0.98, p = 0.011 < αadjusted = 0.017). This result can also be seen in Table 2 that shows that the support among moderately educated smokers increased from 2010 to 2015 by 7.6% whereas the support for highly educated smokers increased by 20.1%. No significant interactions were found between wave and low versus moderate education (OR = 1.07, 95% CI: 0.97, 1.18, p = 0.155 > αadjusted = 0.025), nor between wave and low versus high education (OR = 0.94, 95% CI: 0.84, 1,05, p = 0.238 > αadjusted = 0.05). educated smokers (OR of interaction = 0.87, 95% CI: 0.78, 0.98, p = 0.011 < αadjusted = 0.017). Sample characteristics Table 1 shows characteristics of smokers between 2010 and 2015. The smokers’ educational level increased over the years. Statistical analysis This result can also be seen in Table 2 that shows that the support among moderately educated smokers increased from 2010 to 2015 by 7.6% whereas the support for highly educated smokers increased by 20.1%. No significant interactions were found between wave and low versus moderate education (OR = 1.07, 95% CI: 0.97, 1.18, p = 0.155 > αadjusted = 0.025), nor between wave and low versus high education (OR = 0.94, 95% CI: 0.84, 1,05, p = 0.238 > αadjusted = 0.05). aEstimates were weighted for gender and age Support for a PoS cigarette display ban between 2010 and 2015 A separate analysis including interaction terms with educational level and all independent variables did only show almost significant interactions. There was a nearly significant interaction between attitude towards quitting smoking and moderate versus high education (OR of interaction = 0.49, 95% CI: 0.25, 0.96, p = 0.038 > αadjusted = 0.025), as well as a nearly significant interaction be- tween attitude towards quitting smoking and moderate versus low education (OR of interaction = 0.46, 95% CI: 0.24, 0.90, p = 0.024 > αadjusted = 0.017). Attitude towards quitting smoking was a stronger predictor for high edu- cated smokers (OR = 2.07, p = 0.008, 95% CI: 1.21, 3.55), and for low educated smokers (OR = 2.10, p = 0.006, 95% CI: 1.23, 3.57) as compared to moderate educated smokers (OR = 0.97, p = 0.886, 95% CI: 0.64, 1.46). van Mourik et al. Support for a PoS cigarette display ban between 2010 and 2015 BMC Public Health (2018) 18:740 Page 6 of 9 Table 3 Bivariate t-tests and Chi-square tests of predictor variables in 2014 and support in 2015a,b Support Yes (n = 343) No (n = 501) T-value or χ2 Socio demographics Gender Male (%) 40.2 59.8 χ2 = 0.43 Female (%) 40.9 59.1 p = 0.836 Age group 15-24 years (%) 34.1 65.9 χ2 = 3.91 25-39 years (%) 45.8 54.2 p = 0.271 40-54 years (%) 39.1 60.9 55 years and older (%) 40.4 59.6 Educational level Low (%) 38.8 61.2 χ2 = 0.77 Moderate (%) 39.3 60.7 p = 0.681 High (%) 43.3 56.7 Awareness variables Knowledge health risks of smoking (mean, SD) 0.65 (0.27) 0.54 (0.30) t = −5.62, p < 0.001 Noticing anti-tobacco information (mean, SD) 2.41 (0.96) 2.29 (0.95) t = −1.57, p = 0.116 Perceived susceptibility (mean, SD) 3.14 (0.96) 3.09 (0.84) t = −0.82, p = 0.413 Perceived severity (mean, SD) 3.54 (1.03) 3.16 (0.99) t = −5.42, p < 0.001 Motivational variables Attitude towards quitting smoking (mean, SD) 4.21 (0.70) 3.86 (0.79) t = −6.80, p < 0.001 Social norms for quitting smoking(mean, SD) 4.41 (0.74) 4.10 (0.77) t = −5.73, p < 0.001 Self-efficacy for quitting smoking (mean, SD) 2.45 (1.07) 2.23 (1.00) t = −2.95, p = 0.003 Intention to quit smoking (mean, SD) 2.77 (1.15) 2.32 (1.06) t = −5.85, p < 0.001 aEstimates were weighted for gender and age bImputed data Table 3 Bivariate t-tests and Chi-square tests of predictor variables in 2014 and support in 2015a,b indicated by an increasing perceived societal disap- proval of smoking [48]. Discussion Despite recommendations from the WHO dating back to 2003, the Dutch government only recently (January 2017) decided to implement a PoS cigarette display ban. In this paper we examined predictors of, and trends in support for this tobacco control measure among Dutch smokers. The second research question aimed to identify factors that predict support among smokers for a PoS cigarette display ban. In line with previous research [25], a higher intention to quit smoking predicted support for a PoS display ban of cigarettes. The theory of self-control [49] provides a possible explanation for why smokers with a high intention to quit smoking support this tobacco con- trol measure. A cigarette display ban may be perceived as a welcome external limit on future behavior (smok- ing) since leaving tobacco products out of sight at PoS will prevent smokers to get tempted to start smoking again. This may help smokers who want to quit doing this successfully. This explanation can also be used to explain the other predictors we found, since intention to quit smoking is associated with more knowledge about the health risks of smoking [50], more perceived severity [51], a more positive attitude towards quitting smoking [52], stronger social norms about quitting smoking [35], and higher self-efficacy for quitting smoking [53]. The first research question was whether there were changes in support for a PoS cigarette display ban among Dutch smokers over time. We found a signifi- cant increase in support among smokers between 2010 (28.9%) and 2015 (42.5%). This is consistent with findings from other research that tobacco con- trol measures are also popular among smokers, and support tends to increase over time especially after measures have been implemented [15, 47]. If this trend continues, it seems like a matter of time before the majority of Dutch smokers support this tobacco control measure. The increase in support for a PoS cigarette display ban may also be consequence of a general societal denormalisation of smoking, which is Page 7 of 9 van Mourik et al. Implications for policy To increase support further, educational campaigns may focus on explaining the upcoming PoS tobacco display ban as well as on improving knowledge about the health risks of smoking, increasing perceived severity, stimulat- ing a more positive attitude towards quitting smoking, changing social norms about quitting smoking, increas- ing self-efficacy for quitting smoking, and stimulating quitting. Such educational campaigns may be especially important in the Netherlands where the knowledge and general concern about the health risks of smoking and secondhand smoke in smokers is low compared to other countries [48]. Smokers may thus be aware that exposure to PoS to- bacco displays obstructs smokers to decrease or quit smoking [7–12]. Knowledge, perceived severity, attitude, social norms, and self-efficacy were independent predic- tors for support and should therefore be considered as important points of engagement for future campaigns. Limitations Limitations Several limitations should be taken into account when interpreting the results. First, the sample differed over the years on age, educational level, level of addiction to tobacco, and ever having made a quit attempt which may indicate that the sample is not entirely representa- tive of the Dutch population of smokers. Therefore, we adjusted for these sample characteristics in the analyses and applied weights. Second, to cope with the high num- ber of missing values we applied multiple imputation procedures to increase the statistical power. There is never complete certainty about the correctness of the imputation model but a rather substantial set of vari- ables was used in this model and care was taken to take a large number of imputations to maximize the effi- ciency of the pooled estimates. Third, the measurements of wave 9 took place from November to December 2015, whereas in prior years measurements took place from May to June. This difference can give a somewhat dis- torted image of the trends in support. Fourth, this paper addressed support for a ban of cigarette displays. Since the ITC Netherlands survey did not include a question on support for a complete ban of tobacco displays, we could not study a display ban of other tobacco or nico- tine products. Discussion BMC Public Health (2018) 18:740 Page 7 of 9 Table 4 Multiple logistic regression analysis of predictors associated with support for a PoS cigarette display ban (N = 844, where N = 343 for the supportive group, N = 501 for the non- supportive group)a,b,c OR 95% CI Socio demographics Gender Male 0.99 Female 1.01 (0.88, 1.16) Age group 25-39 years vs.15-24 years (ref) 1.59 (0.92, 2.74) 15-24 years vs. 55+ (ref) 0.77 (0.46, 1.27) 25-39 years vs. 55+ (ref) 1.22 (0.84, 1.77) 40-54 years vs. 15-24 years (ref) 1.24 (0.79, 2.14) 40-45 years vs. 25-30 years (ref) 0.78 (0.53, 1.14) 40-45 years vs. 55+ (ref) 0.95 (0.71, 1.27) Educational level Low vs. high (ref) 0.88 (0.60, 1.29) Moderate vs. low (ref) 1.00 (0.70, 1.43) Moderate vs high (ref) 0.88 (0.64, 1.23) Awareness variables Knowledge health risks of smoking 3.97*** (2.25, 7.00) Noticing anti-tobacco information 1.12 (0.96, 1.32) Perceived susceptibility 0.91 (0.74, 1.11) Perceived severity 1.39*** (1.20, 1.61) Motivational variables Attitude towards quitting smoking 1.44** (1.11, 1.87) Social norms for quitting smoking 1.29* (1.02, 1.64) Self-efficacy for quitting smoking 1.31** (1.12, 1.53) Intention to quit smoking 1.23** (1.06, 1.43) aEstimates were weighted for gender and age bImputed data cAssociations are between independent variables on survey wave 8 and support on survey wave 9 *p < 0.05; **p < 0.01; ***p < 0.001 OR Odds ratio, CI Confidence interval, ref Reference category we found that attitude towards quitting was a stronger predictor for high, and low educated smokers as com- pared to moderate educated smokers. Competing interests h h d l h 17. World Health Organization. WHO Framework Convention on Tobacco Control. 2003. http://apps.who.int/iris/bitstream/10665/42811/1/9241591013. pdf?ua=1. Accessed 5 Oct 2017. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. 18. van Rijn MJ. Letter from the state secretary for Public Health Welfare and Sport (Originally Dutch: Brief van de staatssecretaris van Volksgezondheid Welzijn en Sport). 2017. https://www.rijksoverheid.nl/ministeries/ministerie- van-volksgezondheid-welzijn-en-sport/documenten/kamerstukken/2017/07/ 05/kamerbrief-over-tabak-en-rookwaren. Accessed 10 Oct 2017. Authors’ contributions 13. Braun S, Kollath-Cattano C, Barrientos I, Mejía R, Morello P, Sargent J, et al. Assessing tobacco marketing receptivity among youth: integrating point of sale marketing, cigarette package branding and branded merchandise. Tob Control. 2015;25(6):648–55. DJvM, GN, HdV, and MW devised the basic idea for the manuscript. DJvM drafted the manuscript with substantial contributions from GN. MC performed the statistical analyses with substantial contributions from DJvM. DJvM, MC, GN, MW, GF, KH, BvdP, and HdV contributed to the interpretation of the analyses and revised the manuscript critically. DJvM, MC, GN, MW, GF, KH, BvdP, and HdV read and approved the final manuscript. 14. Henriksen L, Schleicher NC, Feighery EC, Fortmann SPA. Longitudinal study of exposure to retail cigarette advertising and smoking initiation. Pediatrics. 2010;126(2):232–8. 15. McNeill A, Lewis S, Quinn C, Mulcahy M, Clancy L, Hastings G, et al. Evaluation of the removal of point-of-sale tobacco displays in Ireland. Tob Control. 2011;20(2):137–43. Funding This work was supported by the Netherlands Organisation for Health Research and Development (ZonMw; survey wave 4-8; grant numbers 121010008 and 200130002), and the Dutch Cancer Society (KWF Kankerbes- trijding; survey wave 9; grant number UM 2014-7210). Additional support was provided to Geoffrey T. Fong from a Senior Investigator Award from the Ontario Institute for Cancer Research and a Prevention Scientist Award from the Canadian Cancer Society Research Institute. 5. Wakefield M, Morley C, Horan JK, Cummings KM. The cigarette pack as image: new evidence from tobacco industry documents. Tob Control. 2002; 11(Suppl 1):i73–80. 6. Pollay RW. More than meets the eye: on the importance of retail cigarette merchandising. Tob Control. 2007;16(4):270–4. 7. Carter OBJ, Mills BW, Donovan RJ. The effect of retail cigarette pack displays on unplanned purchases: results from immediate postpurchase interviews. Tob Control. 2009;18(3):218–21. References 1 F i h 1. Feighery EC, Ribisl KM, Clark PI, Haladjian HH. How tobacco companies ensure prime placement of their advertising and products in stores: interviews with retailers about tobacco company incentive programmes. Tob Control. 2003;12(2):184–8. 1. Feighery EC, Ribisl KM, Clark PI, Haladjian HH. How tobacco companies ensure prime placement of their advertising and products in stores: interviews with retailers about tobacco company incentive programmes. Tob Control. 2003;12(2):184–8. Abbreviations CI: Confidence interval; FCTC: Framework Convention on Tobacco Control; GEE: Generalized estimating equations; I-Change Model: Integrated change model; ITC: International Tobacco Control; OR: Odds ratio; PoS: Point-of-sale; TAPS: Tobacco advertising, promotion, and sponsorship; WHO: World Health Organization 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. Availability of data and materials 8. Hoek J, Gifford H, Pirikahu G, Thomson G, Edwards R. How do tobacco retail displays affect cessation attempts? Findings from a qualitative study. Tob Control. 2010;19(4):334–7. Data from the ITC Policy Evaluation Project are available to approved researchers 2 years after the date of issuance of cleaned data sets by the ITC Data Management Centre. Researchers interested in using ITC data are required to apply for approval by submitting an International Tobacco Control Data Repository (ITCDR) request application and subsequently to sign an ITCDR Data Usage Agreement. To avoid any real, potential, or perceived conflict of interest between researchers using ITC data and tobacco-related entities, no ITCDR data will be provided directly or indirectly to any researcher, institution, or consultant that is in current receipt of any grant monies or in-kind contribution from any tobacco manufacturer, dis- tributor, or other tobacco-related entity. The criteria for data usage approval and the contents of the Data Usage Agreement are described online (http:// www.itcproject.org). 9. Li L, Borland R, Fong GT, Thrasher JF, Hammond D, Cummings KM. Impact of point-of-sale tobacco display bans: findings from the international tobacco control four country survey. Health Educ Res. 2013;28(5):898–910. 9. Li L, Borland R, Fong GT, Thrasher JF, Hammond D, Cummings KM. Impact of point-of-sale tobacco display bans: findings from the international tobacco control four country survey. Health Educ Res. 2013;28(5):898–910. 10. MacKintosh AM, Moodie C, Hastings G. The association between point- of-sale displays and youth smoking susceptibility. Nicotine Tob Res. 2012;14(5):616–20. 11. Siahpush M, Shaikh RA, Cummings KM, Hyland A, Dodd M, Carlson L, et al. The association of point-of-sale cigarette marketing with cravings to smoke: results from a cross-sectional population-based study. Tob Control. 2016;25(4):402–5. 12. Wakefield M, Germain D, Henriksen L. The effect of retail cigarette pack displays on impulse purchase. Addiction. 2008;103(2):322–8. Acknowledgements h h f ll The authors gratefully acknowledge the assistance of several members of the ITC Project team at the University of Waterloo in all stages of conducting the ITC Netherlands survey. In particular, the authors thank Thomas Agar and Anne Quah. 2. Harper T. Why the tobacco industry fears point of sale display bans. Tob Control. 2006;15(3):270–1. 2. Harper T. Why the tobacco industry fears point of sale display bans. Tob Control. 2006;15(3):270–1. 3. Lavack AM, Toth G. Tobacco point-of-purchase promotion: examining tobacco industry documents. Tob Control. 2006;15(5):377–84. 3. Lavack AM, Toth G. Tobacco point-of-purchase promotion: examining tobacco industry documents. Tob Control. 2006;15(5):377–84. 4. Paynter J, Edwards R. The impact of tobacco promotion at the point of sale: a systematic review. Nicotine Tob Res. 2009;11(1):25–35. 4. Paynter J, Edwards R. The impact of tobacco promotion at the point of sale: a systematic review. Nicotine Tob Res. 2009;11(1):25–35. Ethics approval and consent to participate The ITC Netherlands Surveys received ethics clearance from the University of Waterloo’s Office of Research Ethics. We received permission from the ITC Policy Evaluation Project to use this study’s data. 16. Scheffels J, Lavik R. Out of sight, out of mind? Removal of point-of-sale tobacco displays in Norway. Tob Control. 2013;22(e1):e37–42. Conclusions Support among Dutch smokers for a PoS cigarette dis- play ban increased between 2010 and 2015, and in- creased faster among highly educated smokers than among moderately educated smokers. The findings from the present study showed that predictors of support among smokers for a PoS cigarette display ban were more knowledge about the health risks of smoking, higher perceived severity, more positive attitude towards quitting smoking, stronger social norms to quit smoking, The third research question was whether the findings from the first two research questions differed for smokers with low, moderate and high educated levels. First, support for a PoS cigarette display ban was higher and increased more among the highly educated group than among the moderately educated group. This could again be explained by the fact that less educated smokers tend to have lower intentions to quit [28, 29]. Second, Page 8 of 9 van Mourik et al. BMC Public Health (2018) 18:740 Page 8 of 9 Page 8 of 9 Page 8 of 9 higher self-efficacy for quitting smoking, and stronger intentions to quit smoking. To increase support, educa- tional campaigns may focus on improving knowledge about the health risks of smoking, and encouraging quitting. Netherlands. 5Netherlands Expertise Center for Tobacco Control (NET), Trimbos Institute, Utrecht, the Netherlands. 6Department of Psychology, University of Waterloo, Waterloo, Canada. 7Ontario Institute for Cancer Research, Toronto, Canada. 8School of Public Health and Health Systems, University of Waterloo, Waterloo, Canada. 9Department of Communication, University of Amsterdam (ASCoR), Amsterdam, the Netherlands. Netherlands. 5Netherlands Expertise Center for Tobacco Control (NET), Trimbos Institute, Utrecht, the Netherlands. 6Department of Psychology, University of Waterloo, Waterloo, Canada. 7Ontario Institute for Cancer Research, Toronto, Canada. 8School of Public Health and Health Systems, University of Waterloo, Waterloo, Canada. 9Department of Communication, University of Amsterdam (ASCoR), Amsterdam, the Netherlands. Received: 1 November 2017 Accepted: 4 June 2018 Author details 1 19. Monshouwer K, Verdurmen J, Ketelaars T, van Laar M. Points of sale of tobacco products: synthesis of scientific and practice based knowledge on the impact of reducing the number of points of sale and restrictions on tobacco product. Displays 2014.https://assets.trimbos.nl/ docs/fc34ad60-2cec-4100-8a9f-dc49bb88373e.pdf. Published in 2014. Accessed 5 Oct 2017. 1Department of Health Promotion, Maastricht University (CAPHRI), PO Box 616, 6200, MD, Maastricht, the Netherlands. 2Department of Methodology and Statistics, Maastricht University (CAPHRI), Maastricht, the Netherlands. 3Department of Family Medicine, Maastricht University (CAPHRI), Maastricht, the Netherlands. 4IVO Addiction Research Institute, The Hague, the Page 9 of 9 Page 9 of 9 van Mourik et al. BMC Public Health (2018) 18:740 Page 9 of 9 20. Willemsen MC. Smoking in the Netherlands. The downside of tolerance (originally Dutch: Roken in Nederland. De keerzijde van tolerantie). Maastricht: Oce Business Services; 2011. 40. Enders CK. Applied missing data analysis. New York: Guilford Press; 20 41. White IR, Royston P, Wood AM. Multiple imputation using chained equations: issues and guidance for practice. Stat Med. 2011;30(4):377–99. 21. Hummel K. It comes and goes in waves; evaluation of inconsistently implemented tobacco control policies in the Netherlands [dissertation]. Maastricht: Datawyse; 2017. 42. Driezen P, Thompson M. Comparing policy measures across multple ITC countries: adjusting for time-in sample. 2011. http://www.itcproject.org/ files/ITC_Technical_Report_time-in-sample-adjustment_Dec2011.pdf. Accessed 5 Oct 2017. 22. European Commision. Special Eurobarometer 429. Attitudes of Europeans towards tobacco and electronic cigarettes. 2014. http://ec.europa.eu/ commfrontoffice/publicopinion/archives/ebs/ebs_429_en.pdf. Accessed 5 Oct 2017. 43. Ballinger GA. Using generalized estimating equations for longitudinal data analysis. Organ Res Meth. 2004;7(2):127–50. 44. Schisterman EF, Cole SR, Platt RW. Overadjustment bias and unnecessary adjustment in epidemiologic studies. Epidemiology. 2009;20(4):488–95. https://doi.org/10.1097/EDE.0b013e3181a819a1. 23. Connolly GN, Behm I, Healton CG, Alpert HR. Public attitudes regarding banning of cigarettes and regulation of nicotine. Am J Public Health. 2012; 102(4):e1–2. g 45. von Hippel, PT. Regression with missing Ys: an improved strategy for analyzing multiply imputed data. Sociol Methodol 2017;37(1):83-117. pp , g g p gy analyzing multiply imputed data. Sociol Methodol 2017;37(1):83-117. 46. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;6(2):65–70. 24. Farley SM, Coady MH, Mandel-Ricci J, Waddell EN, Chan C, Kilgore EA, et al. Public opinions on tax and retail-based tobacco control strategies. Tob Control. 2013;24(e1):e10–3. 46. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;6(2):65–70. 47. Fong GT, Hyland A, Borland R, Hammond D, Hastings G, McNeill A, et al. Author details 1 Reductions in tobacco smoke pollution and increases in support for smoke- free public places following the implementation of comprehensive smoke- free workplace legislation in the Republic of Ireland: findings from the ITC Ireland/UK survey. Tob Control. 2006;15(Suppl 3):iii51–8. 25. Brown A, Boudreau C, Moodie C, Fong GT, Li GY, McNeill A, et al. Support for removal of point-of-purchase tobacco advertising and displays: findings from the international tobacco control (ITC) Canada survey. Tob Control. 2012;21(6):555–9. 26. Fong GT, Kummings MC, Borland R, Hastings G, Hyland A. Giovino, et al. the conceptual framework of the international tobacco control (ITC) policy evaluation project. Tob Control. 2006;15(Suppl 3):iii3–11. 48. ITC Project. ITC Netherlands national report. Findings from the wave 1 to 8 survey (2008-2014). 2015. http://itcproject.org/files/ITC_Netherlands_ National_Report-02Sept2015-printfinal.pdf. Accessed 5 Oct 2017. 27. de Vries H. An integrated approach for understanding health behavior; the I-change model as an example. Psychol Behav Sci Int J. 2017; https://doi. org/10.19080/PBSIJ.2017.02.555585. 49. Thaler RH, Shefrin HM. An economic theory of self-control. J Political Eco. 1981;89(2):392–406. 50. Sansone GC, Raute LJ, Fong GT, Pednekar MS, Quah ACK, Bansal-Travers M, et al. Knowledge of health effects and intentions to quit among smokers in India: findings from the tobacco control policy (TCP) India pilot survey. Int J Environ Res Public Health. 2012;9(2):564. 28. Reid JL, Hammond D, Boudreau C, Fong GT, Siahpush M, ITC Collaboration. Socioeconomic disparities in quit intentions, quit attempts, and smoking abstinence among smokers in four western countries: findings from the international Tobobacco control four country survey. Nicotine Tob Res. 2010;12(Suppl 1):S20–33. 51. Janz NK, Becker MH. The health belief model: a decade later. Health Ed Quar. 1984;11(1):1–47. 29. Siahpush M, McNeill A, Borland R, Fong GT. Socioeconomic variations in nicotine dependence, self-efficacy, and intention to quit across four countries: findings from the international tobacco control (ITC) four country survey. Tob Control. 2006;15(Suppl 3):iii71–5. 52. Rise J, Kovac V, Kraft P, Moan IS. Predicting the intention to quit smoking and quitting behaviour: extending the theory of planned behaviour. Br J Health Psychol. 2008;13(2):291–10. 53. Floyd DL, Prentice-Dunn S, Rogers RW. A meta analysis of research on protection motivation theory. J Appl Soc Psychol. 2000;30(2):407–29. 30. Nagelhout GE, Willemsen MC, Thompson ME, Fong GT, van den Putte B, de Vries H. Is web interviewing a good alternative to telephone interviewing? Findings from the international tobacco control (ITC) Netherlands survey. BMC Public Health. 2010;10:351–61. 31. Author details 1 Hyland A, Borland R, Li Q, Yong H-H, McNeill A, Fong GT, et al. Individual- level predictors of cessation behaviours among participants in the international tobacco control (ITC) four country survey. Tob Control. 2006; 15(Suppl 3):iii83–94. 32. Zethof D, Nagelhout GE, de Rooij M, Driezen P, Fong GT, van den Putte B, et al. Attrition analysed in five waves of a longitudinal yearly survey of smokers: findings from the ITC Netherlands survey. Eur J Pub Health. 2016; 26(4):693–9. 33. Hammond D, Fong GT, McNeill A, Borland R, Cummings KM. Effectiveness of cigarette warning labels in informing smokers about the risks of smoking: findings from the international Tob control (ITC) four country survey. Tob Control. 2006;15(Suppl 3):iii19–25. 34. Hammond D, Fong GT, Zanna MP, Thrasher JF, Borland R. Tobacco denormalization and industry beliefs among smokers from four countries. Am J Prev Med. 2006;31(3):225–32. 35. van den Putte B, Yzer MC, Brunsting S. Social influences on smoking cessation: a comparison of the effect of six social influence variables. Prev Med. 2005;41(1):186–93. 35. van den Putte B, Yzer MC, Brunsting S. Social influences on smoking cessation: a comparison of the effect of six social influence variables. Prev Med. 2005;41(1):186–93. 36. Hummel K, Candel MJJM, Nagelhout GE, Brown J, van den Putte B, Kotz D, et al. Construct and predictive validity of three measures of intention to quit smoking: findings from the international tobacco control (ITC) Netherlands survey. Nicotine Tob Res. 2017; https://doi.org/ 10.1093/ntr/ntx092. 36. Hummel K, Candel MJJM, Nagelhout GE, Brown J, van den Putte B, Kotz D, et al. Construct and predictive validity of three measures of intention to quit smoking: findings from the international tobacco control (ITC) Netherlands survey. Nicotine Tob Res. 2017; https://doi.org/ 10.1093/ntr/ntx092. 37. Heatherton TF, Kozlowski LT, Frecker RC, Rickert W, Robinson J. Measuring the heaviness of smoking: using selfreported time to the first cigarette of the day and number of cigarettes smoked per day. Br J Addict. 1989;84(7):791. 38. Thompson ME, Fong GT, Hammond D, Boudreau C, Driezen P, Hyland A, et al. Methods of the international tobacco control (ITC) four country survey. Tob Control. 2006;15(Suppl 3):iii12–8. 38. Thompson ME, Fong GT, Hammond D, Boudreau C, Driezen P, Hyland A, et al. Methods of the international tobacco control (ITC) four country survey. Tob Control. 2006;15(Suppl 3):iii12–8. 39. Diggle PJ, Heagerty P, Liang K-Y, Zeger SL. Analysis of longitudinal data. Oxford: Oxford University Press; 2002. 39. Author details 1 Diggle PJ, Heagerty P, Liang K-Y, Zeger SL. Analysis of longitudinal data. Oxford: Oxford University Press; 2002.
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MRSA Causing Infections in Hospitals in Greater Metropolitan New York: Major Shift in the Dominant Clonal Type between 1996 and 2014
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* tomasz@rockefeller.edu RESEARCH ARTICLE OPEN ACCESS Citation: Pardos de la Gandara M, Curry M, Berger J, Burstein D, Della-Latta P, Kopetz V, et al. (2016) MRSA Causing Infections in Hospitals in Greater Metropolitan New York: Major Shift in the Dominant Clonal Type between 1996 and 2014. PLoS ONE 11 (6): e0156924. doi:10.1371/journal.pone.0156924 MRSA Causing Infections in Hospitals in Greater Metropolitan New York: Major Shift in the Dominant Clonal Type between 1996 and 2014 Maria Pardos de la Gandara1, Marie Curry1, Judith Berger2, David Burstein3, Phyllis Della- Latta4, Virgina Kopetz3, John Quale5, Eric Spitzer6, Rexie Tan7, Carl Urban8, Guiqing Wang9, Susan Whittier10, Herminia de Lencastre1,11, Alexander Tomasz1* 1 Laboratory of Microbiology & Infectious Diseases, The Rockefeller University, New York, New York, United States of America, 2 Division of Infectious Diseases, Saint Barnabas Hospital, Bronx, New York, New York, United States of America, 3 Department of Pathology, Richmond University Medical Center, Staten Island, New York, United States of America, 4 Department of Pathology and Cell Biology, Columbia University Medical Center, New York Presbyterian Hospital, New York, New York, United States of America, 5 Division of Infectious Diseases, SUNY Downstate Medical Center and Kings County Hospital, Brooklyn, New York, United States of America, 6 Department of Pathology, Stony Brook Health Services Center, Stony Brook, New York, United States of America, 7 Department of Microbiology, Saint Barnabas Hospital, Bronx, New York, United States of America, 8 The Dr. James J. Rahal Jr. Division of Infectious Diseases, New York- Presbyterian Queens, Flushing, New York, United States of America, 9 Department of Pathology and Clinical Laboratories, Westchester Medical Center and New York Medical College, Valhalla, New York, United States of America, 10 Clinical Microbiology Service, Columbia University Medical Center, New York Presbyterian Hospital, New York, New York, United States of America, 11 Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica António Xavier (ITQB/UNL), Oeiras, Portugal a1111 Abstract Editor: Paul J Planet, Columbia University, UNITED STATES A surveillance study in 1996 identified the USA100 clone (ST5/SCCmecII)–also known as the “New York/Japan” clone—as the most prevalent MRSA causing infections in 12 New York City hospitals. Here we update the epidemiology of MRSA in seven of the same hospi- tals eighteen years later in 2013/14. Most of the current MRSA isolates (78 of 121) belonged to the MRSA clone USA300 (CC8/SCCmecIV) but the USA100 clone–dominant in the 1996 survey–still remained the second most frequent MRSA (25 of the 121 isolates) causing 32% of blood stream infections. The USA300 clone was most common in skin and soft tissue infections (SSTIs) and was associated with 84.5% of SSTIs compared to 5% caused by the USA100 clone. Our data indicate that by 2013/14, the USA300 clone replaced the New York/Japan clone as the most frequent cause of MRSA infections in hospitals in Metropoli- tan New York. In parallel with this shift in the clonal type of MRSA, there was also a striking change in the types of MRSA infections from 1996 to 2014. Received: October 29, 2015 Accepted: May 20, 2016 Published: June 7, 2016 Copyright: © 2016 Pardos de la Gandara 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: © 2016 Pardos de la Gandara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Introduction Staphylococcus aureus has remained a leading cause of infections in hospitals and continues to represent the most frequently identified antibiotic-resistant nosocomial pathogen in many parts of the world [1]. S. aureus accounted for 12% of all nosocomial infections in the US in 1996, and it still accounts for over 10% of these infections in 2014, in spite of public health con- trols established in hospitals [2,3]. Moreover, while methicillin-resistant S. aureus (MRSA) accounted for close to 35% of staphylococcal infections in 1996, this number increased to 60% of all nosocomial S. aureus infections in more recent years [2–5]. Almost twenty years ago–in 1996 –our laboratory organized a multicenter study to investi- gate the molecular epidemiology of MRSA in 12 hospitals in Metropolitan New York [2]. At that time, the most prevalent clone, recovered from 113 of 270 MRSA infections (42%), was the ‘New York/Japan’ clone (USA100/ST5/SCCmecII), which was also predominant in the neighboring states of Pennsylvania, New Jersey and Connecticut [6]. Subsequently, the same clonal type of MRSA was also identified in hospital infections in Japan [7]. In the United States, this clone has been the predominant MRSA clone in hospitals and healthcare institutions (HA-MRSA) countrywide over the last fifteen years [8,9]. According to the National Nosocomial Infections Surveillance, the rates of MRSA infection in hospitals in New York City have been increasing from 619 cases in 1997 (35% of all S. aureus infections in hospitals) to 3,470 cases in 2004 (60% of all S. aureus infections in hospitals) [10]. In parallel, the frequency of MRSA infections in hospitals due to community-associated MRSA (CA-MRSA) clones has also increased from 18% in 1997 to 25% in 2004 [9,11]. On the other hand, the New York State Department of Health reported a decrease in the number of hospital MRSA infections during the past seven years: MRSA infections in colon surgery decreased from 84 cases in 2008 to 74 in 2013; coronary bypass infections decreased from 55 in 2008 to 24 in 2013; and central-catheter-associated bloodstream infections in chil- dren and adults decreased from 73 in 2008 to 20 in 2013. However, the number of central-cath- eter-associated bloodstream infections in neonatal intensive care units due to MRSA increased from three in 2008 to ten in 2013 [12,13]. Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: Partial support for this study was provided by a US Public Health Service Award 2 RO1 AI457838-15 to ATand by funds from the RB Roberts Bacterial Antibiotic Resistance Group (BARG) to AT. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 MRSA in Hospitals in NYC in 2014 Introduction The latest report from the Active Bacterial Core Surveillance Program (ABCSP) described a reduction in the national incidence of hospital acquired MRSA invasive infections between 2005 and 2011. However, the authors described an increased risk of recurrence among health- care-associated community-onset infections and a very limited change in the rate of commu- nity-associated infections [14]. In the present study we report on the clonal types of MRSA isolates recovered from infec- tions in several of the same hospitals in New York City that participated in the 1996/98 surveillance. Materials and Methods The study was reviewed and approved by the Institutional Review Board (IRB) at The Rockefel- ler University. MRSA in Hospitals in NYC in 2014 Fig 1. Geographic location of hospitals that participated in the two surveillance studies. Numbers from I to XII represent the 12 hospitals that collaborated with the Rockefeller University in the 1996 surveillance study and circled in gray are the 8 hospitals that collaborated again in 2013/14. Hospital XIII was added to the participating hospitals in 2013/14. Fig 1. Geographic location of hospitals that participated in the two surveillance studies. Numbers from I to XII represent the 12 hospitals that collaborated with the Rockefeller University in the 1996 surveillance study and circled in gray are the 8 hospitals that collaborated again in 2013/14. Hospital XIII was added to the participating hospitals in 2013/14. doi:10.1371/journal.pone.0156924.g001 eight hospitals (all except Kings County Hospital) also participated in the previous surveillance study in 1996. Hospital network The eight hospitals that participated in this study and their location within the greater New York City area are shown in Fig 1. These hospitals were as follows: VA Hospital (IV) and Kings County Hospital (XIII) in Brooklyn; Columbia University Medical Center (II) in Man- hattan; New York-Presbyterian Queens (VI) in Queens; St. Barnabas Hospital (IX) in the Bronx; Richmond University Medical Center (X) in Staten Island; Stony Brook Health Sciences Center (XI) in Long Island; and Westchester Medical Center (XII) in Valhalla. Seven of these 2 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 MRSA in Hospitals in NYC in 2014 Table 1. MRSA specimens provided by the participating hospitals. Table 1. MRSA specimens provided by the participating hospitals. 1996 2013/14 Hospital Location (Fig 1) Neighborhood # MRSA isolates analyzed Beds # MRSA isolates analyzed Beds Columbia University Medical Center II Manhattan 20 1475 39 1200 Kings County Hospital XIII Brooklyn — — 9 700 VA Hospital IV Brooklyn 15 324 2 340 New York-Presbyterian Queens Center VI Queens 28 487 9 539 St. Barnabas Hospital IX Bronx 18 458 8 417 Richmond University Medical Center X Staten Island 27 638 35 450 Stony Brook Health Sciences Center XI Stony Brook 19 536 10 597 Westchester Medical Center XII Valhalla 17 639 9 635 Total 144 4557 121 4878 Location: Roman numbering identifies the particular hospitals in Fig 1 and also corresponds to the listing in the 1996 surveillance. Some hospitals that collaborated at that time did not participate in 2013/14, due to closure or association with other institutions. # MRSA isolates analyzed: isolates of methicillin-resistant S. aureus provided by each hospital. # Beds: number of inpatient beds per hospital. doi:10 1371/journal pone 0156924 t001 2013/14 1996 Location: Roman numbering identifies the particular hospitals in Fig 1 and also corresponds to the listing in the 1996 surveillance. Some hospitals that collaborated at that time did not participate in 2013/14, due to closure or association with other institutions. # MRSA isolates analyzed: isolates of methicillin-resistant S. aureus provided by each hospital. # Beds: number of inpatient beds per hospital. doi:10.1371/journal.pone.0156924.t001 guidelines [15]. All isolates were grown overnight on Mannitol Salt Agar plates (MSA, Difco, BBL, Becton Dickinson, Franklin Lakes, NJ, USA) and were tested for coagulase agglutination (Staphaurex, Thermo Fisher Scientific, Lenexa, KS, USA) to confirm species identification. Antimicrobial susceptibility testing MRSA antibiograms were performed by either micro-dilution, E-test or disk-diffusion meth- ods, following the Clinical and Laboratory Standards Institute (CLSI) recommendations [16]. All specimens were tested for susceptibility to a number of antibiotics at the particular hospital providing the strains. Since not all hospitals tested the same antibiotics, in order to allow com- parisons, additional tests were performed at the Rockefeller University so all specimens were tested at least with the following antibiotics: penicillin, oxacillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, rifampicin, tetracycline, trimethoprim/sulfamethoxazole, linezolid, nitrofurantoin, chloramphenicol, daptomycin, vancomycin and mupirocin. Source of MRSA isolates MRSA isolates were obtained from the Pathology Departments of the participating hospitals. Individual centers were asked to submit single patient MRSA isolates obtained from inpatient cultures during an extended period from January 2013 to August 2014. The number of speci- mens provided by each institution was a convenience figure, calculated to be proportional to the sample size of 1996, the rates of sterile site infections in 2013 and the number of inpatient beds in each hospital (Table 1). The 121 bacterial isolates were inoculated on BHI, chocolate slants or blood agar plates for room temperature transportation to the Laboratory of Microbi- ology and Infectious Diseases at The Rockefeller University on the same day, following the U.S. Department of Transportation Pipeline and Hazardous Materials Safety Administration 3 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 Results Of the 12 New York City area hospitals that participated in the original surveillance study in 1996, seven also took part in the study described here. The list of participating hospitals is in Table 1 and their location is shown in Fig 1. Four of the hospitals that were part of the original study no longer exist: they closed or merged with other institutions. The molecular epidemiol- ogy of MRSA in the New York Presbyterian Hospital/Cornell Medical Center will be described in a separate communication. p The total number of beds in the participating hospitals was 4,878, ranging from 1,200 beds in hospital II to 340 in hospital IV. A total of 121 MRSA isolates were obtained in the surveil- lance study. With a median of ten samples per hospital, each center provided MRSA isolates ranging in number from eight to 39 (Table 1). Most MRSA (26%) was recovered from Medical services (Internal Medicine, Neurology, Cardiology, Oncology and Nephrology) and the sec- ond most frequent source was Pediatrics (25%). Information regarding the service attended at the time of the sample collection was not available for 12 of the samples: three samples origi- nating from the VA Hospital in Brooklyn and nine samples from the New York-Presbyterian Queens Hospital in Flushing. The age of patients ranged from 0 (sample taken at the time of delivery) to 94 years with a median age of 54 years old; 32% of patients were >60 years old but the age-group most often involved with MRSA infections was the 0–9 years group (21 cases). For a single patient the age was not available. Isolates were recovered from a variety of sources: 58 SSTIs (skin and soft tissue infections), 50 blood cultures, four respiratory samples, two sam- ples from a placental infection (mother and child), one urine sample, one cerebrospinal fluid sample, and one synovial fluid extraction. No biological source was available for three samples from the VA Hospital in Brooklyn and for one sample from the Richmond University Medical Center in Staten Island at the time of the molecular analysis. Of the 121 isolates characterized 86 (71%) presented a multi-drug resistant (MDR) pheno- type, i.e, they were resistant to at least three different classes of antibiotics. These MDR isolates included 20 of the 25 (80%) USA100 strains and 54 of the 78 (69%) USA300 strains. Molecular identification: spa typing, MLST, PFGE, SCCmec typing Molecular characterization of the 121 MRSA isolates was performed initially by spa typing as described [17] and using the RIDOM web server (http://spaserver.ridom.de/) for assignment of spa types. The spa server was also used to predict sequence types (ST). MLST was performed as previously described [18] when the spa server and the bibliography did not provide STs to the spa types obtained. Assignment of STs was done by DNA amplification and sequencing of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, yqiL) using the online MLST data- base (http://www.mlst.net/). Clonal Complexes were determined for the STs [19]. PFGE was performed to further confirm the relatedness of MRSA isolates belonging to the same clonal complex. Bacterial DNA was restricted with SmaI enzyme and the resulting frag- ments were separated by electrophoresis [20]. Band patterns were compared manually follow- ing guidelines to confirm classification [21,22]. The classification of staphylococcal cassette chromosome mec (SCCmec) carried by the iso- lates was determined using multiplex PCR, following previous guidelines [23,24]. Ambiguous results were further tested by amplification of the ccrB gene [25] and comparing the sequences 4 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 MRSA in Hospitals in NYC in 2014 obtained with the online database available at the Laboratory of Molecular Genetics at Instituto de Tecnologia Quimica e Biologica (ITQB) in Portugal. SCCmec was considered non-typable (NT) when it was not possible to ascertain a class of mec complex and/or a type of ccrB. SCCmec type IV subtyping was also performed by multiplex PCR as previously described [26]. Molecular characterization: detection of mecA, PVL, and ACME The mecA gene, responsible for resistance to oxacillin and other beta-lactam antibiotics and the lukS and lukF genes (which encode PVL, the Panton-Valentine leukocidin) were identified by PCR [27,28]. The arginine catabolic mobile element (ACME) element was identified and typed using primers that target its two main loci (arcA and opp3) in USA300 strain FPR3757 [29] and clas- sified according to its structure: type I (arc and opp3 operons), type II (arc operon only) and type III (opp3 operon only)[30]. Results One iso- late belonging to the USA100 clone was resistant to seven different antibiotics. Isolates belong- ing to clone USA100 were more frequently than USA300 isolates resistant to quinolones (92% vs 72%), clindamycin (84% vs 17%), gentamicin (8% vs 0%), rifampicin (8% vs 1%), daptomy- cin (12% vs 5%), and trimethoprim/sulfamethoxazole (12% vs 4%), while isolates of the USA300 clone were more often resistant to tetracycline (3% vs 0%) and to mupirocin (13% vs 8%). Both clones showed similar proportion of isolates resistant to erythromycin (92% of USA100 vs 90% of USA300 isolates). No isolates resistant to linezolid, nitrofurantoin, 5 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 AP: daptomycin; LZD: linezolid; NIT: nitrofurantoin; VAN: vancomycin; CHL: chloramphenicol. MRSA in Hospitals in NYC in 2014 Table 2. Antimicrobial resistance profiles of strains characterized in the study. TOTAL USA100 USA300 Others n No. R % R n No. R % R n No. R % R n No. R % R OXA 121 121 100% 25 25 100% 78 78 100% 18 18 100% CIP 121 90 74% 25 23 92% 78 56 72% 18 11 61% CLI 121 37 31% 25 21 84% 78 13 17% 18 3 17% ERY 121 104 86% 25 23 92% 78 70 90% 18 11 61% GEN 121 3 2% 25 2 8% 78 0 0% 18 1 6% RIF 121 4 3% 25 2 8% 78 1 1% 18 1 6% TET 121 5 4% 25 0 0% 78 2 3% 18 3 17% SXT 121 11 9% 25 3 12% 78 3 4% 18 5 28% MUP 121 13 11% 25 2 8% 78 10 13% 18 1 6% DAP 121 7 6% 25 3 12% 78 4 5% 18 0 0% LZD 121 0 0% 25 0 0% 78 0 0% 18 0 0% NIT 121 0 0% 25 0 0% 78 0 0% 18 0 0% VAN 121 0 0% 25 0 0% 78 0 0% 18 0 0% CHL 121 0 0% 25 0 0% 78 0 0% 18 0 0% OXA: oxacillin; CIP: ciprofloxacin; CLI: clindamycin; ERY: erythromycin; GEN: gentamicin; RIF: rifampin; TET: tetracycline; SXT: trimethoprim/ sulfamethoxazole; MUP: mupirocin; DAP: daptomycin; LZD: linezolid; NIT: nitrofurantoin; VAN: vancomycin; CHL: chloramphenicol. No. R: number of isolates resistant to a specific antibiotic; n: number of strains analyzed; %R: percentage of resistant isolates in each group. Highlighted are those antibiotics to which either USA100 or USA300 are more prone to be resistant. Table 2. Antimicrobial resistance profiles of strains characterized in the study. OXA: oxacillin; CIP: ciprofloxacin; CLI: clindamycin; ERY: erythromycin; GEN: gentamicin; RIF: rifampin; TET: tetracycline; SXT: trimethoprim/ sulfamethoxazole; MUP: mupirocin; DAP: daptomycin; LZD: linezolid; NIT: nitrofurantoin; VAN: vancomycin; CHL: chloramphenicol. No. R: number of isolates resistant to a specific antibiotic; n: number of strains analyzed; %R: percentage of resistant isolates in each group. Highlighted are those antibiotics to which either USA100 or USA300 are more prone to be resistant. sulfamethoxazole; MUP: mupirocin; DAP: daptomycin; LZD: linezolid; NIT: nitrofurantoin; VAN: vancomycin; CHL: chloramphenicol. No. R: number of isolates resistant to a specific antibiotic; n: number of strains analyzed; %R: percentage of resistant isolates in each group. Highlighted are those antibiotics to which either USA100 or USA300 are more prone to be resistant. doi:10.1371/journal.pone.0156924.t002 chloramphenicol or vancomycin were detected. Ten of the 13 isolates showing high resistance against mupirocin (MIC  1024 μg/ml) belonged to the USA300 clone. The antibiotic resis- tance patterns of MRSA isolates are shown in Table 2. The majority of the MRSA isolates (86 of the total of 121) belonged to a variety of commu- nity-associated MRSA clones, most of these (78 isolates) belonged to the USA300 clone (ST8/ SCCmecIV/PVL±/ACME±). Three were representatives of USA700 (ST72/SCCmecIV/ PVL-/ ACME—); two were USA1100 (ST30/SCCmecIV/PVL+/ACME—); one was USA400 (ST1/ SCCmecIV/PVL+/ACME—); one was USA1000 (ST59/SCCmecIV/PVL—/ACME—); and one isolate was ST88/SCCmecIVa/PVL+/ACME—. Molecular characterization of MRSA identified twenty-eight different spa types, which could be assigned to seven ST types. Of the total of 121 MRSA characterized, 35 belonged to typical hospital-associated clones: twenty-three of these were representatives of the USA100 (‘New York/Japan’) clone (ST5/SCCmecII/PVL—/ACME—) and two additional isolates shared the same PFGE/MLST/PVL/ACME profile but had a non-typable SCCmec cassette. There were three isolates belonging to the USA800 (‘Pediatric’) clone (ST5/SCCmecIV/PVL—/ ACME—); six isolates belonged to the USA500 clone (ST8/SCCmecIV/PVL—/ACME—) and one isolate had the same PFGE/MLST/PVL/ACME profile as USA500 but carried a non-typ- able SCCmec cassette. All but six isolates (72 of 78) belonging to the USA300 clone had the ACME virulence deter- minant but no strains belonging to the other clones carried ACME. The great majority (74 of 78) of USA300 isolates encoded the PVL toxin; only four isolates belonging to clones other than USA300 carried these genetic determinants. These were two isolates of the USA1100, one ST88 isolate and one USA400 isolate. None of the 35 HA-MRSA isolates carried either ACME or PVL determinants. 6 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 MRSA in Hospitals in NYC in 2014 Table 3. Distribution of MRSA clones and the different spa types and SCCmec types identified in this study. MRSA Clones na associated spa types b SCCmec types USA100 (NY/ Japan) 25 t002 (20), t062 (1), t071 (1), t088 (1), t306 (1), t856 (1) II, NTd (t002) USA300 78 t008 (62), t024 (1), t051 (1), t068 (2), t121 (3), t211 (3), t351 (1), t723 (1), t1635 (1), t2229 (1), t3908 (2) IVa USA400 1 t128 (1) IVa USA500 7 t008 (1), t064 (2), t211 (1), t394 (1), t1774 (1), t13975 (1)c IVg, NT (t211) USA700 3 t126 (1), t901 (1), t1346 (1) IVa (t126), IVh (t901), NT (t1346) USA800 3 t002 (3) IVh, IVnste USA1000 1 t216 (1) IVa USA1100 2 t665 (2) IVa ST88 1 t692 (1) IVa Total: 121 a Number of MRSA isolates belonging to a particular clone b Numbers in parenthesis represent the number of isolates with a particular spa type c t13975 was a new spa type identified for the first time in this study d Non-typable e Non-sub-typable doi:10.1371/journal.pone.0156924.t003 Table 3. Distribution of MRSA clones and the different spa types and SCCmec types identified in this study. doi:10.1371/journal.pone.0156924.t003 The currently major USA300 clone was represented by several spa variants (11 spa types) with t008 as the most prevalent type (63 of 78 isolates, 80.77%). The previously dominant MRSA clone USA100 (‘New York/Japan’ clone) was represented by 25 isolates, 20 of which (80%) had the t002 spa type. The spa types of the remaining clones are shown in Table 3. The staphylococcal cassette chromosome (SCCmec) was also characterized: all isolates car- ried variants of the type IV cassette, except the USA100 (‘New York/Japan’) isolates, which car- ried the SCCmec-II characteristic of this clone; the cassette was non-typable for two isolates belonging to the USA100 clone (t002) and one USA500 isolate (t211) (Table 3). Discussion The total number of beds in the hospitals collaborating in the 1996 surveillance study was 5,117 as compared to the 4,878 beds in the hospitals participating in the current (2013/14) sur- veillance. The total number of MRSA isolates characterized in 1996 was 270 and 121 in 2013/ 14. In the study described here, the patients were younger than in the previous surveillance: while 58% of patients were >60 years old in the 1996 study, that age group represented only 32% in the current in 2013/14 study. Medical services are still frequently affected by MRSA infections. In 1996 this group had 71% of all isolates although in the current surveillance this number was reduced to 26%. The second most frequently affected service—Pediatrics—had 25% of the MRSA cases in the present study. Comparison of the results of the surveillance study described here to the surveillance con- ducted 15 years earlier, in 1996 [2] show several striking differences. In 1996 a single clone, USA100 (‘New York/Japan’) was responsible for 42% of all MRSA infections in 12 New York City hospitals [2]. In the study described here, with MRSA isolates collected in 2013/14, a dif- ferent clone—USA300—was involved with most (64.4%) MRSA infections (Table 4). The other strains collected in 2013/2014 belonged to three different HA-MRSA clones: 25 were 7 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 MRSA in Hospitals in NYC in 2014 ones USA100, USA300 and other clonal types in New York City area hospitals during the two surveillance ion of MRSA clones USA100, USA300 and other clonal types in New York City area hospitals during the two Table 4. Representation of MRSA clones USA100, USA300 and other clonal types in New York City area hospitals during the two surveillance periods. USA100 (‘NY/Japan clone) ST5, SCCmecII USA300 clone ST8, SCCmecIVa Other Clonal Types Total No of MRSA isolates* 1996 2013/14 Hospital Location** No %# No %# No %# No(ǂ) %# No Columbia Presbyterian Medical Ctr II 5 25 1 2.5 34 87.0 4 (3) 10.2 39 Kings County Hospital XIII — — 3 37.5 2 80.0 3 (2) 37.5 8 VA Hospital IV 9 60 1 33.3 2 66.6 0 — 3 New York-Presbyterian Queens VI 15 53.6 1 11.1 5 55.5 3 (3) 33.3 9 St. Barnabas Hospital IX 5 27.8 2 25.0 5 62.5 1 12.5 8 Richmond University Medical Ctr X 9 33.3 11 31.4 20 57.1 4 (3) 11.4 35 Stony Brook Health Sciences Center XI 14 73.7 5 50.0 3 30.0 2 (2) 20.0 10 Westchester Medical Center XII 8 47.1 1 10.0 7 80.0 1 10.0 9 TOTAL 113 41.9 25 20.5 78 64.7 18 14.7 121 *Total number of MRSA isolates recovered and tested in 2013/14 surveillance **See Fig 1 # Clonal type in percentage of all MRSA identified in the hospitals (ǂ) Numbers in parentheses indicate the number of different clonal types of MRSA identified in the particular hospital. Table 4. Representation of MRSA clones USA100, USA300 and other clonal types in New York City area hos periods Fig 3. PFGE profiles of hospital isolates representing the major clonal types of MRSA identified in the 1996 (A) and the 2013/14 study (B). (A) PFGE of USA100 isolates in 1996 [2]. (B) PFGE of USA300 isolates in 2013/14. Migration on TBE-agarose gel after digestion with SmaI restriction enzyme. MRSA in Hospitals in NYC in 2014 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 9 / 13 MRSA in Hospitals in NYC in 2014 Hospital *Total number of MRSA isolates recovered and tested in 2013/14 surveillance # Clonal type in percentage of all MRSA identified in the hospitals (ǂ) Numbers in parentheses indicate the number of different clonal types of MRSA identified in the particular hospital. # Clonal type in percentage of all MRSA identified in the hospitals (ǂ) Numbers in parentheses indicate the number of different clonal types of MRSA identified in the particular hospital. representatives of the USA100 clone, seven isolates of USA500 and three to the USA800 clone. The remaining 86 isolates belonged to six community-acquired MRSA lineages: USA300 (78 isolates), USA700 (three isolates), USA1100 (two isolates), USA400 (one isolate), USA1000 (one isolate) and one ST88 isolate. The change in clonal type of MRSA was also accompanied by a change in the type of infec- tions. While in 1996 MRSA was mainly recovered from the respiratory tract (44%), most of the MRSA infections in 2013/14 were skin and soft tissues (SSTIs) (48%). Also, bacteremia/sepsis has increased from 17.5% of MRSA infections in 1996 to 41.3% in 2013/14. Fig 2 illustrates the change in the types of lesions and Fig 3 shows the predominant MRSA clones in 1996 and in 2013/14. Fig 2. Change in the clinical sources of MRSA from 1996 to 2014. ‘Respiratory tract’: lower respiratory tract (including sputum and bronch-alveolar lavage) and sinusitis and pleural fluid. ‘Other’: any other biological specimen from which MRSA was isolated at any of the hospitals including the urinary tract, cerebro-spinal fluid, synovial fluid, placental biopsy and unlisted specimens. doi:10.1371/journal.pone.0156924.g002 Fig 2. Change in the clinical sources of MRSA from 1996 to 2014. ‘Respiratory tract’: lower respiratory tract (including sputum and bronch-alveolar lavage) and sinusitis and pleural fluid. ‘Other’: any other biological specimen from which MRSA was isolated at any of the hospitals including the urinary tract, cerebro-spinal fluid, synovial fluid, placental biopsy and unlisted specimens. doi:10 1371/journal pone 0156924 g002 doi:10.1371/journal.pone.0156924.g002 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 8 / 13 MRSA in Hospitals in NYC in 2014 USA100 (‘New York/Japan’) was the predominant clone in 1996, and the second most frequent clone in 2013–14. The roman numbers on each strain indicate the hospital in which they were isolated. doi:10.1371/journal.pone.0156924.g003 doi:10.1371/journal.pone.0156924.g003 While the MRSA clone USA300 is known to have a strong association with SSTIs [31,32], there is no specific association known between the USA100 clone and respiratory infections. A recent report from the CDC (Emerging Infections Program Healthcare-Associated Infections and Antimicrobial Use Prevalence Survey Team) concluded that S. aureus (together with Kleb- siella oxytoca and K. pneumoniae) were currently the main pathogens responsible for respira- tory tract and skin and soft infections [33]. As to the possible mechanism that has led to the changed clonal types between the current and the previous (1996) surveillance, we hypothesize that the infection control mechanisms introduced in the healthcare system may have succeeded in controlling the spread of HA-MRSA clones like USA100, while the entry and spread of community-associated MRSA strains in hospitals may be responsible for the increase of SSTIs and secondary bacteremia/sep- sis [9,34]. Recent reports indicate that the number of invasive MRSA infections decreased in the United States in 2011 as compared to 2005, but more MRSA infections occurred in the community than during hospitalization in 2011 [35,36]. The hospital as a source of MRSA infections seems to have become better controlled, but the frequency of MRSA infections in the community appears to be increasing. Several other studies have documented the “escape” of MRSA clones from hospitals to the healthy community and to public transportation [37,38]. New clones circulating in both the community and hospitals also come with a change in patient demographics, i.e., younger patients presenting with different types of infections and with an increase in SSTIs and blood infections and decrease in respiratory infections caused by MRSA [3,39,40]. The change in clonal type of MRSA strains in hospitals between the two studies in 1996 and 2013/14 is also associated with a change in the pattern of antibiotic resistance. In the study per- formed in 1996, where the USA100 clone was predominant, up to 96% of strains analyzed were resistant to ciprofloxacin. In 2013/14 only 74% of strains showed this resistance phenotype. PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 Fig 3. PFGE profiles of hospital isolates representing the major clonal types of MRSA identified in the 1996 (A) and the 2013/14 study (B). (A) PFGE of USA100 isolates in 1996 [2]. (B) PFGE of USA300 isolates in 2013/14. Migration on TBE-agarose gel after digestion with SmaI restriction enzyme. Fig 3. PFGE profiles of hospital isolates representing the major clonal types of MRSA identified in the 1996 (A) and the 2013/14 study (B). (A) PFGE of USA100 isolates in 1996 [2]. (B) PFGE of USA300 isolates in 2013/14. Migration on TBE-agarose gel after digestion with SmaI restriction enzyme. Fig 3. PFGE profiles of hospital isolates representing the major clonal types of MRSA identified in the 1996 (A) and the 2013/14 study (B). (A) PFGE of USA100 isolates in 1996 [2]. (B) PFGE of USA300 isolates in 2013/14. Migration on TBE-agarose gel after digestion with SmaI restriction enzyme. PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 9 / 13 MRSA in Hospitals in NYC in 2014 98 out of the 121 hospital isolates studied here were harboring the type IV SCCmec cassette, compared to the 23 isolates belonging to USA100 which carried the SCCmec cassette type II. The smaller size of the type IV cassette has been postulated as an evolutionary advantage for MRSA clones carrying this cassette [8,9,31]. 98 out of the 121 hospital isolates studied here were harboring the type IV SCCmec cassette, compared to the 23 isolates belonging to USA100 which carried the SCCmec cassette type II. The smaller size of the type IV cassette has been postulated as an evolutionary advantage for MRSA clones carrying this cassette [8,9,31]. The virulence factors ACME and PVL are often carried by CA-MRSA strains even when these clones are recovered in the hospital setting. In our study, ACME was exclusively carried by USA300 strains while the PVL was present in other CA-MRSA clones as well. Several factors may have contributed to the shift in the clonal type of MRSA from the “New York/Japan clone” dominant in the 1996 surveillance to the MRSA clone USA300 most preva- lent in 2013/14. These factors may include the smaller (type IV) SCCmec cassette carried by the USA300 clone and also the presence of virulence factors like ACME and PVL in the MRSA clone USA300, which seems to have emerged as the most prevalent clone both in hospitals and in the community [11,31,43]. Author Contributions Conceived and designed the experiments: MPG HdL AT. Performed the experiments: MPG. Analyzed the data: MPG HdL AT. Contributed reagents/materials/analysis tools: MC JB DB PDL VK JQ ES RT CU GW SW. Wrote the paper: MPG AT. PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 This may be due to the current predominance of strains belonging to the USA300 clone (72% of them resistant to ciprofloxacin), as resistance to quinolones reached 92% of strains belong- ing to the USA100 clone. Similarly, resistance to clindamycin affected 88% of strains in 1996, while only 31% of strains in 2013/14 were resistant to this antibiotic, and 84% of the clindamy- cin resistant strains belonged to the USA100 clone. In addition, in 1996 it was reported that 58% of the characterized strains were resistant to gentamicin while in 2013/14 only three iso- lates showed this phenotype. Following the recommendations of the Infectious Diseases Society of America [41] vanco- mycin has been the antibiotic of choice against MRSA invasive infections in hospitalized patients in each of the hospitals participating in this study. All MRSA isolates characterized in this study were susceptible to this antibiotic. Other therapeutic options such as daptomycin, clindamycin, rifampin, gentamicin or trimethoprim/sulfamethoxazole have also been used– alone or in combination–and the high rates of resistance observed in our study to these antibi- otics underlines the importance of keeping track of the drug resistance mechanisms of MRSA strains circulating in a hospital–in order to prevent possible treatment failures. It should also be noted that a number of isolates belonging to the currently predominant MRSA clone USA300 –also show resistance to mupirocin (see Table 2)–an agent frequently used for decolo- nization and topical treatment [42]. It is interesting that the SCCmec type IV, common in community-associated MRSA (CA-MRSA), is also present in some HA-MRSA clones like USA500 or USA800. As many as 10 / 13 PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 References New York State HOSPITAL-ACQUIRED INFECTION REPORTING SYSTEM 2008. New York State Department of Health; 2009 May pp. 1–132. 13. 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PLOS ONE | DOI:10.1371/journal.pone.0156924 June 7, 2016 References JAMA Intern Med. 2013; 173: 1970–1978. doi: 10.1001/jamainternmed.2013.10423 PMID: 24043270 36. David MZ, Boyle-Vavra S, Zychowski DL, Daum RS. Methicillin-susceptible Staphylococcus aureus as a predominantly healthcare-associated pathogen: a possible reversal of roles? PLoS One. 2011; 6: e18217. doi: 10.1371/journal.pone.0018217 PMID: 21533238 37. Espadinha D, Faria NA, Miragaia M, Lito LM, Melo-Cristino J, de Lencastre H, et al. Extensive dissemi- nation of methicillin-resistant Staphylococcus aureus (MRSA) between the hospital and the community in a country with a high prevalence of nosocomial MRSA. PLoS One. 2013; 8: e59960. doi: 10.1371/ journal.pone.0059960 PMID: 23593155 38. Simões RR, Aires-de-Sousa M, Conceição T, Antunes F, da Costa PM, de Lencastre H. High preva- lence of EMRSA-15 in Portuguese public buses: a worrisome finding. PLoS One. 2011; 6: e17630. doi: 10.1371/journal.pone.0017630 PMID: 21407807 39. Deleo FR, Otto M, Kreiswirth BN, Chambers HF. Community-associated meticillin-resistant Staphylo- coccus aureus. Lancet. 2010 ed. 2010; 375: 1557–1568. doi: 10.1016/S0140-6736(09)61999-1 PMID: 20206987 40. David MZ, Cadilla A, Boyle-Vavra S, Daum RS. Replacement of HA-MRSA by CA-MRSA Infections at an Academic Medical Center in the Midwestern United States, 2004–5 to 2008. PLoS One. 2014; 9: e92760. doi: 10.1371/journal.pone.0092760 PMID: 24755631 41. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, et al. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary. Clin Infect Dis. 2011; 52: 285–292. doi: 10.1093/cid/cir034 PMID: 21217178 42. Chadha P, Mariano N, LaBombardi V. In Vitro Activities of Mupirocin, Tigecycline, Ceftaroline, Vanco- mycin, Linezolid and Daptomycin in Clinical Isolates of Methicillin-Resistant Staphylococcus by E-Test Methodology. Open Journal of Medical Microbiology. 2015; 12–16. 43. Pardos de la Gandara M, Raygoza Garay JA, Mwangi M, Tobin JN, Tsang A, Khalida C, et al. Molecular Types of MRSA and MSSA Strains Causing Skin and Soft Tissue Infections and Nasal Colonization— Identified in Community Health Centers in New York City. J Clin Microbiol. 2015; 2648–2658. doi: 10. 1128/JCM.00591-15 PMID: 26063853 13 / 13
https://openalex.org/W1555376292
https://respiratory-research.biomedcentral.com/track/pdf/10.1186/rr-2001-68572
English
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Influenza A infection interferes with the development of asthma
Respiratory research
2,000
cc-by
665
PublisherInfo PublisherName : BioMed Central PublisherLocation : London PublisherImprintName : BioMed Central PublisherInfo PublisherName : BioMed Central PublisherLocation : London PublisherImprintName : BioMed Central PublisherInfo PublisherName : BioMed Central PublisherLocation : London PublisherImprintName : BioMed Central Influenza A infection interferes with the development of asthma ArticleInfo ArticleID : 1640 ArticleDOI : 10.1186/rr-2001-68572 ArticleCitationID : 68572 ArticleSequenceNumber : 51 ArticleCategory : Paper Report ArticleFirstPage : 1 ArticleLastPage : 1 ArticleHistory : RegistrationDate : 2001–9–19 Received : 2000–9–29 Accepted : 2001–9–19 OnlineDate : 2001–9–19 ArticleCopyright : Biomed Central Ltd2001 ArticleGrants : ArticleContext : 129312211 Influenza A infection interferes with the development of asthma Context The effects of viral infections of the respiratory tract on the development of respiratory allergen sensitization and asthma are discussed. Several epidemiological studies provide evidence that viral infections in early childhood protect against the development of asthma and allergies, however, other studies have shown that viral infections enhance the risk of asthma development in children. In a murine model of asthma, inhalation of antigen induces CD4 T cell tolerance and prevents airway hyper- reactivity (AHR) and the production of antigen-specific IgE. This study used the same murine model to investigate the effects of influenza A virus infection on the induction of antigen tolerance to ovalbumin (OVA) and the development of AHR. Keywords Allergen sensitization, asthma, bronchial hyper-reactivity, influenza A virus infection, mouse model Aff1 Wellcome Trust Centre for Human Genetics, Oxford, UK Aff1 Wellcome Trust Centre for Human Genetics, Oxford, UK Influenza A infection interferes with the development of asthma Influenza A infection interferes with the de ArticleInfo ArticleID : 1640 ArticleDOI : 10.1186/rr-2001-68572 ArticleCitationID : 68572 ArticleSequenceNumber : 51 ArticleCategory : Paper Report ArticleFirstPage : 1 ArticleLastPage : 1 ArticleHistory : RegistrationDate : 2001–9–19 Received : 2000–9–29 Accepted : 2001–9–19 OnlineDate : 2001–9–19 ArticleCopyright : Biomed Central Ltd2001 ArticleGrants : ArticleContext : 129312211 Andrea Heinzmann,Aff1 Corresponding Affiliation: Aff1 Aff1 Wellcome Trust Centre for Human Genetics, Oxford, UK Significant findings Mice exposed to OVA intranasally three days after infection with influenza A virus showed an increased production of interleukin (IL)-4, IL-5, IL-13, IFN-? and OVA-specific IgE and subsequently developed AHR. Neutralization of both IL-4 and IL-5 did not abrogate the induction of AHR, but greatly reduced lung eosinophilia. In contrast, respiratory exposure to OVA 15-30 days after infection abrogated the induction of an allergen-specific tolerance but led to a Th1-biased immune response with little or no production of IL-4, IL-5 and IL-13. Moreover it protected from the development of AHR. This protection was abrogated by neutralizing IFN-?. The authors suggest that the time interval between viral infection and respiratory allergen exposure is crucial in determining whether viral infection will enhance, or protect against, the development of respiratory allergen sensitization and asthma. Methods BALB/c mice, whole body plethysmograph, broncoalveolar lavage and cell count, lung histology, in vitro proliferation assay, cytokine assay, ELISA Comments Several earlier studies used murine models of asthma to investigate the effect of influenza A infection on allergen sensitization and AHR. However, this is the first to report on the relationship between the phase of influenza A viral infection and antigen exposure on the development of AHR. The results suggest that early exposure to respiratory allergen during viral infection biases towards a Th2-permissive immune response, with the development of significant AHR and eosinophilia. In contrast, exposure in late stages of infection leads to a Th1-predominant response and decreased airway reactivity. This might at least partially explain the conflicting findings of earlier studies cited above; however, it should be noted that the study has been performed in a rodent model and still needs confirmation in humans. Furthermore, it might prove useful in future studies to investigate the composition of inflammatory cells in the bronchial mucosa at different stages of viral infection and their potential influence on the sensitization outcome. References 1. Tsitoura DC, Kim S, Dabbagh K, Berry G, Lewis DB, Umetsu DT: Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens. J Immunol. 2000, 165: 3484-4391.
https://openalex.org/W2238801732
http://www.sciedupress.com/journal/index.php/ijfr/article/download/8709/5240
English
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An Analysis on the Behaviour of Corporate Social Responsibility towards Profitability of Islamic Banks: Asean and Europe
International journal of financial research
2,015
cc-by
8,504
International Journal of Financial Research International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 1; 2016 Accepted: December 29, 2015 Accepted: December 29, 2015 An Analysis on the Behaviour of Corporate Social Responsibility towards Profitability of Islamic Banks: Asean and Europe Akhmad Affandi Mahfudz1, Muhammad Safizal Abdullah2, Arman Hadi Abdul Manaf3 & Abdullah Osman4 1 College of Business, Universiti Utara Malaysia, Sintok, Kedah, Malaysia 2 School of Business Innovation & Technopreneurship, Kangar, Perlis, Malaysia 3 Faculty of Business Administration, Kanagawa University, Hiratsuka-City, Japan 4 Kulliyyah Muamalat, Insaniah University College, Kuala Ketil, Kedah, Malaysia Correspondence: Arman Hadi Abdul Manaf, Faculty of Business Administration, Kanagawa University, Hiratsuka-City, 259-1293, Japan. Tel: 81-463-59-4111 ext. 2311. mber 29, 2015 Online Published: January 11, 2016 URL: http://dx.doi.org/10.5430/ijfr.v7n1p154 Received: December 9, 2015 doi:10.5430/ijfr.v7n1p154 Received: December 9, 2015 doi:10.5430/ijfr.v7n1p154 Published by Sciedu Press Abstract This study aims to investigate the stability of profitability of Islamic banks resulted from the behaviour of corporate social responsibility in the short term as well as long term taking Indonesia as representative for Asean perspective and Turkey for Europe perspective. The study employs Vector Autoregression (VAR) and followed by Vector Error Correction Models (VECM) if there is co-integration. The IRF (Impulse Response Function) denotes different findings whilst Variance Decomposition emphasizes the most affected profitability variables resulted from the behaviour of corporate social responsibility. IRF result shows only return on asset of Islamic bank in Turkey found to have stability whether in the short terms or long terms. The other variables concluded to have similar pattern for Turkey and Indonesia as they tend to decline even very sharp in the long terms except return on equity for Indonesia has positive response where it tends to increase regardless of the changes in the behaviour or shock of corporate social responsibility. The behaviour of corporate social responsibility in Indonesia mostly influenced return on asset while it influences greatly towards return on equity for Islamic banks in Turkey. The application of corporate social responsibility varies depending on the policy of respective banks that linked to normative and perception of the bank and appears to be more important in disclosing of non-financial information in the annual report. The findings reveal that quite a few challenges lie ahead in shaping proper behaviour of corporate social responsibility that affect profitability of Islamic banks in Indonesia and Turkey. This needs to be taken on promptly by management teams of Islamic banks especially in Indonesia that focus on corporate social responsibility for Muslim society. While profitability variables in Turkey would affect the proper function of corporate social responsibility that aimed for social benefit. This paper is one of few studies which employ VAR/VECM model to investigate and forecast the shock or behaviour of corporate social responsibility towards profitability of Islamic banks in a country who adopt dual banking systems. Keywords: corporate social responsibility, Islamic bank, vector error-correction model, vector auto-regression 1 i 1.1 Background ere were also 23 Islamic Business Unit and 154 Islamic Rural Banks as demonstrated in Table 1: In addition, there were also 23 Islamic Business Unit and 154 Islamic Rural Banks as demonstrated in Table 1: ere were also 23 Islamic Business Unit and 154 Islamic Rural Banks as demonstrated in Table 1: Table 1. Network of Islamic banking 2005 2006 2007 2008 2009 2010 Sept’11 Islamic Banks Islamic Commercial Bank 3 3 3 5 6 11 11 Islamic Business Unit 19 20 26 27 25 23 23 Islamic Rural Bank 92 105 114 131 138 150 154 Office Network Islamic Commercial Bank 304 349 401 581 711 1.215 1349 Islamic Business Unit 154 183 196 241 287 262 300 Islamic Rural Bank 92 105 185 202 225 286 362 Source: Directorate of Islamic Banking, Bank Indonesia 2011 Meanwhile, the situation differs profoundly in Turkey whilst it makes sense for the Islamic financial institutions to foster growth and to regularly measure themselves from the very start in 1985 with Albaraka Türk, the Turkish participation banks (before called Special Finance Houses) were poised to aim at the Turkish market as a whole. Participation banks did not really target the small niche of Muslim population in particular. This of course influenced marketing and product development. As a consequence of this strategy (and compared to Indonesia) the current market share of participation banks in Turkey is a mere 6%. It is noted that the strong growth of the sector that outperforms the conventional counterpart now for 8 consecutive years. The total assets of participation banks in 2010 increased by 25% reaching $28.1 billion; deposits increased by 22% to reach $21.9 billion, of which 66% were in Turkish lira and 34% in foreign currencies; financing allocated by the participation banks grew by 25% to reach $20.8 billion; total shareholders' equity increased by 19% to $3.5 billion; net income however increased by 4% to reach $491.6 million; and the number of branches and number of staff of participation banks totaled 607 and 12,694 respectively growing at a year-on-year rate of 8%. Similarly, the market share of participation banks assets is 4.31%, deposits is 5.4% and financing 6%. The financing to deposit ratio of participation banks in 2010 was 96% compared to 83% for the conventional banking sector in Turkey. In terms of non-performing loans, participation banks also fared better. Published by Sciedu Press 1.1 Background The historical development of Islamic banking in Indonesia started in the 1980s whilst various discussions have been emerged on the establishment of Islamic banking. However, the implementation of these discussions being materialized in 1992 through the establishment of the first Islamic bank, namely Bank Muamalat Indonesia. The Stretch of Islamic banking became increasingly crowded after severe economic crisis had rocked Indonesia in 1998. Having proved that Islamic banking is more resistant to the shocks of economic crises, these conditions have encouraged conventional banks to establish Islamic banks or open an Islamic business unit (Affandi, 2007:2). As from the very start, products and marketing were directly aimed at precisely group of Muslim clients. Foreign banks are not really visible yet on the Indonesian Islamic finance market. HSBC pioneered as first big international institution with a full dedicated Shari'ah head office in Jakarta besides Albaraka Group had started to open their branch offices. It is recorded that the total assets of Islamic banking industry has increased significantly each year from IDR 1.79 ISSN 1923-4023 E-ISSN 1923-4031 ISSN 1923-4023 E-ISSN 1923-4031 154 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 1; 2016 trillion in 2000, became IDR 49, 6 trillion by the end of 2008. In November 2009 it rose to IDR 61.4 trillion and dramatically increased in 2010 by IDR 89 trillion. Asset growth sustained its upward momentum every year which is recorded 46.3%. trillion in 2000, became IDR 49, 6 trillion by the end of 2008. In November 2009 it rose to IDR 61.4 trillion and dramatically increased in 2010 by IDR 89 trillion. Asset growth sustained its upward momentum every year which is recorded 46.3%. However, in 2009 the growth of Islamic banking was only 26.5% with a market share of 2.4% and rebound again with a growth of 46% in 2010. High asset growth is characterized by a growing number of conventional banks to establish Islamic Banks and Syariah Business Unit. It is recorded that in 2011, there were 11 Islamic Commercial Banks like Bank Muamalat, Bank Syariah Mandiri, Bank Rakyat Indonesia Syariah, Bank Mega Syariah, Bank Syariah Bukopin, and most recently Bank Victoria Syariah and Bank Panin Syariah. 2.1 Theoretical Foundation Several theories are relevant in the sphere of corporate social responsibility namely, stakeholder theory, legitimacy theory and proprietary cost theory. The distinction among these theories is often unclear and it appears that there is a great deal of overlap because all the theories are concerned with the interplay between the corporations and its stakeholders. The main difference between them appears to be the viewpoint from which they are observed and tested (Mia & Al-Mamun, 2011). Since the notion of corporate social responsibility varies with respect of concept, application and disclosures, thus it is crucial to review Islamic principles and structured definition of corporate social responsibility in the Islamic financial institutions (IFIs). Standard setting bodies such as the AAOIFI have the ability to influence the activities of Islamic Financial Institutions by giving them flexible Islamic corporate social responsibility provisions that they can adopt depending on their underlying economic capacity. It is hoped that these standards will provide the impetus, long desired in the Islamic community, for IFIs to incorporate Islamic social responsibility principles in all aspects of their activities (Farook, 2007). Information asymmetry problems are present in every relationship characterized by parties with information differences and conflicting interests (Akerlof, 1970). Since information asymmetry exists between the insiders of a company, i.e. the management team and the outsiders of the company, i.e. stakeholders, voluntary disclosure can be seen as originated from a principal-agent problem (Jensen & Mecklingm 1976). Thus to reduce information asymmetry and, thereby, mitigate agency problems a company can choose to disclose voluntary information that exceeds mandatory disclosure regulations. In the social and environmental reporting practices, banks are recognized as forefront players because their dual interest. First, in accessing disclosures from the corporation with which they do business and second as disclosures of information for their own stakeholders (Crawford & Williams, 2010). Stakeholders also demanding more accountability from their corporate investment while banks also demanding more information from their own investors, creating and responding simultaneously to transparency pressures. The Islamic concept of corporate governance is in line with the concept of corporate social responsibility proposed by scholars (Bhatti & Bhatti, 2009). Promoting social justice and accountability is included in the Islamic financial institution’s objectives of complying with the Shari’ah (Farook & Lanis 2005). From the Islamic perspective, the moral objectives of society must be integrated into the objectives and business strategies of Islamic financial institutions. 2.1 Theoretical Foundation Thus, for Islamic financial institutions, corporate social responsibility is a major condition of their business activity (Hassan & Abdul Latiff 2009). In fact, the AAOIFI standard defines corporate governance to include corporate social responsibility disclosures. This is reflected by the inclusion of the corporate social responsibility standard in the AAOIFI’s governance standard (Governance Standard for Islamic Financial Institutions No. 7: Corporate Social Responsibility Conduct and Disclosure for Islamic Financial Institutions). In determining the appropriate corporate governance structure for Islamic financial institutions, Islamic law, Islamic economic and financial principles must be considered. The principles of Islamic finance for instance, zakat, prohibition on riba, prohibition on gharar, prohibition on hoarding and economic systems based on the profit and loss sharing, will affect the structure of corporate governance of Islamic financial institutions. Bhatti & Bhatti (2010) claim that the structure of Islamic corporate governance is, more or less, similar to the conventional corporate governance structure, for instance, the OECD Principles of Corporate Governance, but within the religious-based moral codes of Islam. Published by Sciedu Press 1.1 Background The NPL for participation banks in 2010 was 341 million Turkish liras or 0.34% compared to 379 million liras or 0.47% in 2009. As such Turkish participation banks are increasingly exploring the possibilities of raising funds through a Sukuk issuance, albeit dependent on the quality and volume of available Ijara assets that can be securitized. However, there is no specific government aid to the sector and no (government or corporate) Islamic bond, no access to the money market as well as no Islamic windows and therefore foreign market players can only enter the market through shareholdings in the existing participation banks (or have to start their own participation bank from scratch). On the other side, the corporate social responsibility of Islamic banks in Indonesia and Turkey marked by different contributions, philanthropy, understanding and concept. As for Indonesia, the concept of Zakat, Shadaqah, Infaq and other charity benefits are inherent as part of social contribution, in fact they establish ‘Amil of Zakat institution in every bank to manage and disburse these funds to the destitute and the needy. Apart from this, corporate social responsibility also implemented through economic empowerment program to upgrade the life of the poor, extending financial assistance to the victim of natural disasters and other social activities. Meanwhile, the implementation of corporate social responsibility in Turkey not specifically addressed to the Muslim population. The contribution mostly dedicated for social activities such as giving sponsorship for university students, main sponsor for national sports, culture and arts sponsorship. The concept of corporate social contribution viewed as an investment regardless of social background and furthermore, it is not implicitly mentioned in the annual financial statement of every participation bank. ISSN 1923-4023 E-ISSN 1923-4031 155 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com 1.2 Research Question What is the influence of corporate social responsibility behavior towards profitability of Islamic banks in the short terms and long terms in Indonesia and Turkey? 1.3 Objective of Study To examine the influence of behavior or shocks in corporate social responsibility towards profitability of Islamic banks in the short term as well as long term in both countries. 2.3 Corporate Social Responsibility and Financial Performance in Islamic Banks The finding of this study indicates that corporate social responsibility disclosures in annual reports (in the form of disclosure on the Shari’ah Supervisory Board and disclosure on zakat) are still limited. 2.2 Disclosure of Corporate Social Responsibility Corporate social responsibility is prominent and evident more than ever due to the emphasis laid down on business regarding environmental, social and ethical issues. The level of corporate social responsibility activities of the firm is made known to public only through the disclosures. There are two kinds of disclosures, mandatory and voluntary. Voluntary disclosures are given by companies to improve the firm’s performance and reputation. This disclosure affects the firm’s corporate social responsibility and financial performance. There are various factors which determine the amount of disclosures like the size of the firm, industry and high visibility (Kavitha & Anita, 2011). ISSN 1923-4023 E-ISSN 1923-4031 156 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com Since the mid-1990s there has been a general increase in the demand for greater corporate accountability as evidenced by the numerous stakeholder requests for more transparent information (Anonymous, 2002; Owen et al., 2001; Swift, 2001). There are several reasons; First, stakeholders demanding more transparent financial report. Second, there is an increased of public awareness that climate change represents real environmental risks for corporation and addressed as company’s strategic planning. Third, big corporation such as Nike and Wal-Mart have been the public target of activists and suffered material reputation and brand damage as result of their mismanagement of social issues. Since the mid-1990s there has been a general increase in the demand for greater corporate accountability as evidenced by the numerous stakeholder requests for more transparent information (Anonymous, 2002; Owen et al., 2001; Swift, 2001). There are several reasons; First, stakeholders demanding more transparent financial report. Second, there is an increased of public awareness that climate change represents real environmental risks for corporation and addressed as company’s strategic planning. Third, big corporation such as Nike and Wal-Mart have been the public target of activists and suffered material reputation and brand damage as result of their mismanagement of social issues. A study on the disclosure of non-financial information in the annual report was revealed by (Arvidsson, 2011). The findings indicate that the voluntary disclosure compensates for the deficiencies of financial statements to properly disclose intangible asset. This may lessen the risk of the argued impairment of the efficient allocation of resources on the stock market. 2.3 Corporate Social Responsibility and Financial Performance in Islamic Banks 2.3 Corporate Social Responsibility and Financial Performance in Islamic Banks Specific study on the relationship of corporate social responsibility towards profitability of the banks in Indonesia was conducted by (Cahya, 2010) taking the sample of 226 local banks. The study employs multiple regressions through several tests like t-test, F-test, coefficient of determination and the test of classical problems in multiple regressions. The result of t-test showed that corporate social responsibility has no relation with return on asset while the result of F-test revealed that return on asset and leverage have close relation with corporate social responsibility. The topic of social responsibility and ethical banking is of relevance especially for those involved in Islamic banking and finance who regard their ethics and social responsibility as being more enduring since they are ultimately based on divine revelation, whereas ethics derived from secularist morality are inevitably transitory. The issue of corporate social responsibility not foreign to Islamic banking. Rather, it shows the relevance of corporate social responsibility as a globally accepted practise to Islamic banks. Indeed, Islamic banking should endeavour to be the epicentre in the financial galaxy of promoting good corporate social responsibility practises. In this respect, an assimilation of corporate social responsibility and other Islamic ideals in fulfilling stakeholders’ expectations deserves utmost consideration as it represents a fundamental difference between Islamic and conventional banking, and has the potential to propel Islamic banking to greater heights in securing stakeholders’ recognition and acceptance (Dusuki & Dar, 2007). The Board's role in assuring stakeholders that the Islamic financial institution has complied with the Shari’ah (Islamic law) is crucial. Hence, some of the most important information to be presented in the annual reports of Islamic financial institutions is transparent disclosures about the activities of the Shari’ah Supervisory Board. The study on Shariah supervisory board disclosure as part of corporate social responsibility. It examines the extent of disclosure on the Shari’ah Supervisory Board as well as the content of the Board's report in the annual reports of the 18 Islamic banks in Malaysia and four Islamic banks in Indonesia. The study also investigates the disclosure on zakat (Islamic levy) which is regarded as an important corporate social responsibility disclosure of an Islamic organization. The finding of this study indicates that corporate social responsibility disclosures in annual reports (in the form of disclosure on the Shari’ah Supervisory Board and disclosure on zakat) are still limited. 3.1 Sources of Data Secondary data and time series data of this study were generated from respective Islamic banks of Indonesia and Turkey in the form of monthly data from January 2006 to December 2010. In this regard, Islamic banks in Indonesia represented by Bank Muamalat Indonesia, Bank Syariah Mandiri and Bank Syariah Mega Indonesia. As for Turkey, participation banks represented by Bank Asya, Albaraka Turk Participation Bank and Turkeye Finans Katilim Bankasi. By way of generating time series data from respective countries (2006-2010) comprising Return on Asset, Return on Equity, Operating Efficiency Ratio and Corporate Social Responsibility, in a mathematic form these data can be formulated as follow: Indonesia:           OER ROE ROA CSR 3 2 1 0 (1) Turkey:           OER ROE ROA CSR 3 2 1 0 (2) (1) (2) (1) (2) However, in order to have equality and the data can be compared, then the original time series data in nominal terms, such as CSR transformed in real terms. While the data in the form of percentage such as return on asset, return on equity and operating efficiency ratio are generally not necessary to be transformed. Therefore the above model then becomes: Indonesia:           OER ROE ROA CSR 3 2 1 0 ln (3) Turkey:           OER ROE ROA CSR 3 2 1 0 ln (4) (3) (4) (3) (4) Published by Sciedu Press 157 ISSN 1923-4023 E-ISSN 1923-4031 157 ISSN 1923-4023 E-ISSN 1923-4031 ISSN 1923-4023 E-ISSN 1923-4031 157 ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press Published by Sciedu Press IInternational Jouurnal of Financiaal Research Vol. 7, No. 1; 2016 http://ijfr.sciiedupress.com 3.2 Hypothheses 3.2 Hypoth The prelim hypothesis heses minary conclusi are as follow: on from this reesearch will bee conducted bassed on theory aand previous research studiess using The prelim hypothesis minary conclusi are as follow: on from this reesearch will bee conducted bassed on theory aand previous research studiess using The prelim hypothesis minary conclusi are as follow: on from this reesearch will bee conducted bassed on theory aand previous research studiess using Ho = NNo relationshipp between corpporate social reesponsibility annd protabililty of Islamic Bannks. Ho = NNo relationshipp between corpporate social reesponsibility annd protabililty of Islamic Bannks. H1 = TThere is relatioonship betweenn corporate soccial responsibillity and profitab ability of Islamiic b H1 = TThere is relatioonship betweenn corporate soccial responsibillity and profitab ability of Islamiic banks. Ho = T i The relationshi is same as in T ip between cor Turkey. rporate social responsibility aand profitabilityy of Islamic baanking in Indonnesia Ho = T i The relationshi is same as in T ip between cor Turkey. rporate social responsibility aand profitabilityy of Islamic baanking in Indonnesia H2 = T i The relationshi is not the same ip between cor e as in Turkey. rporate social responsibility aand profitabilityy of Islamic baanking in Indonnesia H2 = T i The relationshi is not the same ip between cor e as in Turkey. rporate social responsibility aand profitabilityy of Islamic baanking in Indonnesia 3.3 Analysiis of Method 3.3.2.2 Selection of Lag Optimum 3.3.2.2 Selection of Lag Optimum To determine the amount of lag (order) to be used in the VAR, can be determined based on the Akaike Information Criterion (AIC), Schwarz Information Criterion (SIC) or Quinnon Hannan (HQ). Lag selected in this research model is a model with the smallest HQ. In this stage, also the stability of the VAR model being tested. The criteria that are used in this test are as follows: Akaike information crieterion (AIC): െ2 ቀ ଵ ்ቁ൅2ሺ݇൅ܶሻ (6) Schwarz Information Criterion (SIC): െ2 ቀ ଵ ்ቁ൅݇ሺ ୪୭୥ሺTሻ T ሻ (7) Hannan-Quinn Information Criterion (HQ): െ2 ቀ ଵ ்ቁ൅2݇ܮܱܩሺ ୪୭୥ሺTሻ T ሻ (8) Akaike information crieterion (AIC): െ2 ቀ ଵ ்ቁ൅2ሺ݇൅ܶሻ (6) (6) Schwarz Information Criterion (SIC): െ2 ቀ ଵ ்ቁ൅݇ሺ ୪୭୥ሺTሻ T ሻ (7) Hannan-Quinn Information Criterion (HQ): െ2 ቀ ଵ ்ቁ൅2݇ܮܱܩሺ ୪୭୥ሺTሻ T ሻ (8) Schwarz Information Criterion (SIC): െ2 ቀ ଵ ்ቁ൅݇ሺ ୪୭୥ሺTሻ T ሻ (7) (7) ் T Hannan-Quinn Information Criterion (HQ): െ2 ቀ ଵ ்ቁ൅2݇ܮܱܩሺ ୪୭୥ሺTሻ T ሻ (8) Hannan-Quinn Information Criterion (HQ): െ2 ቀ ଵ ்ቁ൅2݇ܮܱܩሺ ୪୭୥ሺTሻ T ሻ (8) (8) Subject to: 1 = the value of lag likelihood is the same as െ ் ଶ൬1 ൅logሺ2ߨሻ൅݈݋݃ ቀ ఌ"ఌᇱ ்ቁ൰ ; ߝ"ߝ where it is sum of squared 1 = the value of lag likelihood is the same as െ ் ଶ൬1 ൅logሺ2ߨሻ൅݈݋݃ ቀ ఌ"ఌᇱ ்ቁ൰ ; ߝ"ߝ where it is sum of squared T = number of observation k = estimated parameter From the result of three criteria, the study will select the smallest value of lag. From the result of three criteria, the study will select the smallest value of lag. From the result of three criteria, the study will select the smallest value of lag. If the phenomenon of stationarity is at the level of first difference or I (1), it is necessary to see the possibility of testing for cointegration. The concept of cointegration is basically to look at long-term balance between the observed variables. Sometimes an individual's data are not stationary, but when connected in a linear data, becomes stationer. This is called that the data is cointegrated. In addition, the cointegration test will also be done by following the Johansen procedure. In the Johansen test, the determination of cointegration in view of the trace statistic and Max Eigenvalue statistics preceded by seeking a long lag to be determined. .3.1 Vectoor Auto Regression (VAR) annd Vector Errorr Correction Moodel (VECM) 3.3.1 Vectoor Auto Regression (VAR) annd Vector Errorr Correction Moodel (VECM) The data in Models (V among var variables c be used in condition m difference The VAR/V n this study ana VECM) if there riables in the ontained in the n the future an must be fulfill stationary (firs VECM process alyzed by empl e is co-integrat system. The m e model are en nd referred to a ed is stationar st difference), t s can be illustra loying Vector A tion. VAR met method was d ndogenous (det as a-theoretica rity or contains then the VAR ated in Figure Auto regression thod is called developed in 1 termined by th al model (not s no unit root. model will be 1. n (VAR) and fo non-structured 1980 by Sims he model). So i based on theo However, wh combined wit followed by Ve d approach tha where the as it is not surpris ory). In the VA hen new data i th error correct ctor Error Corr at describes ca ssumption is t se if this meth AR method, th is used after th tion model (VE rection ausality that all hod can he first he first ECM). Stationary VAR   y at level [I(0)] R Level No Fi D IRF Ye igure 1. The flo Data Informatio V Opti Cointeg Innova F Unit Root Yes es ow of VAR/VE on VECM imal Order gration Rank ation Accounting ECM Model Data Explo Stationary at fir Cointegra FEVD oration rst difference ation N VAR First Dif No fference DData Informatioon Data Explooration No Unit Root Stationaryy at level [I(0)] Cointegraation VARR Level Yees VAR First Diffference Optiimal Order FEVD FEVD Fiigure 1. The floow of VAR/VEECM Model ISSN 1923-40223 E-ISSN 19223-4031 1588 Published byy Sciedu Press Vol. 7, No. 1; 2016 International Journal of Financial Research http://ijfr.sciedupress.com 3.3.2.2 Selection of Lag Optimum Value trace and Max Eigenvalue statistics that exceeds the critical value indicate that there is cointegration in model. 3.3.2 Stages of Data Processing 3.3.2.1 Unit of Root Test or Stationarity Test 3.3.2.1 Unit of Root Test or Stationarity Test At this stage, the test must be conducted to see the data stationarity of each variable through the Augmented Dickey Fuller (ADF) and Phillips-Peron (PP). ADF and PP test results then compared with McKinnon Critical Value. If the t-statistic < McKinnon critical value, it can be concluded that the results are significant, which means there is no Unit Root. But if the opposite happens, then the data is in the first difference. If the result becomes stationer and there is cointegration, then VECM method is applied, however if there is no cointegration, then VAR method is employed. According to (Gujarati, 2003:817) the equation of stationarity test can be simplified with ADF analysis into the following equation: (5) ∆ܻ௧ൌ ߙ଴ ൅yY୲ିଵ൅β୧∑ ∆Y୲ି୧ାଵ ୮ ୧ୀଵ ൅ߝ௧ ∆ܻ௧ൌ ߙ଴ ൅yY୲ିଵ൅β୧∑ ∆Y୲ି୧ାଵ ୮ ୧ୀଵ ൅ߝ௧ (5) Where: ∆ܻ௧ = form of first different ߙ଴ = intercept ܻ = variables of stationarity test P = long of lag used in model ߝ௧ = error term 3.3.2.2 Selection of Lag Optimum ∆ܻ௧ = form of first different ߙ଴ = intercept ܻ = variables of stationarity tes P = long of lag used in model ߝ௧ = error term 3.3.2.2 Selection of Lag Optimum ∆ܻ௧ = form of first different ߙ଴ = intercept ܻ = variables of stationarity test Published by Sciedu Press 3.3.2.4 Vector Error Correction Model (VECM) VECM is a restricted Vector Autoregression form. This additional restriction must be given due to the existence of a stationarity data but cointegrated. VECM cointegration restriction then uses the information into the specification. That is why VAR VECM is often called the design for the series that has a relationship non-stationer cointegration. ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press 159 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com After the discovery of a cointegration test, the process is then performed using the method of error correction. If there is a degree of integration among different variable test, the test is performed simultaneously (jointly) between long-term equation with the equation error correction, given that cointegration occurs in a variable. The difference in degree of integration for cointegrated variable called Lee and Granger as multi-cointegration. But if not encountered the phenomenon of cointegration, the test continued using the first difference variables. 4. Empirical and Finding Analysis 4. Empirical and Finding Analysis 3.3.2.5 VAR/VECM Instruments In conducting analysis, the VAR/VECM has a specific instrument that has a specific function in explaining the interactions between the variables in the model. Instruments include the Impulse Response Function (IRF) and Forecast Error variance Decomposition (FEVD), or commonly called Variance decompositions (VD). IRF vector moving average is an application that aims to see how long the shock of one variable affects another variable. While the VD in the VAR function is to analyze how much the shock of a variable affect other variables. 4. Empirical and Finding Analysis 4.1 Unit of Root Test or Stationarity Test Times series data in this study must pass stationarity data test or unit of root test. This test employs Augmented Dickey Fuller (ADF). The result of test statistic can be compared with MacKinnon critical value. If the value of ADF smaller than critical value, then the data is not stationer, but if ADF value is greater than critical value, then the data is stationer. During the first stage, the data is tested at level. If the result of this test shows that the data non stationer yet, then the test will proceed to first difference level. The result of unit of root test can be seen in Tables 2 and 3 as follows: Published by Sciedu Press 160 ISSN 1923-4023 E- Table 2. The Result of Unit of Root Test at First Difference for Indonesia Null Hypothesis: D(LCSR) has a unit root Exogenous: Constant Lag Length: 0 (Automatic based on SIC, MAXLAG=11) t-Statistic Prob.* Augmented Dickey-Fuller test statistic -7.448785 0.0000 Test critical values: 1% level -3.350096 5% level -2.133549 10% level -2.793501 *MacKinnon (1996) one-sided p-values. Source: E-Views (generated data) Table 3. The Result of Unit of Root Test at First Difference for Turkey Null Hypothesis: D(LCSR) has a unit root Exogenous: Constant Lag Length: 0 (Automatic based on SIC, MAXLAG=11) t-Statistic Prob.* Augmented Dickey-Fuller test statistic -7.643209 0.0000 Test critical values: 1% level -3.550312 5% level -2.913549 10% level -2.594521 *MacKinnon (1996) one-sided p-values. Source: E-Views (generated data) Table 2. The Result of Unit of Root Test at First Difference for Indonesia Table 3. The Result of Unit of Root Test at First Difference for Turkey Null Hypothesis: D(LCSR) has a unit root Exogenous: Constant Lag Length: 0 (Automatic based on SIC, MAXLAG=11) t-Statistic Prob.* Augmented Dickey-Fuller test statistic -7.643209 0.0000 Test critical values: 1% level -3.550312 5% level -2.913549 10% level -2.594521 *M Ki (1996) id d l ISSN 1923-4023 E-ISSN 1923-4031 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com Tables 2 and 3 indicate that the data for Indonesia and Turkey found to have unit root or the data are stationer at first difference level. Although each table presenting one variable only for stationarity test but the rest of variable revealed the same result for both countries. 4.1 Unit of Root Test or Stationarity Test Both tables indicate that ADF value for Indonesia -7.448785 is greater than critical values with probability value of 0.0000 while ADF value for Turkey show -7.643209 which is greater than critical value with probability of 0.0000. Table 4. VAR Stability Test for Indonesia 4.2 VAR Stability Test Before conducting further test, VAR stability test must be done first because if VAR is not stable, IRF (Impulse Response Function) and Variance Decomposition become not valid. VAR stability model can be seen from its modulus where VAR assumed to be stable if the value of modulus less than 1 and at optimum point then VAR can be said as stable. Tables 4 and 5 reveal the same result that the VAR of Islamic banks in both countries found to be stable with no root lies outside the unit circle. The data also shows that both countries have the modulus value lesser than 1. In other words, VAR satisfies the stability condition. Table 4. VAR Stability Test for Indonesia Root Modulus 0.909170 - 7.91e-05i 0.909170 0.909170 + 7.91e-05i 0.909170 0.909012 - 7.92e-05i 0.909012 0.909012 + 7.92e-05i 0.909012 -5.72e-05 - 5.73e-05i 8.10E-05 -5.72e-05 + 5.73e-05i 8.10E-05 5.72e-05 - 5.72e-05i 8.09E-05 5.72e-05 + 5.72e-05i 8.09E-05 No root lies outside the unit circle. VAR satisfies the stability condition. Source: E-Views (generated data) Table 4. VAR Stability Test for Indonesia Table 4. VAR Stability Test for Indonesia Table 5. VAR Stability Test for Turkey Root Modulus 0.909271 0.909271 0.909091 - 0.000180i 0.909091 0.909091 + 0.000180i 0.909091 0.908911 0.908911 -7.17e-05 - 7.19e-05i 0.000102 -7.17e-05 + 7.19e-05i 0.000102 7.17e-05 - 7.14e-05i 0.000101 7.17e-05 + 7.14e-05i 0.000101 No root lies outside the unit circle. VAR satisfies the stability condition. Source: E-Views (generated data) Table 5. VAR Stability Test for Turkey No root lies outside the unit circle. Source: E-Views (generated data) ISSN 1923-4023 E-ISSN 1923-4031 161 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com 4.3 Determination of Optimum Lag In determining lag optimum, the table undertakes information of Aike Information Criterion (AIC) and Hannan-Quinn (HQ), Schwarz Criterion (SC). Based on optimum lag test conducted with these three methods, the table produces lag optimum at first lag at Aike Information Criterion (AIC), and Hannan-Quinn (HQ), Schwarz Criterion (SC). The above three model recommends that only first lag can avoid the problem of autocorrelation among variable of ROA, ROE, OER and CSR. Tables 6 and 7 for Indonesia and Turkey depict that at first lag, these variables influenced corporate social responsibility. In determining lag optimum, the table undertakes information of Aike Information Criterion (AIC) and Hannan-Quinn (HQ), Schwarz Criterion (SC). Based on optimum lag test conducted with these three methods, the table produces lag optimum at first lag at Aike Information Criterion (AIC), and Hannan-Quinn (HQ), Schwarz Criterion (SC). The above three model recommends that only first lag can avoid the problem of autocorrelation among variable of ROA, ROE, OER and CSR. Tables 6 and 7 for Indonesia and Turkey depict that at first lag, these variables influenced corporate social responsibility. In determining lag optimum, the table undertakes information of Aike Information Criterion (AIC) and Hannan-Quinn (HQ), Schwarz Criterion (SC). Based on optimum lag test conducted with these three methods, the table produces lag optimum at first lag at Aike Information Criterion (AIC), and Hannan-Quinn (HQ), Schwarz Criterion (SC). The above three model recommends that only first lag can avoid the problem of autocorrelation among variable of ROA, ROE, OER and CSR. Tables 6 and 7 for Indonesia and Turkey depict that at first lag, these variables influenced corporate social responsibility. Table 6. Table Optimum Lag for Indonesia Lag LogL LR FPE AIC SC HQ 0 -224.1602 NA 0.076070 8.775392 8.925487 8.832935 1 -17.35189 373.8458* 4.96e-05* 1.436611* 2.187089* 1.724327* 2 -16.70458 1.070546 9.05e-05 2.027099 3.377960 2.544988 3 -15.92066 1.175889 0.000167 2.612333 4.563577 3.360394 4 -14.95170 1.304365 0.000315 3.190450 5.742076 4.168683 5 -13.72333 1.464597 0.000613 3.758590 6.910599 4.966995 6 -12.11502 1.670162 0.001239 4.312116 8.064508 5.750695 7 -9.917695 1.943791 0.002647 4.842988 9.195763 6.511739 * indicates lag order selected by the criterion LR: sequential modified LR test statistic (each test at 5% level) FPE: Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, HQ: Hannan-Quinn information criterion Source: E-Views (generated data) Table 7. 4.3 Determination of Optimum Lag Table Optimum Lag for Turkey Lag LogL LR FPE AIC SC HQ 0 -15.18282 NA 2.46e-05 0.737801 0.887896 0.795344 1 191.6255 373.8458* 1.60e-08* -6.600980* -5.850501* -6.313264* 2 192.2728 1.070546 2.92e-08 -6.010492 -4.659630 -5.492603 3 193.0567 1.175889 5.40e-08 -5.425258 -3.474014 -4.677197 4 194.0257 1.304365 1.02e-07 -4.847141 -2.295514 -3.868908 5 195.2540 1.464597 1.98e-07 -4.279001 -1.126992 -3.070595 6 196.8623 1.670162 4.00e-07 -3.725475 0.026917 -2.286896 7 199.0597 1.943791 8.55e-07 -3.194603 1.158172 -1.525852 * indicates lag order selected by the criterion FPE: Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, Source: E-Views (generated data) Published by Sciedu Press 4.3 Determination of Optimum Lag Table Optimum Lag for Turkey Lag LogL LR FPE AIC SC HQ 0 -15.18282 NA 2.46e-05 0.737801 0.887896 0.795344 1 191.6255 373.8458* 1.60e-08* -6.600980* -5.850501* -6.313264* 2 192.2728 1.070546 2.92e-08 -6.010492 -4.659630 -5.492603 3 193.0567 1.175889 5.40e-08 -5.425258 -3.474014 -4.677197 4 194.0257 1.304365 1.02e-07 -4.847141 -2.295514 -3.868908 5 195.2540 1.464597 1.98e-07 -4.279001 -1.126992 -3.070595 6 196.8623 1.670162 4.00e-07 -3.725475 0.026917 -2.286896 7 199.0597 1.943791 8.55e-07 -3.194603 1.158172 -1.525852 * indicates lag order selected by the criterion LR: sequential modified LR test statistic (each test at 5% level) FPE: Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, HQ: Hannan-Quinn information criterion S E Vi ( t d d t ) Table 6. Table Optimum Lag for Indonesia Lag LogL LR FPE AIC SC HQ 0 -224.1602 NA 0.076070 8.775392 8.925487 8.832935 1 -17.35189 373.8458* 4.96e-05* 1.436611* 2.187089* 1.724327* 2 -16.70458 1.070546 9.05e-05 2.027099 3.377960 2.544988 3 -15.92066 1.175889 0.000167 2.612333 4.563577 3.360394 4 -14.95170 1.304365 0.000315 3.190450 5.742076 4.168683 5 -13.72333 1.464597 0.000613 3.758590 6.910599 4.966995 6 -12.11502 1.670162 0.001239 4.312116 8.064508 5.750695 7 -9.917695 1.943791 0.002647 4.842988 9.195763 6.511739 Table 6. Table Optimum Lag for Indonesia * indicates lag order selected by the criterion LR: sequential modified LR test statistic (each test at 5% level) FPE: Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, HQ: Hannan-Quinn information criterion Source: E-Views (generated data) * indicates lag order selected by the criterion : Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, Table 7. Table Optimum Lag for Turkey Lag LogL LR FPE AIC SC HQ 0 -15.18282 NA 2.46e-05 0.737801 0.887896 0.795344 1 191.6255 373.8458* 1.60e-08* -6.600980* -5.850501* -6.313264* 2 192.2728 1.070546 2.92e-08 -6.010492 -4.659630 -5.492603 3 193.0567 1.175889 5.40e-08 -5.425258 -3.474014 -4.677197 4 194.0257 1.304365 1.02e-07 -4.847141 -2.295514 -3.868908 5 195.2540 1.464597 1.98e-07 -4.279001 -1.126992 -3.070595 6 196.8623 1.670162 4.00e-07 -3.725475 0.026917 -2.286896 7 199.0597 1.943791 8.55e-07 -3.194603 1.158172 -1.525852 * indicates lag order selected by the criterion LR: sequential modified LR test statistic (each test at 5% level) FPE: Final prediction error, AIC: Akaike information criterion, SC: Schwarz information criterion, HQ: Hannan-Quinn information criterion Source: E-Views (generated data) Table 7. Table Optimum Lag for Turkey Table 7. 4.4 Cointegration Test Cointegration test conducted to know the relationship between variables of profitability of Islamic banks and corporate social responsibility in the short term and long term. The result of cointegration based on trace statistic which must be higher than critical value. Since there is cointegration for both countries, then the model continued to ISSN 1923-4023 E-ISSN 1923-4031 ISSN 1923-4023 E-ISSN 1923-4031 162 ISSN 1923-4023 E-ISSN 1923-4031 Published by Sciedu Press International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 1; 2016 VECM. Table 8 shows that only one variable among profitability of Islamic in Indonesia has influence towards corporate social responsibility while Table 9 shows that there are three variables of profitability of Islamic banks that have influence with corporate social responsibility in Turkey in the short and long term. Both tables also indicate that cointegrations are significant at 5% level. VECM. Table 8 shows that only one variable among profitability of Islamic in Indonesia has influence towards corporate social responsibility while Table 9 shows that there are three variables of profitability of Islamic banks that have influence with corporate social responsibility in Turkey in the short and long term. Both tables also indicate that cointegrations are significant at 5% level. Table 8. Cointegration Test for Indonesia Unrestricted Cointegration Rank Test (Trace) Hypothesized Trace 0.05 No. of CE(s) Eigenvalue Statistic Critical Value Prob.** None * 0.492732 68.75545 63.87610 0.0184 At most 1 0.200000 34.14096 42.91525 0.2821 At most 2 0.200000 22.76064 25.87211 0.1163 At most 3 0.200000 11.38032 12.51798 0.0769 Trace test indicates 1 cointegrating eqn(s) at the 0.05 level * denotes rejection of the hypothesis at the 0.05 level **MacKinnon-Haug-Michelis (1999) p-values Table 9. Cointegration Test for Turkey Unrestricted Cointegration Rank Test (Trace) Hypothesized Trace 0.05 No. of CE(s) Eigenvalue Statistic Critical Value Prob.** None * 0.333333 61.45265 47.85613 0.0016 At most 1 * 0.333333 41.58486 29.79707 0.0014 At most 2 * 0.333333 21.71707 15.49471 0.0051 At most 3 0.037037 1.849276 3.841466 0.1739 Trace test indicates 3 cointegrating eqn(s) at the 0.05 level * denotes rejection of the hypothesis at the 0.05 level **MacKinnon-Haug-Michelis (1999) p-values Source: E Views (generated data) Table 8. Cointegration Test for Indonesia 4.5 Impulse Response Function The study proceeds to analyze IRF (Impulse Response Function) since the cointegration result revealed that Indonesia and Turkey have cointegration. Figure 2 shows that the behavior of corporate social responsibility in Indonesia toward return on asset only fluctuates in the short term. In the long terms, return on asset tend to decline starting from the period of 3. The response of corporate social responsibility towards return on equity shows different situation where it tends to increase in the long term. However, operating efficiency ratio predicted to decline very sharp in the long terms. Meanwhile, Figure 3 indicates that the behavior or shock of corporate social responsibility in Turkey towards return on asset differ compared to its counterparts. Return on asset seems to fluctuate in a small quantity in the short terms but it tends to be stable in the long terms. The fluctuation starts from the period of 4 and remain stable throughout the period. As for return on equity, it tends to decline in the long term resulted from the behavior corporate social responsibility while operating efficiency ratio experienced the same pattern as it is predicted to decline very sharp in the long term even started from the beginning. ISSN 1923-4023 E-ISSN 1923-4031 163 Published by Sciedu Press Published by Sciedu Press IInternational Jouurnal of Financiaal Research Vol. 7, No. 1; 2016 http://ijfr.sciiedupress.com 6 Foreca he object esponsibil nfluenced nd 21.41% i l st Error Varian tive of FEVD lity in both cou by operating e % respectively. ibili i h Figure Figur nce Decompos is to predict th untries in the lo efficiency ratio Meanwhile, in h l e 2. Impulse Re re 3. Impulse R ition (FEVD) he behavior or ong term. As d o which is 24.5 n Turkey, retur T bl 11 h esponse Functi Response Funct r shock of prof depicted by Ta 56% in the peri rn on equity is h i h on for Indones tion for Turkey fitability variab able 10 that cor riod of 10 comp the most influ i d f 10 sia y bles resulted f rporate social r mpared to ROA uenced by the b i ib from corporate responsibility m and ROE only behavior of cor 61 71% f ll e social mostly y 6.9% rporate d b Figuree 2. Impulse Reesponse Function for Indonessia Figuree 2. Impulse Reesponse Function for Indonessia Figuree 2. 4.5 Impulse Response Function Impulse Reesponse Function for Indonessia Fi 3 I l RR Figurre 3. Impulse RResponse Functtion for Turkeyy Figurre 3. Impulse RResponse Functtion for Turkeyy Figurre 3. Impulse RResponse Functtion for Turkeyy 4.6 Forecast Error Variannce Decomposition (FEVD) Amid the resurrection of Islam 5 0.258129 4.378083 15.88742 29.50494 50.22956 6 0.283478 4.797598 17.06205 28.48229 49.65806 7 0.307291 5.278510 18.20429 27.46241 49.05478 8 0.329878 5.798604 19.31230 26.46424 48.42486 9 0.351443 6.342939 20.38391 25.49749 47.77566 10 0.372135 6.901119 21.41732 24.56777 47.11379 Choleky Ordering: ROAROE OER LCSR Source: E-Views (generated data) Table 11. FEVD Turkey Period S.E. ROA ROE OER LCSR 1 0.053629 0.058327 35.57855 30.64757 33.71556 2 0.074889 0.093474 36.69719 29.60630 33.60304 3 0.090668 0.139185 37.78283 28.59098 33.48701 4 0.104054 0.122992 40.90849 26.55274 32.41577 5 0.115884 0.102556 44.68949 24.22410 30.98385 6 0.126597 0.089014 48.57446 21.90223 29.43430 7 0.136475 0.083416 52.30796 19.74509 27.86353 8 0.145698 0.084849 55.76832 17.81117 26.33566 9 0.154386 0.091642 58.90616 16.11154 24.89066 10 0.162626 0.102185 61.71648 14.63318 23.54815 Cholesky Ordering: ROA ROE OER LCSR Source: E-Views (generated data) Source: E-Views (generated data) Published by Sciedu Press 4.6 Forecast Error Variannce Decomposition (FEVD) 4.6 Forecast Error Variannce Decomposition (FEVD) The object responsibil influenced and 21.41% social resp OER which tive of FEVD lity in both cou by operating e % respectively. onsibility in th h is 14.63% an is to predict th untries in the lo efficiency ratio Meanwhile, in he long terms a nd ROA 10.21% he behavior or ong term. As d o which is 24.5 n Turkey, retur as Table 11 sho %. r shock of prof depicted by Ta 56% in the peri rn on equity is ows that in the fitability variab able 10 that cor riod of 10 comp the most influ e period of 10, bles resulted f rporate social r mpared to ROA uenced by the b it contributes from corporate responsibility m and ROE only behavior of cor 61.71% follow e social mostly y 6.9% rporate wed by Published by Table 10. F Perio 1 2 3 4 y Sciedu Press FEVD Indonesi d ia S.E. 0.12030 0.16686 0.20071 0.23075 164 ROA 07 4.697 61 4.298 19 3.936 52 4.058 4 ROE 247 10.8 921 12.1 156 13.4 216 14.6 E OE 81164 32 11994 32 43445 31 68002 30 ISSN 1923-402 ER L 2.68085 2.03428 1.38395 0.49065 23 E-ISSN 192 LCSR 51.81027 51.54686 51.24545 50.77111 23-4031 Table 10. FFEVD Indonesiia 1644 International Journal of Financial Research http://ijfr.sciedupress.com Vol. 7, No. 1; 2016 5 0.258129 4.378083 15.88742 29.50494 50.22956 6 0.283478 4.797598 17.06205 28.48229 49.65806 7 0.307291 5.278510 18.20429 27.46241 49.05478 8 0.329878 5.798604 19.31230 26.46424 48.42486 9 0.351443 6.342939 20.38391 25.49749 47.77566 10 0.372135 6.901119 21.41732 24.56777 47.11379 Choleky Ordering: ROAROE OER LCSR Source: E-Views (generated data) Table 11. FEVD Turkey Period S.E. ROA ROE OER LCSR 1 0.053629 0.058327 35.57855 30.64757 33.71556 2 0.074889 0.093474 36.69719 29.60630 33.60304 3 0.090668 0.139185 37.78283 28.59098 33.48701 4 0.104054 0.122992 40.90849 26.55274 32.41577 5 0.115884 0.102556 44.68949 24.22410 30.98385 6 0.126597 0.089014 48.57446 21.90223 29.43430 7 0.136475 0.083416 52.30796 19.74509 27.86353 8 0.145698 0.084849 55.76832 17.81117 26.33566 9 0.154386 0.091642 58.90616 16.11154 24.89066 10 0.162626 0.102185 61.71648 14.63318 23.54815 Cholesky Ordering: ROA ROE OER LCSR Source: E-Views (generated data) 5. Conclusion It is interesting to note that Indonesia and Turkey are adopting dual banking system. 5. Conclusion These findings reveal that the behavior of corporate social responsibility in Turkey resulted from the behavior of stakeholders compared to Indonesia where the behavior of corporate social responsibility mostly influenced return on asset. References Affandi, A. (2007). Performance Evaluation of Islamic Commercial in Indonesia after the Financial Crisis. Journal of Tazkia Islamic Business Review, 2(1), 27-45. Affandi, A. (2007). Performance Evaluation of Islamic Commercial in Indonesia after the Financial Crisis. Journal of Tazkia Islamic Business Review, 2(1), 27-45. Akerlof, G. A. (1970). The Market for Lemons Quality Uncertainty and the Market Mechanism. The Quarterly Journal of Economics, 84(3), 488-500. http://dx.doi.org/10.2307/1879431 Anonymous. (2002). Should the SEC Expand Non-Financial Disclosure Requirements? Harvard Business Law Review, 115, 1433-55. Arvidsson, S. (2011). Disclosure of Non-Financial Information in the Annual Report: A Management-Team Perspective. Journal of Intellectual Capital, 12(2), 277-300. http://dx.doi.org/10.1108/14691931111123421 Bhatti, M., & Bhatti, M. I. (2009). Development in legal Issues of Corporate Governance in Islamic Finance. Journal of Economic & Administrative Sciences, 25, 67-91. http://dx.doi.org/10.1108/10264116200900004 ———. (2010). Toward Understanding Islamic Corporate Governance Issues in Islamic Finance. Asian Politics & Policy, 2, 25-38. http://dx.doi.org/10.1111/j.1943-0787.2009.01165.x Cahya, B. A. (2010). An Analysis on the Influence of Financial Performance towards Corporate Social Responsibility. Thesis Undergraduate Faculty of Economics University of Diponegoro Indonesia, 40-58. Crawford, E. P., & Williams, C. C. (2010). Should Corporate Social Reporting be Voluntary or Mandatory? Evidence from the Banking Sector in France and the United States. Journal of Corporate Governance, 10(4), 512-526. http://dx.doi.org/10.1108/14720701011069722 Dusuki, A. W., & Dar, H. (2007). Stakeholder’s Perception of Corporate Social Responsibility of Islamic Banks: Evidence from Malaysian Economy. Retrieved 7th March 2012, from http://www.dinarstandard.com/maqasid/23%20Asyraf%20Wajdi%20Dusuki%20&%20Humayon%20Dar%20St akeholders.pdf Farook, S. (2007). On Corporate Social Responsibility in Islamic Financial Institutions. Islamic Economics Studies, 15(1), 31-46. Farook, S., & R. Lanis. (2005). Banking on Islam? Determinants of Corporate Social Responsibility Disclosure. Paper presented at the 2005 AFAANZ Conference, Melbourne, Australia. Hassan, A., & Abdul Latiff, S. (2009). Corporate Social Responsibility of Islamic Financial Institutions and Businesses: Optimizing Charity Value. Humanomics, 25, 177-188. http://dx.doi.org/10.1108/08288660910986900 Jensen, M. C., & Meckling, W. H. (1976). Theory of the Firm: Managerial Behaviour, Agency Costs and Ownership Structure. Journal of Financial Economics, 3(4), 305-60. http://dx.doi.org/10.1016/0304-405X Kavitha W., & Anita P. (2011). Disclosures about CSR Practices: A Literature Review. The IUP Journal of Corporate Governance, X(1), 43-53. Mia, P., & Al-Mamun, A. (2011). Corporate Social Disclosure during the Global Financial Crisis. International Journal of Economics and Finance, 3(6), 175-177. http://dx.doi.org/10.5539/ijef.v3n6p174 Owen, D. L., Swift, T., & Hunt, K. (2001). Questioning the Role of Stakeholder Engagement in Social and Ethical Accounting, Auditing and Reporting. Accounting Forum, 25(4), 264-82. http://dx.doi.org/10.1111/1467-6303.00066 Swift, T. (2001). 5. Conclusion It is interesting to note that Indonesia and Turkey are adopting dual banking system. Amid the resurrection of Islamic banking industry in the world, Turkey performed relatively better in terms of Islamic banking market share in the country compared to its counterparts. With regard to the behavior of Islamic banks in performing corporate social responsibility of the two countries revealed some differences regardless the perception, disclosures and application of corporate social responsibility. The cointegration result revealed that only one profitability variable of Islamic bank in Indonesia that found to have cointegration, in other words, only one variable that has shock resulted from the behavior of corporate social responsibility. Meanwhile, there are three profitability variables of Islamic banks in Turkey that have influence resulted from the changes of behavior in corporate social responsibility. The other findings reveal that IRF (Impulse Response Function) result shows only return on asset of Islamic bank in Turkey found to have stability whether in the short terms or long terms. In other words, regardless of the behavior in corporate social responsibility, return on asset will remain stable and resilient. The other variables concluded to have similar pattern for Turkey and Indonesia tend to decline even very sharp in the long terms except, return on equity for Indonesia has positive response where it tends to increase regardless of the changes in the behavior or shock of corporate social responsibility. Meanwhile, the table of FEVD or variance decomposition also shows different findings between Indonesia and Turkey. The behavior of corporate social responsibility in Indonesia mostly influenced profitability of Islamic banks in terms of operating efficiency ratio followed by return on equity and return on asset. However, profitability of ISSN 1923-4023 E-ISSN 1923-4031 165 International Journal of Financial Research Vol. 7, No. 1; 2016 http://ijfr.sciedupress.com Islamic banks (participation banks) in Turkey greatly influenced by the behavior of corporate social responsibility only on return on equity followed by operating efficiency ratio and return on asset respectively. These findings reveal that the behavior of corporate social responsibility in Turkey resulted from the behavior of stakeholders compared to Indonesia where the behavior of corporate social responsibility mostly influenced return on asset. Islamic banks (participation banks) in Turkey greatly influenced by the behavior of corporate social responsibility only on return on equity followed by operating efficiency ratio and return on asset respectively. References Trust, Reputation and Corporate Accountability to Stakeholders. Business Ethics: A European Review, 10(1), 16-26. http://dx.doi.org/10.1111/1467-8608.00208 ISSN 1923-4023 E-ISSN 1923-4031 166 Published by Sciedu Press
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Review of: "Signboards prohibiting tobacco sale within 100 yards of educational institutes: The appraisal of prohibition compliance and on-ground status of the COTPA Act, 2003 in Chanakyapuri Division of New Delhi Revenue District, the administrative precinct of India."
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Qeios ID: 701VUE · https://doi.org/10.32388/701VUE Qeios, CC-BY 4.0 · Review, November 29, 2022 Review of: "Signboards prohibiting tobacco sale within 100 yards of educational institutes: The appraisal of prohibition compliance and on-ground status of the COTPA Act, 2003 in Chanakyapuri Division of New Delhi Revenue District, the administrative precinct of India." Balaji Sekar Potential competing interests: No potential competing interests to declare. The Author did not mention the name of the 4 institutes where the Tobacco shops are very closely located(less than 100 yards). It will be helpful for the law enforcement if the author specifically mention the institutes name and relative distance of shop from the institute with proper proof(photographs). Similarly the Author must mention the name of of 11 institutes where there was no proper sign board. milarly the Author must mention the name of of 11 institutes where there was no proper sign board. Qeios ID: 701VUE · https://doi.org/10.32388/701VUE 1/1
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Objects, Relations, Potential and Change
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Object-Oriented Ontology and Its Critics Bart Nooteboom* Objects, Relations, Potential and Change https://doi.org/10.1515/opphil-2020-0004 Received July 19, 2019; accepted October 10, 2019 https://doi.org/10.1515/opphil-2020-0004 Received July 19, 2019; accepted October 10, 2019 Abstract: This article attempts to develop further the conception of dynamics in Object-Oriented Ontology (OOO): its model of how objects develop and change. Objects are affected by relations between them, and have the potential both to produce and undergo effects, as realised in interaction with other objects. To elaborate on the change of objects in OOO, an idea is adopted from transcendental ontology. A key Hegelian question in this article is how the realisation of existing potential can produce new potential (Schelling: potentialisation, going from the actual to the possible). Stated differently: how can objects change to the point of breaking their identity and generating a new object? One needs to consider that objects are nested at different levels, and that the degree of how radical change may be depends on the perspective of any given level. To address this issue, the article employs the notion of a script: a structure of nodes, each with its own subscripts. The analysis is applied and developed further through a comparative analysis of change in evolution, economics, a theory of discovery, and linguistics. The dual intention of this is to see if OOO can help us understand those phenomena, and to see in turn if those phenomena can inform the further development of OOO. Keywords: Object-Oriented Ontology, transcendental ontology, transformation, evolution, economics, discovery, meaning Keywords: Object-Oriented Ontology, transcendental ontology, transformation, evolution, economics, discovery, meaning   Open Philosophy 2020; 3: 53–67 Open Access. © 2019 Bart Nooteboom, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 Public License. y 2 Bhaskar, A Realist Theory of Science; DeLanda, Assemblage Theory; Bryant, The Democracy of Objects. 3 Gabriel, Transcendental Ontology, 71. 1 Harman, Object-Oriented Ontology; Garcia, Form and Object. 2 Bhaskar, A Realist Theory of Science; DeLanda, Assemblage Theory; Bryant, The Democracy of Objects. 3 Gabriel, Transcendental Ontology, 71. 1 Harman, Object-Oriented Ontology; Garcia, Form and Object. Keywords: Object-Oriented Ontology, transcendental ontology, transformation, evolution, economics, discovery, meaning *Corresponding author: Bart Nooteboom, Tilburg University, Tilburg, Netherlands; E-mail: bart.nooteboom@gmail.com 2 Relations Harman claims that objects are not fully determined by their outside relations.6 In this way he objects to the Actor Network Theory (ANT) of Bruno Latour, with its claim that objects are constituted by their actions in relations with other objects in networks. This, Harman claims, neglects the ongoing identity of objects, regardless of their actions and relations. New actions or relations actually yield new objects. Also, objects do not have relations with all other objects; relations are selective and constrained in their effects. It is part of the aim of this article to investigate further what those constraints are. An object in a network –say, a person lodged in economic and social networks– has access through the networks to resources it needs to maintain its existence, resources provided by objects in the networks. Not all objects provide what is required. The person can survive with a range of different menus, obtained differently in different networks, but she is restricted as to what she can absorb. A well-known characteristic of life, even the very definition of it, is that a living organism (human, animal, plant, microbe….) resists the natural law of closed systems, in which they lose their coherence and structure through increasing entropy, eventually decaying into an undifferentiated mass. To stay alive, the organism needs to go against this law of increasing entropy, and to that end it needs be open to its environment, with ‘things going in and things coming out’ (Garcia). It must maintain homeostasis in its metabolism, keeping certain variables within distinct boundaries, such as humidity, acidity, salinity, sugar, oxygen, CO2, or calcium. Even an inanimate object –say, a stone– cannot remain what it is without a certain safe range of temperatures outside of which it will disintegrate; depending on the weather, in the long run it will erode into sand. Levi R. life, products, and ideas (as in the case of evolution) and innovation, discovery, and meaning (as in poetry). Also, they are areas in which the author has some experience in the areas of economics.4 This orientation towards learning by way of application goes back to American Pragmatism (notably Dewey), but can be traced back further to Schelling’s idea that ideas arise from reality without being reducible to it.5 life, products, and ideas (as in the case of evolution) and innovation, discovery, and meaning (as in poetry). Also, they are areas in which the author has some experience in the areas of economics.4 This orientation towards learning by way of application goes back to American Pragmatism (notably Dewey), but can be traced back further to Schelling’s idea that ideas arise from reality without being reducible to it.5 4 Nooteboom, “From Evolution to Language and Learning”; Nooteboom, “Learning and Innovation.” 5 Schelling, Philosophical Enquiries Into the Nature of Human Freedom and Matters Connected Therewith. 6 Harman, Object-Oriented Ontology, 108. 7 Bryant, The Democracy of Objects, 208. 8 See also DeLanda, Assemblage Theory, 74. 4 Nooteboom, “From Evolution to Language and Learning”; Nooteboom, “Learning and Innovation.” 5 Schelling, Philosophical Enquiries Into the Nature of Human Freedom and Matters Connected Therewith. 6 Harman Object-Oriented Ontology 108 1 Introduction This article builds on what has been developed in OOO as propounded by Graham Harman and Tristan Garcia, among others.1 Objects can be material, non-living, living, or abstract. Objects exist regardless of any perception or understanding by any subject. They have a beginning and an end, an inside with a composition of elements, an outside of relations with other objects, and cannot be reduced to any of these. Features of objects are ‘withdrawn’ (Harman): one cannot know all their features, not only for epistemological reasons (we have no ‘access’ to them) but also for ontological reasons (they are not there to be known). Objects are nested in different levels of objects within objects. Objects have relations with some but not all other objects. They are affected but not fully determined by such relations, and in interaction they retain their identity. Objects have a potential or capacity to ‘act,’ have effects, create differences (Bhaskar, DeLanda, Bryant).2 That is the point of departure for this article, whose focus is on change, on how potential is realised; in particular, we will consider whether and how, and to what extent, the realisation of potential can lead to the rise of a new potential. This question arises already with Hegel and Schelling, as in the latter’s idea of ‘potentialisation,’ meaning a transition from the actual to the potential in transformative change.3 The ideas of change are applied and further developed by application to change in the areas of evolution, economics, and linguistics. These areas are chosen because they entail transformative change of 54   B. Nooteboom 8 See also DeLanda, Assemblage Theory, 74. 7 Bryant, The Democracy of Objects, 208. 2 Relations Bryant notes that in thinking about relations between objects one should consider the nesting, the composition of an object from its parts, its mereology.7 There is ‘upward causation,’ where the elements together produce properties of the whole that the parts do not have (emergence, as discussed by Harman), and ‘downward causation,’ where the whole imposes restrictions on the parts in order to be and remain what it is.8 A stone is composed of molecules, which are composed of atoms, which are composed of nuclei of neutrons and protons plus clouds of electrons, as well as underlying forces and fields that are difficult to understand. Beyond a certain temperature the molecules will disintegrate. Solids can change state and become fluid, and fluids can turn into gases. for the purposes of mereology I use the notion of a script, as a model to reconstruct the nesting process. A script is a network of connected nodes that each has a repertoire of ‘subscripts’ yielding options for actions that fit into the node. Nodes generate the script (upward causation), and are constrained to maintain the integrity of the script (downward causation). The connections between nodes are directed, and may be bi-directional, in interaction. They may indicate temporal succession, causation, logical implication or induction, sharing of resources, or connections between words in a sentence (by grammar or syntax). The claim is not that objects are scripts, but that a script can model the structure of objects, and to some extent their dynamics. The script is not an ontological but a notional category, or a ‘sensual object’ in Harman’s parlance. The classic example is the restaurant script, with successive nodes of entry, seating, ordering, eating, paying and leaving. The restaurant itself is a node in the superscript of the location of the restaurant, which 55  Objects, Relations, Potential and Change  55 includes parking facilities, the supply chain, and institutional conditions such as opening times. Inside a node, there is a variety of potential ways (a repertoire) of performing the activity in the node, each with its own subscript. In entering the restaurant one may be accompanied, or not, by a host. For seating, one may or may not need a prior reservation. Eating can be done with knife-fork-spoon or chopsticks. Along with the waiter there may or may not be a sommelier. Payment can be made by cash, card, or both. 9 Harman, Object-Oriented Ontology, 7. 10 Gabriel, Transcendental Ontology, 135. 11 Manuel DeLanda & Graham Harman, The Rise of Realism, 53. 12 DeLanda, Assemblage Theory, 180. 13 Harman, Object-Oriented Ontology, 52. 14 Ibid., 167. 15 Garcia, Form and Object, 35-36. 2 Relations For an actual restaurant not everything is or can be specified as an element of a script, and much remains tacit or taken for granted. In other words, some features of the restaurant are ‘withdrawn,’ in the sense discussed by OOO.9 This withdrawnness is not just epistemological, in the sense of someone not knowing its features: it is ontological, in that not all features are there to be specified. A host of incompletely determinate, tacitly adopted, unreflected, open-ended presuppositions or ‘background assumptions’ are involved. F.W.J. Schelling referred to this irreducible or indivisible remainder as a nie aufgehobener Rest.10 One is not supposed to throw food around in a restaurant or stuff it into one’s pocket, but these rules are not specified and are perhaps not inescapable (one might try to act this way and see what happens). In some cases doggy bags are provided (as in the United States) while in others (such as European restaurants) they are not. As a result, the script is open-ended: new features may be added, such as having to put one’s phone in silent mode, or being able to pay with a smart phone. As a result, the parts or nodes have a certain unspecified and unspecifiable, open-ended scope for autonomy, unaffected by the overall script. Yet they are not allowed to jeopardize the functioning of the restaurant as a whole. Customers are not allowed into the kitchen to cook their own meals. There could be, however, the innovation of a restaurant where customers are allowed into the kitchen to learn how to cook. 3 Change What is it that drives, allows or facilitates the actions of objects? Manuel DeLanda claims that objects are inherently dynamic: “Processes of genesis and maintenance are what ensure that the individuals are not mere bundles of properties.”11 Also according to DeLanda, an object has actual properties that yield the potential (capacity) to produce features, in cases of interaction with other objects, in the sense that it can both affect them and be affected by them.12 By implication, events are distinguished from objects, as the actions that objects perform in their relations. This contrasts with Harman’s view of events as objects.13 Harman uses the example of a collision between two airplanes.14 First there are two objects (the two planes), then the collision and crash, and finally the debris on the ground. Let’s take the criterion for an object to be a more or less durable coherence of elements of which it is composed, alongside its external relations with other objects. In the crash itself one sees little coherence (let alone durability) of parts, but rather the opposite: a disintegration of parts and a change of their identities. The planes no longer have the capacity to fly, and the passengers perish. Yet fundamentally Harman is right. Even in the collision there is some durability of things, however brief, without which the event could not occur. Yet our intuition tells us that the collision is an event, not an object. What to do with this riddle? Garcia rightly claims that continuity of objects is a matter of scale, in space (macroscopic or microscopic) and time (long or short).15 He uses the example of apiece of slate, which in the long run is also subject to the decay or erosion of particles. And when we dive deeply enough into its molecular structure, we encounter seemingly isolated atoms hanging in space. Since objects may change their features within certain boundaries while maintaining their identity, to project a film of their changes (such as viewing our own lives unfolding at high speed) they would indeed look like events. At low speed, by contrast, an object 56   B. Nooteboom 56 may seem static. Thus, if we present a film of an object at high speed we will only see emergence, change and decay, while if we show it at low speed we will mostly see stasis. 3 Change As concerns a stone, over the brief time of a human lifespan we see no change; as concerns a streaming river it is the opposite– we do not see the constancy of the water molecules. Stones, thoughts and stories are all objects, but in the ordinary temporal order of human life we do not see stones as being in motion, while thoughts may seem to flit in all directions all the time. In sum, the distinction between objects and events depends on the time frame. Within human experience it seems odd not to distinguish events from objects. On the spatio-temporal scale of daily human experience, some objects (material objects) have a spatial and temporal continuity different, and greater, than that of abstractions. On that level of human experience, in terms of temporal and spatial continuity material objects do in fact differ from idea or concepts. With their less visible and slower rate of change, material objects have a special place in human cognition. They represent how we tacitly conceive of existence as a fixed substance, the very paradigm of existence. This article sides with DeLanda in holding that, as concerns the spatio-temporal scale of human experience, events result from the activity of objects, from the interaction between them, in which they create and undergo effects.16 The ability of an object to have effects is enabled and constrained by the object’s potential. The ability of an object to undergo effects, and what the effects are, also depend on internal potential structure: in particular, its ‘absorptive capacity’. The idea of potential and its realisation goes back much further, to Aristotle’s notion of entelechy. An acorn develops into an oak tree. A butterfly miraculously emerges from a caterpillar. This realization is not autonomous and inexorable but contingent: one that depends on conditions, on the relations with the ‘outside’ of the object, such as humidity, wind and temperature. Potential is a ‘virtual’ property, as Deleuze called it, in the sense that at any time potential is not fully exhausted, given that its realization is dependent on conditions that are open to endless possibilities.17 This is consistent with Harman’s claim that the features of an object can never fully be specified, since the realisations of its potential are open-ended. Now, there are several possibilities here. 16 DeLanda & Harman, The Rise of Realism, 67-68. 17 Bryant, The Democracy of Objects, 96; see also DeLanda & Harman, The Rise of Realism, 89. 18 DeLanda & Harman, The Rise of Realism, 68. 19 Ibid., 89 20 Ibid., 101 3 Change One is that the range of possible manifestations is pre- established as a repertoire of possible qualities, from which one is selected according to the context. DeLanda speaks of tendencies, understood as repetitive and limited in variation. Another possibility is the capacity to produce new qualities depending on the context. This is more flexible and adaptive than a tendency. As DeLanda noted, this requires that the capacity to affect is coupled to the capacity to be affected. Harman objected to this model potentialities and capacities because they would yield an excess of possible manifestations, a ‘slum of possibles’ as Harman called it, quoting Quine.18 DeLanda accepts capacities only if one has a way of clearing the slum by separating significant from insignificant manifestations.19 This seems odd. What is significant appears to depend on purpose and context, and thus one would quickly repopulate the slum with possible significances. Yet the slum only arises if one postulates that all possible manifestations have to be there (and where, exactly?) from the start. Possible manifestations are not predetermined but produced in some context, in the interaction between objects, and the range of possible interactions and their effects is open-ended, open to new interactions, and the appearance of new objects and forms of relations. Suppose that someone is about to take a walk, an activity that involves using one’s muscles. How it works out depends on the weather, the quality of one’s shoes, where one happens to be going, and one’s success in not being run over by a tram. Perhaps the newspaper this morning announced a musical performance on a square, and one decided to attend. One does not go to fetch the walk from some repository of pre-existing walks, but produces it on the spot. As DeLanda claims: “it is the open-ended nature of the world, not so much a fundamental withdrawal, that makes dreams of final truth vanish.”20 Yet in fact, both are pertinent: fundamental withdrawal as well  57 Objects, Relations, Potential and Change as open-endedness. The case of the restaurant illustrates this by showing that there are both unspecified and unspecifiable, indeterminate features, as well as openness to new ones. But even while potential is open-ended, it is constrained, and this constitutes at least part of the identity of an object. Can identity be identified with potential? Could it be called the essence of an object? 3 Change An answer to this question will come later, or at least an attempt in that direction. Potential is limited by the structure and properties of the object’s components and those of the objects it interacts with, as well as the laws of nature, logic, and mathematics, not to mention judicial laws and other institutional conditions. One of DeLanda’s proposals is to think of capacity in terms of possible trajectories in the state space of the object.21 The dimensions of that space are features the object can have; there is some process or logic that determines trajectories. DeLanda used the example of water. It has the capacity to be a fluid –which can have different structures– or a piece of ice or even a gas (in the form of steam), depending on outside temperature and atmospheric pressure. Yet it cannot turn into gold. The genome is a good example of a capacity, with genes generating amino acids, yielding cells, building organs, and thereby ‘expressing’ themselves through interacting with each other and with their local metabolic environment, as well as with conditions external to the organism. We return to mereology. If objects are nested, so is change, and the degree of radicality of change is relative to the perspective or level from which one looks. From the perspective of a script (of a restaurant, for example) any change of subscripts in nodes is minor or subordinate (such as changes in methods of payment). By contrast, while a change of the set of nodes themselves and their ordering involves major change in the script’s identity (such as transition to a self-service restaurant). However, any change of a subscript in a node, or an addition of one, is a major change from the perspective of that node. The introduction of payment by card requires the requisite apparatus and internet connection. The transformation of a service restaurant to self-service is a radical change to the point of transformation, with the order of the nodes changing to the following: entry, food selection, paying, seating, eating and leaving. This has consequences for the content of the nodes, for their repertoires of possible subscripts. The act of eating now includes carrying a tray of selected foods. 22 Žižek, The Parallax View; Gabriel, Transcendental Ontology. 21 DeLanda, Assemblage Theory, 169. 4 The Emergence of Identity This question is taken up in the following section. In Aufhebung lies the principle of the creativity of failure; it is through the failure of something that we get to know it better. This point also appeared in Harman’s discussion of Heidegger’s tool-analysis: we take the hammer for granted, unawares, as long as it works, but when it breaks we pay explicit attention to it and its repair or replacement.24 Testing allows reality to shout ‘No!’25 Earlier, Fichte had made use the notion of Anstoß: reality shocks and nudges thought.26 There is a whole world of experience and practice behind this. One sees this also in science, which is –or should be– oriented toward finding out where a theory fails, in order to get from there to an improvement or replacement. To realize oneself, to learn and to transcend one’s ideas, to have a chance of being freed from preconceptions, one should profit from the opposition of others with different views.27 Different kinds of objects exert and undergo different kinds of effects; they change in different ways, through different sorts of processes. For living organisms, evolution is the salient process. The change of ideas or discovery follows a different process, though it is in some ways similar to evolution. The change of meaning in words is different again, though it bears some similarity to the change of ideas. What they have in common is the phenomenon of transformation, of the emergence of new forms from the old. Below, all three of these cases are discussed. All of them can benefit from insights from OOO, and contribute to a further analysis of change as an addition to OOO. 23 Žižek, For They Know Not What They Do. 24 Harman, Tool-Being. 25 Bachelard, La philosophie du ‘non.’ 26 Johnston, Žižek’s Ontology, 16. 27 Nooteboom, Beyond Humanism. This book seeks to find a way between Nietzsche’s immanent transcendence of the self within the self and Levinas’ imperative to open up to “the Other.” 28 Elements of evolutionary theory were also discussed by Harman in Object-Oriented Ontology as well as by DeLanda in Assemblage Theory. 29 Hodgson and Knudsen, Darwin’s Conjecture. 4 The Emergence of Identity OOO opposes the idea that effects of relations on objects are universal (with all objects having an effect on each other) and determinative (fully determining the identity of an object) because that would jeopardize the existence, the continued coherence of elements that constitutes its identity. The question now is: how does new identity emerge? When does change ‘break’ any identity and produce a new one? We have already seen an example: the transition from a full-service to a self-service restaurant. Such a change is frame-breaking, insofar as it does not maintain essential structure (if the term “essential” is appropriate here). How did this occur? By transferring the principle of self-service from supermarkets to restaurants. It is a classic case of innovation by way of ‘novel combinations,’ connecting previously unconnected things. Subsequently, the principle of self-service moved on further, to hotels. Here, a fundamental question arises. Aristotelian entelechy entails the realisation of a given potential. Can it be that with the realisation of potential, a new potential is created? That question appears to be central to the thought of Hegel. Thus, for the further development of OOO, some elements might well be adopted from transcendental ontology.22 Such an effort is the core of the present article. Can it be that in the realisation of potential, a new potential arises? Žižek taught us that Hegel should be read in this way. Hegel’s self-realisation of the ‘Absolute Spirit’ in world history should not be read as an inexorable march towards a pre-established end point or telos of perfection, but on the contrary, as a repeated and unpredictable breaking through the established order: through its failure, again and again, 58   B. Nooteboom 58   B. Nooteboom 58 in the course of history.23 Schelling used the notion of ‘potentialisation,’ a transition from the actual to the possible. Hegel, for his part, spoke of a dialectic of Aufhebung, i.e. elimination of an old synthesis in the process of lifting it to a new one. Is there more to be said about this process? This question is taken up in the following section. in the course of history.23 Schelling used the notion of ‘potentialisation,’ a transition from the actual to the possible. Hegel, for his part, spoke of a dialectic of Aufhebung, i.e. elimination of an old synthesis in the process of lifting it to a new one. Is there more to be said about this process? 29 Hodgson and Knudsen, Darwin’s Conjecture. 5 Evolution How would one look at evolution from the perspective of OOO?28 Is OOO effective in this context? In evolution the salient objects are organisms, with their internal metabolic structure and their external relations of seeking and taking in food, excreting waste, reproducing, seeking and serving as prey, and competing for survival with other objects. A species constitutes a superobject. It arises from the survival and reproduction of organisms, depending on the ‘selection environment.’ DNA would constitute the potential, or core part of it in animals. For green plants it would be the ability to conduct photosynthesis, producing cells from the intake of sunlight and CO2. But what could OOO learn from evolution? Harman already mentions the principle of symbiosis, where different objects complement each other through mutually supplementing materials or mutual protection. Let us see what more OOO could adopt, given that economics and various cultural disciplines have profited from taking evolutionary theory as a model. In evolution there are three fundamental processes: variation, selection and transmission (of the potential of what survives). In ‘generalised Darwinism,’ genes are ‘replicators’ copied in transmission, in inheritance; organisms are the ‘interactors’ that are selected and carry and transmit the replicators.29 This generates species, which cannot breed beyond their own boundaries. Perhaps the most remarkable thing about evolution is that it generates new forms without prior ‘intelligent design.’ There is the ontogenetic development of an individual organism, in the realisation of the potential of genes, which through interaction with each other and their environment produce elements of the organism (cells, organs, blood, 59 Objects, Relations, Potential and Change  hormones, etc.) through ‘gene expression.’ This is, I suggest, a particular case of the notion of emergence in OOO. The variety of organisms arises from chromosome crossover, in sexual reproduction. Could this occur more generally in OOO, with new objects arising from a mixing of potentials? There is also the phylogenetic development of species, through the selection of ‘interactors’ by the environment and the transmission of genes or ‘replicators.’ That is a second type of emergence. Species locked in an isolated environment develop in directions different from those in other environments: think of the koala bear, platypus (egg- laying, duck-billed, beaver-tailed, otter-footed), and kangaroo in Australia. Shifts in phylogeny can arise by (random) change of genes, in mutation, and changes in the selection environment. 5 Evolution The classic example of the latter is the crash of a large meteor on earth that –in blocking the sunlight– eliminated almost all species, such as the highly successful dinosaurs, which eventually opened up the opportunity for new species such as humans. This might suggest that OOO look at changes in any kind of object in terms of evolutionary change, random shifts of potential, and shifts in a given selection environment. To some extent organisms can build or at least affect their selection environment, which is known as ‘co-evolution’: as with beavers building dams to secure their habitat, an example mentioned by Bryant.30 They may also engage in symbiosis with other species, exploiting opportunities of complementarity, to the point of sharing or exchanging elements between them, as taken up by Harman in Immaterialism. Perhaps it can also contribute to the emergence of novel objects. A colourful example of symbiosis is that of small fish that swarm in and out of a shark’s maw to clean its teeth by picking out remnants of food. Some organisms can adapt to changes in the selection environment, such as chameleons that adapt their colour to where they are. Some organisms migrate to different selection environments that offer better conditions of food or safety, such as microbes moving by whipping a tail. How genes work yields an illustration and elaboration for OOO of how the emergence of new features from potential can work. Genes sometimes determine features more or less directly, such as eye colour, as experiments with fruit flies show. But in most cases properties emerge from a constellation of genes: as in intelligence, for example. Often, genes do not produce features directly, whether singly or in concert, but instead produce the potential to develop features depending on the environment. Thus it appears that people have an inborn, instinctive capacity to fear monsters, but whether this applies to snakes, spiders, or alligators depends on where people grow up. Babies have an inborn potential to both fear and welcome strange faces, and which of these develops first or most intensely depends on their educational environment.31 For OOO, this illustrates how the realisation of potential is not deterministic or pre- determined, but depends on the context –which can vary– so that realisation of potential becomes open while remaining constrained. 31 Sheets-Johnstone, The Roots of Morality. 30 Bryant, The Democracy of Objects, 208. 31 Sheets-Johnstone, The Roots of Morality. 30 Bryant, The Democracy of Objects, 208. 60   B. Nooteboom 60   B. Nooteboom In evolution, potential in the form of DNA can be transferred to a new object through reproduction. This raises a puzzle. If potential is seen as an object’s essence, is essence then really transferrable to a new object? That seems odd. What may save the idea is that in replication of DNA variation does occur, in mutation and chromosome crossover, so that it assumes a new constellation and hence a shift of essence. However, with identical twins and clones DNA is transferred identically. The point now is that in their existence the twins or clones go their separate ways, with different realisations of the same potential, and therefore are different objects. So then, identity is not just potential but potential plus its realization. That amounts to an important conclusion for OOO. Also potentially interesting for OOO is the distinction between the development of the individual object through a change of its composition (ontogeny) and its contribution to the wider development of the type or kind to which it belongs (phylogeny). Could this help us with the perennial issue of essence, and the corresponding relation between particulars and universals? This topic will come up in our later section on meaning and its change. How can a new kind of chair yield a shift in the general notion of chairs? Evolution can only build upon what was yielded by previous evolutions. Thus evolution can sometimes become stranded in a dead end, for lack of elements needed for a viable form of life in novel circumstances. What often happens instead is so-called ‘exaptation,’ where what is yielded in previous evolution is adapted to a different function from its original one. Perhaps the human ability to think is an exaptation of our ability to handle objects, providing a basis for logic to emerge. One of the reasons that reason developed in humanoids, then, would be that with their erect posture they had hands available to manipulate things. Is this notion of exaptation useful for OOO? Does it help to explain how objects can survive by novel use of what had developed previously? In evolution, not everything that survives automatically has a useful function in survival: things may survive simply because they do not hinder survival. That seems to apply, for example, to the appendix of our intestine. 60   B. Nooteboom It perhaps also applies to the silly-looking little arms of certain large dinosaurs that seem too small to reach anything. They may once have had the function of crawling, at an earlier stage of evolution, and it is not clear that they had any function at all in fully developed dinosaurs themselves. Another classic example is that of ‘spandrels’: features that arise as a by-product of the evolution of some other characteristic, rather than as a direct product of adaptive selection.32 For OOO this might imply that even if features of objects could be fully enumerated –which for Harman is of course not the case– those features may no longer be relevant. The next point is that, by definition, species are reproductively isolated from each other. That is a good thing for evolution. If all species could breed with all others, all differentiation would fade out, as would competition for survival and symbiosis. If convergence into a single species did occur, evolution would slow down but would not stop. As species spread across the world into different niches, new species would arise from what is called ‘allopatric speciation.’ However, they would then need to be locked up in impenetrable niches. This suggests that the fact that not all objects can affect each other, as proposed by Harman, may yield a basis for explaining the emergence of new objects. In principle evolution is a slow process, taking place over many generations, but sometimes it can happen comparatively fast, in what is called ‘punctuated equilibrium’: a rhythm in which there are long periods with little change but then a relatively fast emergence of new species. This can happen in particular in allopatric speciation, in which new species move into a new environment with different conditions, where they can utilize their latent potential undisturbed by the adverse conditions that ruled in their former habitat. This notion will re-appear in our later discussion of changes of ideas, with the claim that such change requires a shift in the environment or a move to a different one. In sum, the features of evolution may inform the OOO theory of change, with its notions of symbiosis (also employed by Harman), exaptation, allopatric speciation and punctuated equilibria. What follows are some examples of how evolutionary logic can inform fields other than biology. 32 The term arose in architecture to describe the triangular space between two adjacent arches and the ceiling, which occurs by geometrical necessity. These came to be utilized as bonus spaces for paintings, whether decorative or symbolic. 5 Evolution Potential may not directly produce features, but rather the ability to develop features as a function of outside conditions. That further contributes to the idea, proposed by Harman, that features of objects cannot be ‘paraphrased’ or fully enumerated. There was a time in evolutionary theory when it was thought, following Lamarck, that the genes of a child can be affected or formed by the conduct of its mother during pregnancy: such as playing piano to make the foetus more musical. This idea was eventually rejected, though later accepted again in a modified form. Since genes express themselves in interaction with their environment, the process of affecting or forming that environment can affect what the genes eventually produce. However, this is not an effect on the genes but on the realisation of their potential. For OOO, this illustrates that while the identity of an object in the form of potential remains the same, it can yield new and even unpredictable features depending on the environment of the object. To summarize, the relevance of all this for the present article on OOO is as follows. Evolution gives an example of how objects (organisms) have a potential (DNA) that is realized in its selection environment in interaction with other objects (prey, predator, symbiont). Important for OOO now is the question as to when and how the identity of an object can change or break, and whether and how that might yield a new object, with a new identity. Can potential still be seen to constitute identity? And can evolution yield ideas about this? 33 An example is the Journal of Evolutionary Economics, set up by heterodox economists excluded from the professional mainstream. 34 Campbell, “Blind Variation and Selective Retention in Creative Thought as in Other Knowledge Processes”; Boyd and Richerson, Culture and the Evolutionary Process. 35 Morton, Hyperobjects. 36 Lakatos, The Methodology of Scientific Research Programmes. 60   B. Nooteboom The logic of evolution has been utilized in both economic and cultural life, in the evolution of products and ideas. In economics, 61  Objects, Relations, Potential and Change invention and entrepreneurship generate variety, selection is performed by markets and institutions (laws, regulations), and transmission of success takes place through the growth of firms, imitation, and training and education. What is interesting from the perspective of policy and politics is the emergence of forms without the prior intelligent design to which people are stubbornly inclined, even in the wake of communism. This happens even in the field of innovation policy, where one directly intuits that planning is inappropriate there, because it is characterized by surprise, by the unexpected. Instead, the focus of policy should be on research, education, and entrepreneurship. Or, to put it in OOO terms: potential, its realisation, and its transfer. We should also consider the case of perverse markets with entry barriers for novelty. Existing firms have an interest in blocking ‘creative destruction,’ so as to profit a longer time from their established investments. Through lobbying activities they also exercise influence on the institutions that form part of the selection environment. In evolutionary terms: if large firms can shape the selection environment, in co-evolution, evolution stalls. Firms can avoid being selected out by creating or modifying markets and lobbying for their own advantage in laws and regulations (whether concerning the environment, taxes, or anti-monopoly laws),with the threat of taking their employment elsewhere if they do not get their way. Scientists can block the access of rivals to the journals they need to publish in order to obtain prestige, or prevent them from acquiring resources for research; but when locked out, the rivals can still set up their own journal.33 The model of evolution for economics is useful, but misleading; on a number of points there are fundamental differences. This applies as well to cultural evolution, such as the development of the meanings of words or expressions, with ‘memes’ (bits of memory) taking the place of genes.34 For first of all, the production of variety here is still subject to trial and error, but far less randomly than in mutation of genes and crossover of chromosomes. 60   B. Nooteboom With memes, it is partly based on creativity, learning, insight from failures, inference, and experimentation prior to submission to selection in markets; there is also the fact of ‘artificial selection,’ as in breeding cattle, laboratories, computer simulations, and tests with potential users. And second, as noted, selection in markets is often blocked by entry barriers. Existing large firms also buy up emerging innovative small firms, with the purpose of holding up (or ‘freezing’) the innovation. Third and finally, transmission in such cases takes the form of communication rather than replication, but produces deviant interpretations and misunderstanding that are much more fundamental and pervasive than ‘copying errors’ in the transmission of genes. In fact, transmission becomes part of the production of variation, in shifts and re-interpretations of what is communicated. For a good evolutionary account of economics and culture, one must therefore include theories of knowledge, invention and language. All things considered, evolution may be a misleading model in such cases. 6 Evolution of Ideas For OOO, ideas also are objects. Perhaps one can say that their internal constitution is formed by logic and semantics, in processes of sense making and communication; that is a subject for a later section. But the external relations of ideas are with other ideas. In any given theory, ideas cohere in assumptions or axioms that constitute its potential. In this respect, a theory can be viewed as a kind of hyperobject.35 Different theories are included in the sort of higher level object that Lakatos called ‘research programmes,’ with a ‘hard core’ of basic assumptions as its potential which is not up for falsification. In order to maintain its identity, this hard core is surrounded with a ‘protective belt’ of subsidiary assumptions.36 When falsification of a particular theory in a research programme arises, repairs are made to the protective belt, not the core. Can this idea perhaps yield a contribution to OOO, with the idea that the potential of an object is surrounded 62   B. Nooteboom 62 62 by some similar ‘protective belt’ to protect its identity? How far can it go? Can it lead to a transformation by which a new object or new idea arises? As a model of transformation take the butterfly, which ‘pupates’ from a caterpillar. Is that the manifestation of one object constituted of both butterfly and caterpillar? Here I return to Aristotle’s notion of entelechy. In the realisation of the caterpillar’s potential, nothing else could have emerged. An acorn produces an oak tree and cannot produce a butterfly. This leads to what was described earlier as ‘Hegel’s question,’ though it was also put by Schelling. In the realisation of a potential, can a new potential emerge: a new object with a new essence? Here I focus on the evolution of ideas, adopting the analysis from my already published idea of the ‘cycle of discovery,’37 inspired by the ideas of the genetic epistemologist Jean Piaget that cognition develops from ‘assimilating’ experience into existing ideas (‘frames’) and ‘accommodating’ them, developing new frames when this fails.38 I note the connection between this concept and Schelling’s notion that ideas arise from agency in reality. It is, I propose, a Hegelian theory of cognition. The basic idea of the cycle of discovery is that new ideas arise by applying an old idea in new settings. 37 Nooteboom, Learning and Innovation in Organizations and Economies. 38 See Flavell, The Developmental Psychology of Jean Piaget. Piaget’s work goes back to the 1930’s-1950’s, and has met with considerable criticism. This led to the replication and extension of his experiments in variant ways, some of them confirming his claims while others contradicted them. Thus his thought became controversial, but overall it retains a place in modern textbooks of developmental psychology; see Leman et al., Developmental Psychology. The crux of this book is the notion that cognition develops from action, through assimilation and accommodation, a point that does not seem to be contested. 39 Child, “Trust in International Strategic Alliances.” 6 Evolution of Ideas To use an analogy from evolution, bringing an idea into a new selection environment produces a kind of allopatric speciation. It is confronted with new challenges to survival from phenomena that do not fit its previous uses. Thus, the product or idea has to adapt if it is to survive. For example, when Xerox introduced copying machines in Japan, the machine was too high for office personnel to reach the buttons on top while standing, so they had to climb on boxes. This pointed to an easy way to adapt: either lower machines, or place buttons on the side. But if one is so powerful that one does not need to adapt to local circumstances, an opportunity to innovate may be missed. This is what happened to large American multinationals during the earlier economic development of China. They were so powerful in offering employment, technology and access to international markets that they did not have to adapt to local habits, tastes, organisation, labour conditions, etc. Meanwhile, smaller firms (often European ones) were forced to adapt, leading them to engage in alliances with local Chinese companies, and as a result they became more innovative.39 How does change proceed in the new environment? In a first effort to adapt, minor changes will be tried that leave the structure of the script and its nodes intact, by varying the choice of subscripts from existing repertoires, in ‘differentiation.’ When that is insufficient, the next step will be to incorporate, into the existing product/idea, elements from a local product/idea that is successful where one’s own is not. At first this will take the form of adopting new subscripts from nodes of successful local scripts, and then adopting entire nodes but within the existing overall script structure of nodes: a process known as ‘reciprocation.’ Note the similarity here with the evolutionary principle of symbiosis, proposed for use in OOO by Harman. The logic of hybrids is also close to the logic of metaphor: one learns to see something familiar in the new light of something other. An illustration of the adoption of a foreign element is the emergence of kilns for iron, blowing in air to raise the temperature, which was adopted from the smithy. An illustration of symbiosis is Information and Communication Technology (ICT) as a symbiosis of communication and computing. 6 Evolution of Ideas The amalgamation of old and new elements yields hybrids –a familiar phenomenon in innovation– a combination of old and new elements, forced into an old basic design or logic. A present-day example is the introduction of electric propulsion in gasoline-fuelled cars, yielding problems of inconsistency, overlap or duplication. There is overlap in propulsion, given that in the hybrid car there are ducts for both electricity and gasoline with its exhaust fumes. The heat of combustion may melt the casings of the electrical wires. The electric engine adds weight to the combustion engine. And electric propulsion in its own right is hampered by the weight of batteries, its limited range without recharging, and a general lack of recharging stations. The stage of hybridization is important for exploring the potential of foreign elements before making the sacrifice of dumping existing basic logic or design. This can be clarified with the notion of scripts, 63  Objects, Relations, Potential and Change as mentioned earlier. One can experiment with new subscripts in nodes while preserving overall node structure. This stage also allows one to explore where the problems lie in the integration of old and new and what, in the existing structure, limits the full utilization of new potential, and this gives hints as to the direction in which more promising structural change might lie. This in turn may lead to the next stage, where one makes experiments with greater structural change, known as ‘accommodation,’ using different configurations of old and re-configured or new nodes, either in the script itself or in the superscript in which it is lodged. This may not be easy, and in some cases may not even be possible. The problem of hybrid cars cannot be solved without more electric loading points or longer-life batteries without making the car too large and heavy. A competing technology is hydrogen fuel for cleaner combustion, with the additional advantage that it can be transported in the pipes and trucks that presently serve gas stations. Even the potential of ICT was not fully realised until the miniaturisation of computing in the development of microchips, leading eventually to the smart phone. There is still a gap in this portion of the theory, to the extent that it does not quite explain how accommodation is to be achieved. 6 Evolution of Ideas It gives conditions, incentives, and hints for how structural change might occur, but does not show how exactly it is to be achieved. That would seems to require a theory of creativity, in which chance or ‘serendipity’ will play a role. There is a connection here with ontology. Adrian Johnston speaks of “an alteration so radical that the criteria for its representation and recognition aren’t available.”40 In such cases, new criteria may need to be found through trial and error. When a new basic design does emerge, it is initially far from perfect and for a long time may carry residuals from the old logic that hamper efficiency and prevent the full breakthrough of the novelty in question. In the paintings of J.M.W. Turner, one sees the side-by-side existence of new, somewhat lumbering steam boats and elegant old sailing ships. In the transition from wooden bridges to the use of iron, the old constructive principle of ‘swallow tails’ to connect wooden parts was initially maintained, although with iron there is the alternative of welding. In an analysis of artillery practice it was found that upon firing a cannon, soldiers would back up a few steps more than needed to protect themselves from the recoil of the gun. Why? The analysis concluded that this was a left-over from old-time horse-drawn artillery, where upon firing it was necessary to step back to prevent the horses from bolting in fright at the explosion. The breakthrough of a novel design, and its subsequent improvement and refinement, is fast relative to the long process of transferring the old product or idea to a new field, making minor adjustments, creating hybrids, and tinkering with attempts at structural change. This process is reminiscent of punctuated equilibrium in biology. The question before us now is twofold. First, does the logic of change just described give us an answer to ‘Hegel’s question’? Is it a proper and valid example of how an existing potential in its realization can produce a novel potential? If potential constitutes the essence of an object, does this involve a change of potential, the shift of an object and its identity into a new one? Note that the principle of Hegelian logic that novelty emerges from failure appears here as well: the process is driven by the threat of a failure to survive in the new environment. 40 Johnston, Žižek’s Ontology, 100. 7 Meaning Alongside reference, or what is given, Gottlob Frege added the term ‘sense’ to denote how a given object manifests itself (die Art des Gegebenseins, or way in which things are given).42 In analytic philosophy there has been a debate over the sameness of meaning, using the principle (attributed to Leibniz) that two expressions have the same meaning if they can substitute for each other in a proposition salva veritate, or preserving the truth of the proposition. Famously, this does not work in ‘intentional and modal contexts’ (as discussed, notably, by Quine. An example of an intentional context is belief: “Jack believes that Aristotle was a teacher of Alexander the Great.” This cannot be substituted for with “Jack believes that a pupil of Plato’s was a teacher of Alexander the Great,” even though in fact Aristotle was a pupil of Plato, because Jack may not know this. Hence, there is sameness of reference but not sameness of sense. An example of a modal context is necessity: the number of seas (seven) is equal to the number of sins, but that is not necessarily so. So, sameness of reference is no guarantee for the sameness of meaning in all contexts. Concerning necessity, a solution was proposed in ‘possible world semantics’: two expressions have the same meaning if they have the same reference in all possible worlds. But identification across possible worlds is problematic, and it turns out to require a return to essence as what connects an identity across worlds in ‘trans world heir lines.’43 Attempts were made to preserve the reduction of meaning to the logically clear and determinate notion of reference, in order to avoid the more fuzzy, subjective, psychological notion of meaning as sense, or how an object is identified as something. In the present article, by contrast, the aim is to preserve sense: not only because logic requires it, but because it is a source of variety and change of meaning. Here, the meaning of sense is shifted from “how an object manifests itself” to “how people make the identification,” or “how they see something as ...” It is a property not of the object, but of the subject, which means that it is cognitive rather than ontological. The classic example is that of Venus, which can be seen as both ‘the evening star’ and ‘the morning star.’ People observe Venus either as one or the other. 7 Meaning From a OOO standpoint, words, linguistic expressions, and their meanings are all objects.41 Words are contained in sentences, which can be called superobjects by comparison. The sentence, in turn, may be part of a larger discourse. What is the internal structure of a word? Of meaning? The internal structure of a sentence would rest on its grammar and syntax. What is the potential of a word, of a meaning? Words and meanings are related, typically in the context of sentences. What are the distinctive features of words and meanings compared to other objects? When if ever do they break their identity, and how does this occur? Would an analysis of this process make a contribution to OOO? Would features of language have analogues elsewhere in the theory? We ask, first, what is the difference between words and material objects? A material object remains the same, from a human temporal perspective, while moving in time and space. When you move a chair from one room to another it remains the same. But when you move a word from one sentence to another, its meaning changes because that depends on the sentence in which it occurs. It would be as if the chair were to change colour or lose a leg when moved into a different room. In a sentence about dinner, the word ‘chair’ has a different meaning from what it does in a sentence about babies. In communication, meaning does not remain the same but is adapted to fit the absorptive capacity of the receiver. The old idea of meaning was that it is reference. This leads to the issue of knowledge and truth as a correspondence of ideas with reality, which we will not discuss further here. However that may be, it is dubious to claim that words, or linguistic expressions more generally, actually do refer in that sense. Reference, I propose, is intentional rather than ontological: people aim to refer to reality, leaving aside whether, or in what sense and to what extent, they actually do so. Words of course are not always only meant to refer. ‘Speech acts’ may be used performatively, such as to express emotion, utter oaths, or get people to do something or refrain from doing it. 41 Some of the following was also discussed by Gabriel in his Transcendental Ontology. 42 Thiel, Sinn und Bedeutung in der Logik Gottlob Freges. 43 Hintikka, The Intentions of Intentionality and Other New Models for Modalities. 6 Evolution of Ideas Unlike a wooden bridge, an iron bridge cannot burn. But it does expand and contract with temperature more than wood does, and on a hot day it may have to be doused with cooling water to maintain the fit in its berth. It does not require cutting down trees, but does require kilns for making the iron, as well as a different sort of training for bridge builders. Furthermore, it also offers a bridge span and a variety of forms that are impossible in wood. In short, it is indeed a different kind of object. Second, for OOO purposes, is it helpful to think of the change of objects in terms of such shifts of environment? To repeat, these shifts entail the need to adapt by incorporating elements from local objects, then encountering inconsistencies between old and new elements, raising the need for a more fundamental change of the basic ‘logic’ of internal composition. Could this then be seen as a break of identity or essence, and a source of the emergence of a novel object? Just as in the case of evolution, this is a programmatic proposal that cannot fully be developed here. 64   B. Nooteboom 7 Meaning Sense develops from ‘associations’ that people have built up through the course of their lives: such as whether they saw the ‘star’ in the morning or in the evening. Experience can shift how things are identified, and as what they are identified, thereby 65  Objects, Relations, Potential and Change also shifting reference. In Fregean semantics, the reference of a proposition was its truth value. Sense, then, is how one establishes truth. Our space is too limited here to enter into the debate on truth, its meaning, and its forms. But sense is personal, developed from experiences along one’s path of life. For example, one may associate a connotation with the word “chair” based on a memory of granddad’s chair: reclining, with curved armrests of polished mahogany, and upholstery of blue-grey velour fastened with buttons. When sense is widely shared between people, it becomes public. Here, inspiration can be taken from de Saussure’s distinction between parole (the idiosyncratic, diachronic spoken word) and langue (the synchronic, intersubjective order of meaning).44 As Wittgenstein said, there is no private language.45 One needs communication with others for a check that sense fits reference and is consistent rather than flying off in all directions, as happens in psychosis and in dreams. If someone lives on an uninhabited island and calls the stone he hits his toe on “dinger,” and on the next hit calls it “donger,” there is no one to correct this inconsistency. If someone asserts something then they presumably believe it, or they would not assert it, just as we cannot doubt a pain in the moment of having it. For the sake of coherence parole needs langue, but it can also inject change into it. Stated in the terms of OOO: sense and parole as objects are internal to a person (subjective) while reference and langue are external (intersubjective). The sentence in which a word is located is a hyperobject, and these sentences are part of a jargon or language, or “language game” as Wittgenstein would put it. Can the relations between them yield a new or general model of “mereology” (Bryant), of how objects and superobjects may be related? What is the relation between word and sentence? 7 Meaning Frege proposed that the meaning of a sentence is a function (in the mathematical sense) of the meanings of words in the sentence, in ‘upward causation.’ But the reverse applies as well: the meaning of a word depends on the sentence in which it is, as well as the context of action in which the sentence appears, in ‘downward causation.’ When someone hears “He sat on his chair at the desk,” that pretty much –if not totally– settles what kind of chair it is. As Wittgenstein said, if you want to know the meaning of a word, see how it is used. Sentences determine or settle word meaning in two ways. They settle the intended reference of a propositional sentence, picking out specific senses of words in the sentence from their multitudes of sense, which helps to establish the reference of the sentence. Gabriel puts it as follows: “Sense is a medium of difference whereas reference is meant to limit the sheer proliferation of sense without a referent.”46 Parole, or personal usage, is progressive. It can yield new personal sense, with only a small chance that it will adopted by public sense, langue, which is conservative. Perhaps the collapse of a host of possible senses of a word into a specific one in a sentence, can be seen by analogy with how a cloud of probabilities for different possible positions of elementary particles –such as electrons around the nucleus of an atom– collapses into a determinate position when colliding with another particle or object. Here the analogue to such a collision would be that a word gets connected with other words, “bumps into them” in the sentence. How, then, can meaning change? Here is an illustration of the change of sense. Some time ago in a newspaper there was a picture of a man sitting on a cow in his living room. It was a stuffed cow, with a dent in its back serving as the seat. The caption of the picture said: “See him sitting in his cow.” This is an idiosyncratic addition to the sense of a cow. After reading this, whenever one passes a field of cows, one may wonder how it would be to have one of them as a seat, though this sense is unlikely to become part of the public, shared sense of a cow. 44 De Saussure, Cours de linguistique générale. 45 Wittgenstein, Philosophical Investigations. 46 Gabriel, Transcendental Ontology, xiv. 44 De Saussure, Cours de linguistique générale. 45 Wittgenstein, Philosophical Investigations. 46 Gabriel, Transcendental Ontology, xiv. 7 Meaning An available model of meaning change has been offered by the ‘hermeneutic circle,’ for which Roman Jakobson employed the notions of the ‘syntagmatic’ and ‘paradigmatic’ axes.47 There is a connection here with the classic difference between Plato’s view of particulars (meanings in sentences, in the present context) as being reflections of fixed universal concepts that constitute ‘real reality,’ and Aristotle’s view of reality as consisting only of particulars from which universals arise. Here, the Aristotelian side is taken: general concepts may change from their specific use in specific sentences and settings. Parole can shift langue. In the hermeneutic circle, concepts from the paradigmatic axis are selected to come together in a sentence on the syntagmatic axis, and there clouds of sense collapse in specific meanings. Surprising combinations of sense in the sentence, then, may shift what is in the clouds on the paradigmatic axis. The question still remains as to when and how an identity-breaking change of sense occurs. Perhaps this happens when a word adopts a variety of senses that do not cohere well, and then a new coherent set is developed, allowing it to fit in sentences where it could not have made sense before. A new content of a node (word meaning) may open up new scripts (sentences) in which it fits. The stuffed cow as a chair, if adopted widely, may then allow the word “chair” to appear in a new script of producing chairs from stuffed cows, in a spin-off from the ordinary butchery business, as a new branch of the furniture business. But what about the difference between languages? Are different words for more or less the same concept different manifestations of the same meaning, or do they have different linguistic identities? No doubt, most such words –say, the English “chair” and the French chaise– have an overlap of sense. However, for some concepts there are two words in one language and only one in another. In English there are “faith” and “belief” while in Dutch there is only one word. For some words it is difficult to find a proper translation, such as the German Hegelian term Aufhebung. Sentences obtain their coherence or structure from different grammars and syntax. I think they should therefore be seen as different objects. What about essential meaning? 7 Meaning A ‘milk cow,’ by contrast, has become part of the sense of objects like investments or employees, who are seen to be ‘milked.’ The sense of a thing, as a repertoire of features associated with it in memory, is not to be seen as a repository of fixed things waiting to be selected for the purpose of establishing reference. They are not only triggered by the context at hand, but are also already affected by it. Memory is not retrieval, but reconstruction on the occasion of experience in a new context. In this way sense is a potential, with new realizations depending on context. Does sense-making potential now form the identity of (idiosyncratic) meaning? When and how would that identity change? Would it help to apply the ‘cycle of discovery’ in the 66   B. Nooteboom 66 change of knowledge as discussed above? That would mean that a fundamental change of sense could arise by shifting the word to a new context, finding that it fails there in inadequate reference, trying to adapt it by introducing sense from words that work in the new context, and experimenting with hybrids in preparation for a more fundamental or ‘structural’ change. What would that signify here? change of knowledge as discussed above? That would mean that a fundamental change of sense could arise by shifting the word to a new context, finding that it fails there in inadequate reference, trying to adapt it by introducing sense from words that work in the new context, and experimenting with hybrids in preparation for a more fundamental or ‘structural’ change. What would that signify here? Does the model of the script apply? Take the sentence as a script and the word as a node, with connections between the nodes made according to logic, grammar and syntax. In downward causation, the sentence (script) limits what potential meanings of the word (node) are ‘admissible,’ what meanings can ‘work.’ But that does not necessarily leave such admissible sense unaffected: the sentence and its action context may ‘tweak’ the sense. The story about someone sitting at a desk may show that the chair has no backrest. That is unusual, but it can make sense, and may then be added to the repertoire of the sense of the word “chair”’ Even the stuffed cow may make sense. 47 Jakobson, Language in Literature. The syntagmatic ‘axis’ is better seen not as a line but as a collection of sentences, and the ‘paradigmatic axis’ (a bit of a misnomer given the confusion about ‘paradigms’) as concepts with their repertoires of sense. 48 Fry, The Ode Less Travelled.   67 Objects, Relations, Potential and Change  67 What, if any, is the importance of all this for OOO? It raises the following programmatic questions. Can objects other than meanings also change in something like metaphoric or metonymic shifts, incorporating features from other objects? Note the similarity of these shifts to the adaptation of an object by the adoption of features from another object, as discussed earlier with the transformation of ideas as well as with symbiosis in evolution. ­­­­How different would the cases be? Does it help to see the phenomenology of an object, its effects on other objects (such as the meanings of words that meet in a sentence) as varying across settings (such as sentences)? Would it be fruitful to consider how this variation (here on the syntagmatic axis) can affect the identity of the originating object (here on the paradigmatic axis)? In other words, how might an object extend or shift its potential as a function of the variety of its effects? These are deep questions that may merit further thought. References Bachelard, Gaston. La philosophie du non. Paris: Presses Universitaires de France, 1975. Bhaskar, Roy. A Realist Theory of Science. London: Verso, 1975. Bhaskar, Roy. A Realist Theory of Science. London: Verso, 1975. Boyd, Robert and Peter J. Richerson. Culture and the Evolutionary Process. Chicago:Un Boyd, Robert and Peter J. Richerson. Culture and the Evolutionary Process. Chicago:University of Chicago Press, 1983 Bryant, Levi. The Democracy of Objects. Ann Arbor, MI: Open Humanities Press, 2011. Campbell, D.T. ‘Blind Variation and Selective Retention in Creative Thought as in Other Knowledge Processes,’ Psychology Review, 67(6), 1960, 380-400. Child, John. ‘Trust in International Strategic Alliances,’ in C. Lane and R. Bachmann (eds.), Trust Within and Between O zations. Oxford: Oxford University Press, 2001. DeLanda, Manuel. Assemblage Theory. Edinburgh: Edinburgh University Press, 2016. DeLanda, Manuel and Graham Harman. The Rise of Realism. Cambridge, UK: Polity Press, 2017. lavell, John H. The Developmental Psychology of Jean Piage Flavell, John H. The Developmental Psychology of Jean Piaget. London: Van Nostrand, 1963. tephen, The Ode Less Travelled. London: Penguin, 2005. Gabriel, Markus. Transcendental Ontology. New York: Bloomsbury, 2011. Garcia, Tristan. Form and Object: A Treatise on Things, trans. M.A. Ohm & J. Cogburn. Edinburgh: Edinburgh Unive 2014. Harman, Graham. Object-Oriented Ontology: A New Theory of Everything. London: Pelican, 2018. Harman, Graham. Tool-Being: Heidegger and the Metaphysics of Objects. Chicago: Open Court, 2002. Hintikka, Jaakko. The Intentions of Intentionality and Other New Models for Modalities. Dordrecht, The Netherlands: Reidel, 1975. Hodgson, Geoffrey and Thorbjorn Knudsen. Darwin’s Conjecture: The Search for General Principles of Social and Economic Evolution. Chicago: University of Chicago Press, 2010. Hodgson, Geoffrey and Thorbjorn Knudsen. Darwin’s Conjecture: The Search for General Principles of Social and Evolution Chicago: University of Chicago Press 2010 Hodgson, Geoffrey and Thorbjorn Knudsen. Darwin s Conjecture: The Search for General Principles of Social and Evolution. Chicago: University of Chicago Press, 2010. Jakobson, Roman. Language in Literature. Cambridge, MA: Harvard University Press, 1987. Ž Johnston, Adrian. Žižek’s Ontology: A Transcendental Materialist Theory of Subjectivity. Evanston, Il: Northwestern University Press, 2008. Lakatos, Imre. The Methodology of Scientific Research Programmes: Philosophical Papers, Volume 1. Cambridge, UK Lakatos, Imre. The Methodology of Scientific Research Programmes: Philosophical Papers, Volume 1. Cambridge, UK: Cambridge University Press, 1980. Lakatos, Imre. The Methodology of Scientific Research Programmes: Philosophical Papers, Volume 1. Cambridge, UK Cambridge University Press, 1980. an, Patrick, Andy Bremer, Ross D. Parke and Mary Gauvain. Developmental Psychology. 7 Meaning If essence for OOO is indeed the potential of an object to manifest itself, within boundaries and depending on contexts, plus its manifestations as exhibited so far, how would that apply to meaning? Is there an essential chair? There are ‘prototypes,’ or cases taken as ‘typical,’ with other manifestations taken as more or less similar. It seems that for the Dutch the prototype of a bird is the sparrow, while for the British it is the robin. Could this be helpful for OOO? Poetry can shift sense by means of metaphor (where an object is seen in terms of another type of object) and in metonymy (where it is seen in terms of a related object). Fishing for compliments is a metaphor, fishing for pearls is metonymy, since fishing for fish and for pearls share a similar relation to the sea whereas compliments do not. Shifts can also occur also by means of rhyme or assonance, which connects words by sound rather than meaning, yielding a surprising connection that may affect sense.48 Concerning general public concepts, or universals, whether taken as essences or as prototypes, widely used in similar ways by different people and in different contexts, these are subject to new senses arising from language use: as in a shift or tweak of the universal to such an extent that its essence seems dubious. Should the notion of essence be dropped, perhaps? Could “prototype” serve to replace it? References London: McGraw-Hill, 2019. y y p y gy Morton, Timothy. Hyperobjects: Philosophy and Ecology After the End of the World. Minneapolis: University of Minnesota Press, 2013. ton, Timothy. Hyperobjects: Philosophy and Ecology After the End of the World. Minneapolis: University of Minnesota Press, 2013. Press, 2013. teboom, Bart. “From evolution to language and learning,” pp. 41-49, in J. Foster (ed.), Frontiers of Evolutionary Econo Nooteboom, Bart. “From evolution to language and learning,” pp. 41-49, in J. Foster (ed.), Frontiers of Evolutionary Economics Nooteboom, Bart. “From evolution to language and learning,” pp. 41-49, in J. Foster (ed.), Frontiers of Evolutionary Economi Competition Self Organisation and Innovation Policy Cheltenham UK: Edward Elgar 2001 Nooteboom, Bart. From evolution to language and learning, pp. 41 49, in J. Foster (ed.), Frontiers of Ev Competition, Self-Organisation and Innovation Policy. Cheltenham, UK: Edward Elgar, 2001. Competition, Self-Organisation and Innovation Policy. Cheltenham, UK: Edward Elgar, 2001. Nooteboom, Bart. Learning and Innovation in Organizations and Economies. Oxford: Oxford University Press, 2000 Nooteboom, Bart. Beyond Humanism: The Flourishing of Life, Self and Other. London: Palgrave McMillan, 2012. Saussure, Ferdinand de. Cours de linguistique générale. Paris: Payot, 1972. Schelling, F.W.J. Philosophical Enquiries Into the Nature of Human Freedom and Matters Connected Therewith, trans. J. Gutman. Chicago: Open Court, 1936 [1809]. Sheets-Johnstone, Maxine. The Roots of Morality, University Park, PA: Penn State University Press, 2008. Thiel, Christian. Sinn und Bedeutung in der Logik Gottlob Freges. Meisenheim am Glan, Germany: Anton Hain V Wittgenstein, Ludwig. Philosophical Investigations, trans. G.E.M. Anscombe, P.M.S. Hacker, and J. Schulte, revised 4th edition. London: Wiley-Blackwell, 2009. y , Žižek, Slavoj. The Parallax View. Cambridge, MA: MIT Press, 2009. ek, Slavoj. The Parallax View. Cambridge, MA: MIT Press, 2 Žižek, Slavoj. For They Know Not What They Do: Enjoyment as a Political Factor. London: Verso, 2002.
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Editorial: Modulating Cytokines as Treatment for Autoimmune Diseases and Cancer
Frontiers in immunology
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INTRODUCTION Cytokines are key mediators in the regulation of the normal immune response. They are ambivalent molecules which can be either beneficial to the treatment of diseases, but can also be harmful and participate in pathogenesis. Indeed, despite regulatory controls at multiple levels, abnormal immune responses involving cytokines can occur and cause various pathologies, including autoimmunity and inflammation-induced cancer. For these reasons, it is crucial to continue efforts focused on understanding the different modes of action of cytokines with an eye toward the design of new selective drugs that modulate cytokine activities that direct beneficial immune responses. EDITORIAL published: 15 October 2020 doi: 10.3389/fimmu.2020.608636 Edited and reviewed by: Silvano Sozzani, Edited and reviewed by: Silvano Sozzani, Sapienza University of Rome, Italy *Correspondence: Erwan Mortier erwan.mortier@univ-nantes.fr Edited and reviewed by: Silvano Sozzani, Sapienza University of Rome, Italy Deregulation of cytokine expression has a complex role in disease pathogenesis and novel therapeutic agents that neutralize cytokines have been successfully translated into clinical practice. For instance, the use of monoclonal anti-TNF antibodies have greatly improved the health of patients suffering from diseases like inflammatory bowel diseases, rheumatoid arthritis, spondyloarthritis, or psoriasis. Furthermore, additional cytokine blockers such as anti-IL-6R antibodies, IL-12/IL-23 p40 inhibitors and IL-23 p19 blockers have been approved for various immune-mediated diseases. The use of effector cytokines (e.g., IL-2, IFNg) either alone or in combination with other therapeutic reagents, such as checkpoint inhibitors and emerging immunocytokines, is accelerating in cancer immunotherapy. While, IL-2 was approved by the Food and Drug Administration for the treatment of metastatic kidney cancer in 1992 and for metastatic melanoma in 1998, researchers are still working to improve IL-2 efficacy and reduce toxicity. Given the broad range of biological activities of cytokines, the side effects of biologic therapies need to be carefully assessed and warrant the development of new therapeutics with improved specificity of action. Thus, fundamental discoveries on structural features of cytokines in interaction with their different receptor chains could lead to the identification of cytokines with reduced toxicity and increased specificity. *Correspondence: Erwan Mortier ti @ i t f Specialty section: This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology Received: 21 September 2020 Accepted: 28 September 2020 Published: 15 October 2020 Keywords: cytokine, oncology, inflammation, inhibitor, therapy Keywords: cytokine, oncology, inflammation, inhibitor, therapy Editorial: Modulating Cytokines as Treatment for Autoimmune Diseases and Cancer 1 University of Nantes, CNRS, Inserm, CRCINA, Nantes, France, 2 Department of Medicine, University of California, San Francisco, San Francisco, CA, United States, 3 University Hospital Erlangen, University Erlangen-Nürnberg, Erlangen, Germany, 4 Deutsches Zentrum Immuntherapie DZI, Erlangen, Germany Keywords: cytokine, oncology, inflammation, inhibitor, therapy Editorial on the Research Topic Modulating Cytokines as Treatment for Autoimmune Diseases and Cancer Modulating Cytokines as Treatment for Autoimmune Diseases and Cancer Citation: Mortier E, Ma A, Malynn BA and Neurath MF (2020) Editorial: Modulating Cytokines as Treatment for Autoimmune Diseases and Cancer. Front. Immunol. 11:608636. doi: 10.3389/fimmu.2020.608636 In this Research Topic issue entitled “Modulating Cytokines as Treatment of Autoimmune Diseases and Cancer”, we have compiled 6 original research articles, 1 hypothesis and theory and 6 reviews. This collection is divided into four sections. The first section presents recent knowledge for a better understanding of the mode of action and the structural features of the interaction between cytokines and their receptors. The second section describes new targets for the treatment of October 2020 | Volume 11 | Article 608636 Frontiers in Immunology | www.frontiersin.org Editorial: Modulating Cytokines for Therapy Mortier et al. autoimmune diseases. The third is devoted to the use of cytokines to induce tolerance and the last section presents different combinations between cytokines and other therapeutic agents for the treatment of cancer. focused on recent knowledge of the functions of human ILCs and provide a comprehensive view of the major regulators, including cytokines, that selectively support the three ILC subpopulations. A better understanding of the regulation of human ILC functions should help researchers use ILCs and modulate their action under inflammatory conditions in the future. BETTER UNDERSTANDING OF CYTOKINE STRUCTURE/FUNCTION AND THEIR MODES OF ACTION analyzed the literature that used dry eye disease models to find cells and cytokines that could be targeted in this pathology. They show the involvement of Th1 cells as well as IL-1b and TNFa proinflammatory cytokines. This meta-analysis also prompts precaution when using animal models that do not fully recapitulate human pathology. USE OF CYTOKINES TO INDUCE TOLERANCE Autoimmune diseases are characterized by the disruption of tolerance to self antigens. Different approaches have been designed by researchers to restore tolerance, including cell therapy by injecting tolerogenic dendritic cells (Tol-DC) or regulatory T (T-reg) cells. Another approach is to target tolerogenic cells directly in vivo. Cauwels and Tavernier proposed an original strategy to expand endogenous Tol-DC in vivo by the administration of AcTakine molecules. The latter consists of a targeting module (VHH is more commonly used) fused to a mutated cytokine with reduced affinity to its cognate receptor. They engineered a Tol-DC AcTaferon with IFN-I to induce tolerance in autoimmune diseases. Cytokines are primarily described as soluble factors, but they can also be packaged within extracellular vesicles. In their review, Barnes and Somerville provide a new vision of the action of cytokines and lead us to bear in mind that the production of both cytokines and extracellular vesicles play an important role in pathology. These properties could be translated into therapy by engineering extracellular vesicles to deliver immune modulators such as cytokines in pathological conditions. IL-2 is an important cytokine for the development of T-reg cells, which constitutively express IL-2Ra. The latter forms a trimeric receptor with IL-2Rb and the common gamma chain and binds IL-2 with a high affinity allowing T-reg cells to respond to low dose of IL-2. In their study, Ghelani et al. attempt to find the threshold required for IL-2 to selectively expand T-reg cells into effector cells. To this end, they generated a series of IL-2 muteins and found that minimal IL-2 receptor signaling is required to fully expand regulatory T cells and support their immunosuppressive functions. BETTER UNDERSTANDING OF CYTOKINE STRUCTURE/FUNCTION AND THEIR MODES OF ACTION Adipokines are cytokines produced by adipocytes. Among them, visfatin appears to play an important role in the pathogenesis of rheumatoid arthritis (RA) by increasing the adhesion of RA synovial fibroblasts to endothelial cells. Hasseli et al. draw attention to visfatin and other adipokines as potentially interesting targets in the search for RA therapeutics. In generating new reagents, investigators need to assess their efficacy in relevant animal models. In their study, Lio et al. analyzed the literature that used dry eye disease models to find cells and cytokines that could be targeted in this pathology. They show the involvement of Th1 cells as well as IL-1b and TNFa proinflammatory cytokines. This meta-analysis also prompts precaution when using animal models that do not fully recapitulate human pathology. Adipokines are cytokines produced by adipocytes. Among them, visfatin appears to play an important role in the pathogenesis of rheumatoid arthritis (RA) by increasing the adhesion of RA synovial fibroblasts to endothelial cells. Hasseli et al. draw attention to visfatin and other adipokines as potentially interesting targets in the search for RA therapeutics. Increasing knowledge of the structural interactions between a cytokine and its receptor chains is fundamental for generating original selective reagents targeting cytokine’s action. In their review, Markovic and Savvides focused on the structure and the mode of signaling assemblies of two closely related cytokines that share the IL-7Ra chain, IL-7 and TSLP. The review of Metcalfe et al. is devoted to the IL-6 family with a focus on IL-11. The authors of both reviews present structural overviews of the two cytokine systems and their involvements in pathological conditions. They also provide an overview of the broad array of potential therapeutic agents in autoimmune diseases to thwart overexpression of the targeted cytokines including monoclonal antibodies, chemical compounds, soluble receptors, and muteins. Along this line, Holgado et al. focused on an original strategy for inhibiting the action of cytokines by generating cytokine-traps. Their approach is based on the generation of molecules consisting of the fusion of receptors chains to form soluble heterodimers capable of capturing cytokines before interacting with their membrane-bound receptors. They are able to efficiently modulate either IL-33 alone by the IL-33-Trap or two cytokines simultaneously using the dual IL-14/13-Trap to inhibit experimental airway inflammation. In generating new reagents, investigators need to assess their efficacy in relevant animal models. In their study, Lio et al. Frontiers in Immunology | www.frontiersin.org LOOKING FOR NEW TARGETS The research community is always on the lookout for new targets for the design of novel therapeutics. Thus, it is crucial to deepen our knowledge of relevant targets involved in pathology. Brune et al. focused on IRF5, a key transcriptional regulatory factor of type-I interferon. Hyperactivation of IRF5 has been identified as key factor in several autoimmune diseases, including systemic lupus erythematosus. In their review, Brune et al. provide an original perspective on the complex role of IRF5 and focus on T cell functions and polarization. IL-34 is another cytokine with tolerogenic properties. In their study, Bézie et al. show that CD4 and CD8 FoxP3 regulatory T cells increase significantly when cultured in the presence of monocytes differentiated by IL-34. In addition, human CD8 regulatory T cells grown under these conditions suppress the immune response in a humanized model of acute GVHD by effectively increasing the survival of the graft after organ transplantation by acting on T-regs cells and monocytes. These Innate lymphoid cells (ILC) are unique cell populations that play important roles in immune defense in response to chronic inflammatory and autoimmune diseases. Schulz-Kuhnt et al. October 2020 | Volume 11 | Article 608636 Frontiers in Immunology | www.frontiersin.org 2 Editorial: Modulating Cytokines for Therapy Mortier et al. results demonstrate that IL-34 should also be considered for therapy with regard to its property for promoting the development of regulatory T cells. To restrict IL-21 activity on targeted cells and slow down the clearance of the ICK, the investigators mutated the cytokine to decrease its affinity for its cognate receptor. Preliminary preclinical data reveal that the anti-PD1-attenuated-IL-21 immunocytokine shows promise for anti-cancer indications. CYTOKINES IN COMBINATION WITH OTHER THERAPEUTIC AGENTS IN CANCER Collectively, the above studies highlight the potential of recombinant cytokines and their inhibitors to more effectively treat autoimmunity and cancer. These results open new avenues for research and may lead to improved therapeutic options in clinical therapy by precision editing of cytokine responses. IL-15 is a cytokine that shares with IL-2 an important role in supporting the development and functions of effector cells, such as NK and CD8 T cells. Unlike IL-2, IL-15 does not support regulatory T cells. For these reasons, IL-15 was identified early as a potential candidate for use in cancer immunotherapy. However, IL-15 administrated as monotherapy did not show therapeutic efficacy despite a dramatic expansion of NK and CD8 T cells. In their review, Waldmann et al. present assays that use IL-15 in combination with therapeutic monoclonal antibodies such as anti-CD40, anti-CD20, anti-CD52, anti- EGFR, anti-CTLA-4, anti-PD-1 and anti-PDL-1. Preliminary combination studies show better efficacy than individual agents alone and hold promise for the treatment of patients with metastatic malignancy. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. October 2020 | Volume 11 | Article 608636 Frontiers in Immunology | www.frontiersin.org ACKNOWLEDGMENTS We would like to thank all the authors for their contributions to this Research Topic and the reviewers for their insightful comments. 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. Another way to combine cytokines and therapeutic monoclonal antibodies is to fuse them to generate immunocytokines (ICK). In their study, Shen et al. fused anti-PD-1 antibody to IL-21 muteins. The advantages of such molecules are numerous. They improve both the half-life and bioavailability of cytokines. Targeting cytokine therapies to specific cells may better replicate the paracrine activities of physiologically delivered cytokines. This approach can thus enhance efficacy and limit off-target effects. Copyright © 2020 Mortier, Ma, Malynn and Neurath. 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. October 2020 | Volume 11 | Article 608636 Frontiers in Immunology | www.frontiersin.org 3
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Technical evaluation of different respiratory monitoring systems used for 4D CT acquisition under free breathing
Journal of applied clinical medical physics
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a Corresponding author: Christian Heinz, Department of Radiation Oncology, LMU University Hospital, Marchioninistraße 15 D-81377, Munich, Germany; phone: +49 89 4400 76767; fax: +49 89 4400 76770; email: Christian.Heinz@med.uni-muenchen.de Technical evaluation of different respiratory monitoring systems used for 4D CT acquisition under free breathing Christian Heinz,a Michael Reiner, Claus Belka, Franziska Walter, Matthias Söhn Department of Radiation Oncology, LMU University Hospital, D-81377, Munich, Germany Christian.Heinz@med.uni-muenchen.de Received 28 January, 2014; accepted 29 October, 2014 Respiratory monitoring systems are required to supply CT scanners with informa­ tion on the patient’s breathing during the acquisition of a respiration-correlated computer tomography (RCCT), also referred to as 4D CT. The information a respi­ ratory monitoring system has to provide to the CT scanner depends on the specific scanner. The purpose of this study is to compare two different respiratory monitor­ ing systems (Anzai Respiratory Gating System; C-RAD Sentinel) with respect to their applicability in combination with an Aquilion Large Bore CT scanner from Toshiba. The scanner used in our clinic does not make use of the full time dependent breathing signal, but only single trigger pulses indicating the beginning of a new breathing cycle. Hence the attached respiratory monitoring system is expected to deliver accurate online trigger pulse for each breathing cycle. The accuracy of the trigger pulses sent to the CT scanner has to be ensured by the selected respiratory monitoring system. Since a trigger pulse (output signal) of a respiratory monitoring system is a function of the measured breathing signal (input signal), the typical clinical range of the input signal is estimated for both examined respiratory moni­ toring systems. Both systems are analyzed based on the following parameters: time resolution, signal amplitude, noise, signal-to-noise ratio (SNR), signal linearity, trigger compatibility, and clinical examples. The Anzai system shows a better SNR (≥ 28 dB) than the Sentinel system (≥ 14.6 dB). In terms of compatibility with the cycle-based image sorting algorithm of the Toshiba CT scanner, the Anzai system benefits from the possibility to generate cycle-based triggers, whereas the Sentinel system is only able to generate amplitude-based triggers. In clinical practice, the combination of a Toshiba CT scanner and the Anzai system will provide better results due to the compatibility of the image sorting and trigger release methods. PACS numbers: 87.57.Q-, 07.07.Df PACS numbers: 87.57.Q-, 07.07.Df Key words: 4D CT, respiratory monitoring system, free breathing JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 16, NUMBER 2, 2015 JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 16, NUMBER 2, 2015 Technical evaluation of different respiratory monitoring systems used for 4D CT acquisition under free breathing Christian Heinz,a Michael Reiner, Claus Belka, Franziska Walter, Matthias Söhn Department of Radiation Oncology, LMU University Hospital, D-81377, Munich, Germany Christian.Heinz@med.uni-muenchen.de Received 28 January, 2014; accepted 29 October, 2014 Heinz et al.: Evaluation of different respiratory monitoring systems Heinz et al.: Evaluation of different respiratory monitoring systems To minimize these errors, different methods of CT acquisition were developed including respiration-correlated computer tomography (RCCT)(1,2,3) also referred to as 4D CT, respiratory- gated CT acquisition,(4,5) or dynamic volume techniques.(6,7) Applicable methods depend to a certain degree on characteristics of the CT scanner. Dynamic volume techniques are limited to CT scanners with a large-area 2D detector. Such detectors allow effective lengths of the scanned volume up to 160 mm.(6) Thereby a whole volume scan can be acquired in about 0.35 sec. From multiple volume scans at different time points a dynamic volume can be reconstructed. During the acquisition of a respiratory-gated CT, image data are collected only when the patient is in the previously defined respiratory state. The most prominent respiratory states for gated CT techniques are the end-inspiration and the end-expiration states. Another commonly used acquisition method is the 4D CT. A time resolved image dataset is reconstructed by oversam­ pling — each image position is acquired multiple times during at least one complete breathing cycle — and retrospective sorting of all images into image stacks with the same respiratory state. The process of retrospective image sorting can be divided in two main groups using (a) amplitude-based sorting algorithms or (b) phase-based sorting algorithms. A more detailed classification is given by Guckenberger et al.(8) The prerequisite common to all image sort­ ing algorithms is that the images need to be tagged with their corresponding respiratory state. Therefore, respiratory monitoring systems are necessary to deliver information on the patient’s respiratory state to the CT scanner. Inaccurate information on the respiratory state will result in errors during the retrospective image sorting and, consequently, in image artifacts. To avoid errors in image sorting it is important that the respiratory monitoring system delivers the exact information expected by CT scanner. There are numerous respiratory monitoring systems on the market that uses different prin­ ciples of measurement (e.g., Varian’s RPM system; Philips Medical Systems’ pneumatic bel­ lows(9)). The aim of this study is to evaluate the applicability of the newly developed C-RAD Sentinel system (C-RAD AB, Uppsala, Sweden) in combination with the 4D CT mode of a Toshiba CT scanner (Toshiba Medical System Group, Tokyo, Japan). The second respiratory system available in our department — Anzai Respiratory Gating System (Anzai Medical Co. Ltd, Tokyo, Japan) — serves as a reference. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 II. MATERIALS AND METHODS A. Toshiba CT scanner I. INTRODUCTION In several radiotherapy treatment sites (e.g., lung, liver) respiration-induced movements com­ promise the intention to deliver the prescribed dose to the tumor. The initial problem of tumor motion results in motion artifacts, which can be observed in the reconstructed images, errone­ ous Hounsfield unit (HU) values, and potentially insufficient dose coverage caused by incor­ rect motion estimation during delineation of the tumor volume. Hypofractionated stereotactic radiotherapy treatments are especially affected by these problems because of their particular small volumes. 334 334 335 335 Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 A. Toshiba CT scanner The scanner used in this study is a Toshiba Aquilion 16 Large Bore scanner including the AquilionLB Software Ver3.38ER005.(10,11) The 4D CT mode of the Toshiba scanner is derived from an electrocardiography mode (ECG), originally designed for cardiac imaging. With respect to the classification given by Guckenberger et al.,(8) the implemented image sorting method is a special form of phase-based sorting called cycle-based sorting. For each breathing cycle including inspiration and expiration, the CT scanner expects a single trigger from the respiratory monitoring system indicating the beginning of the cycle. Images are then sorted into n equidistant phases between two recognized trigger pulses corresponding to n phases of the breathing cycle (Fig. 1(a)). The trigger pulses delivered to the CT scanner can be manipulated retrospectively, but in clinical use this is impractical and lacks accuracy, as the software does not show the time-resolved breathing curve but solely the trigger pulses. Therefore, an important clinical requirement of each respiratory monitoring system is the ability to deliver cycle-based online trigger pulses with high reliability. Heinz et al.: Evaluation of different respiratory monitoring systems 336 Heinz et al.: Evaluation of different respiratory monitoring systems 336 Fig. 1. Scheme of the cycle-based 4D CT mode as it is implemented in the Toshiba scanner. An exemplary respiratory signal with corresponding triggers delivered by (a) the Anzai System and (b) the Sentinel system is used to reconstruct n = 4 respiratory phases. Triggers are expected to indicate the beginning of a new breathing cycle. Note that the amplitude-based triggers delivered by the Sentinel system in (b) are set incorrect with respect to the length of a breathing cycle, including a complete inspiration and expiration. As a result, the image sorting in (b) is faulty. Fig. 1. Scheme of the cycle-based 4D CT mode as it is implemented in the Toshiba scanner. An exemplary respiratory signal with corresponding triggers delivered by (a) the Anzai System and (b) the Sentinel system is used to reconstruct n = 4 respiratory phases. Triggers are expected to indicate the beginning of a new breathing cycle. Note that the amplitude-based triggers delivered by the Sentinel system in (b) are set incorrect with respect to the length of a breathing cycle, including a complete inspiration and expiration. As a result, the image sorting in (b) is faulty. C. C-RAD Sentinel laser scanner The Sentinel system consists of a laser which sweeps within a few seconds over the surface of the patient and a camera to detect the laser projections (Fig. 3(a)). From multiple projection lines, the software generates a static three-dimensional model of the patient’s surface. In addition to assistance in patient positioning, the Sentinel system contains a software module for 4D CT acquisition (c4D Version 4.5.0). By spotting a user-defined region on the surface of a patient (Fig. 3(b)) and subsequent triangulation, an online surrogate signal of the patient’s respiration can be acquired, which bases on the anterior/posterior movement (Z position) of the selected region. The resulting respiratory signal is scaled absolute and displayed in millimeters. By the use of two threshold values for the respiratory signal a window can be specified. Trigger events are released either on entrance or exit of the specified window or a combination of both. Since triggers are released at defined threshold values, the Sentinel system is an amplitude-based triggering system (compare Fig. 1(b)). The c4D software does not provide any option to trigger on inspiration or expiration peaks. p p p There are at least two problems that have to be addressed to assure the accuracy of the recorded respiratory signal. First, the laser has to track the user-specified measurement region correctly during couch movements. Therefore, the Sentinel system requires a wire ruler (Fig. 3(c)) that delivers the couch position to the laser positioning controller. Second, a CT couch typically consists of carbon fibers and is mounted with a slight pretension in positive Z direction to compensate for deflections under the load of a patient. Depending on the patient’s weight and the actual couch position, different deflections in Z direction can be observed, which are superimposed on the respiratory signal. In order to correct this error, the Sentinel system makes use of a couch calibration profile. During the calibration process two profiles are recorded. The first one is used for daily check purposes only and, despite the Sentinel’s QA Phantom, no additional weights are placed on the CT couch. The second couch calibration profile is recorded with the Sentinel’s QA Check Phantom and additional calibration weights of 80 kg. The weights are placed along the couch to reproduce a typical patient load. This profile is used in the clinical mode for all patients regardless of their actual weight. B. Anzai Respiratory Gating System y g y The Anzai Respiratory Gating System (AZ-733V Version 2.2H) consists of a fixation belt which is used to position a pressure transducer at the right upper quadrant of a patient’s abdo­ men (Fig. 2(a)). Due to expansion and contraction of the belt during breathing, the pressure transducer delivers a digital voltage signal, which is amplified and then evaluated by the Anzai software. The system includes two pressure transducers (low/high) with different sensitivities for patients with shallow vs. deep respiration amplitudes, as well as four different sized fixation belts used to compensate different circumferences of different patients (Fig. 2(b)). Three LEDs indicate whether the pressure signal is optimal, too low, or too high, corresponding to a correct or incorrect application of the fixation belt (Fig. 2(c)). By selecting appropriate values for off­ set and gain of the electrical amplifier, the user is able to optimize the baseline and amplitude range of the breathing signal. Hence the monitored respiratory signal is a relative signal, which depends on the choice of the fixation belt and its application, as well as the used offset and gain values of the electrical amplifier. The software is able to trigger on different events such as inspiration-peak, expiration-peak or derived events, so that the system can be classified as a phase-based triggering system (Fig. 1(a)). To detect these events correctly, a prediction model is implemented in the Anzai software. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 7    Heinz et al.: Evaluation of different respiratory monitoring systems 337 Fig. 2. Application and components of the Anzai system: (a) pressure transducer placed at a patient’s abdomen, attached by the fixation belt; (b) fixation belts in different sizes and both pressure transducers (low/high); (c) electrical amplifier with signal quality indicators for a low, good or high input signal. Fig. 2. Application and components of the Anzai system: (a) pressure transducer placed at a patient’s abdomen, attached by the fixation belt; (b) fixation belts in different sizes and both pressure transducers (low/high); (c) electrical amplifier with signal quality indicators for a low, good or high input signal. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 C. C-RAD Sentinel laser scanner The calibration weight of 70 kg recommended in the Sentinel’s manual is increased by 10 kg to reproduce a representative patient weight in our clinic. Heinz et al.: Evaluation of different respiratory monitoring systems 338 Fig. 3. Installation and application of the Sentinel system: (a) Sentinel unit mounted at the ceiling; (b) patient setup and coordinate system; (c) the wire ruler (red) measures the couch position in Y direction and is mounted at the foot of the CT couch. Fig. 3. Installation and application of the Sentinel system: (a) Sentinel unit mounted at the ceiling; (b) patient setup and coordinate system; (c) the wire ruler (red) measures the couch position in Y direction and is mounted at the foot of the CT couch. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 E. Analysis parameters y p To be able to compare both respiratory systems, comparable parameters must be defined and extracted for each system. Figure 4 gives an overview of the analysis parameters, as well as short information on the methods that are used to extract them. To be able to compare both respiratory systems, comparable parameters must be defined and extracted for each system. Figure 4 gives an overview of the analysis parameters, as well as short information on the methods that are used to extract them. Fig. 4. Overview of analysis parameters and methods to extract them for both respiratory monitoring systems. Fig. 4. Overview of analysis parameters and methods to extract them for both respiratory monitoring systems. D. Patient data In this study two different patient datasets were analyzed. Set A contains 72 patient breathing curves and triggers recorded during clinical 4D CT sessions under free breathing using the Anzai system. The mean evaluated length of a respiratory signal was 46.8 sec, which corresponds to the mean time slot to acquire a 4D CT dataset using the Toshiba scanner. For all patients, a proper fixation belt was chosen and applied in such a way that the pressure transducer deliv­ ered an optimal signal corresponding to the LED indicators of the Anzai system. All breathing curves were recorded using the “low” sensor, which was applied at the right upper quadrant of a patient’s abdomen. Set B contains breathing curves and triggers of 19 patients simultaneously recorded with the Anzai and the Sentinel system. This dataset was recorded during clinical 4D CT acquisition sessions using the Anzai system as trigger system for the CT scanner. The mean evaluated length of a respiratory signal was 43.1 sec. All application parameters for the Anzai system are the same as in Set A. Ideally the Sentinel’s measurement region would have been placed at the same position as the Anzai pressure transducer. To avoid interference with the fixa­ tion belt, the individual measurement regions were placed in close proximity to the fixation belt. 339 339 Heinz et al.: Evaluation of different respiratory monitoring systems E.1  Time resolution Both respiratory monitoring systems must be able to sample respiratory signals without alias­ ing artifacts. Consequently, the sampling time should at least be half of the shortest respiration cycle, whereas a respiration cycle contains one complete inspiration end expiration phase (i.e., inspiration peak to inspiration peak). In practice, the sampling time should be less or equal than a tenth of the respiration cycle time. The latter is estimated in terms of the minimal respiration cycle times present in patient Set A, where the interpatient variability of minimal respiration cycle lengths ranged from 1.3 sec up to 8.7 sec. Consequently, the sampling time of both respiratory monitoring systems should match ideally 100 msec or less, as requirement. The actual sample times of the Anzai system and the Sentinel system are extracted from Set B. E.2  Signal amplitude The signal amplitude detected by the Anzai system is basically defined by the application of the fixation belt. The initial pressure signal is adapted by a tighter or looser application of the belt, and the mechanical pressure applied to the pressure transducer can only be estimated. In an ideal case the adapted pressure signal covers the whole input range of the pressure transducer. However, the only information about the input signal quality of the transducer is given by three Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Heinz et al.: Evaluation of different respiratory monitoring systems 340 340 indicator LEDs. To quantify the peak-to-peak amplitude of the Anzai system, the input range of each pressure transducer is determined by loading the transducer with increasing weights as long as the quality indicator is green. In a second step, it is assumed that the adaption done by the fixation belt is not ideal, but the signal applied to the transducer covers only a 1/3 of the input range. This assumption is somehow arbitrary because there is no way to determine the actual coverage of the input range beside the quality indicator LED’s. For purposes of noise measurements, the input range was determined twice for each pressure transducer with different signal amplification values (gain) of 1 and 10. g pi (g ) Typical signal amplitudes for the Sentinel system are derived from patient Set B. For each patient, the respiratory signal is divided manually into inspiration and expiration phases. For each phase, the peak-to-peak amplitude between the inspiration peak and the expiration peak is calculated. The peak-to-peak signal amplitude for the Sentinel system is estimated by averaging the peak-to-peak amplitudes over all patients. E.3  Noise Both respiratory monitoring systems are exposed to different noise contributions. The noise amplitude of the Anzai system is determined during signal amplitude measurements. The measurement using the maximal signal amplification of 10 provides the maximal signal resolu­ tion and minimal input range. Hence, any noise contribution should be maximal and the noise amplitude is estimated from that measurement. To examine different noise contributions on the Sentinel signal, the system is supposed to measure the surface (Z position) of the Sentinel’s QA Check Phantom (see Fig. 5) in the clini­ cal mode under the following conditions: A1) stationary couch without additional weights, A2) couch movement without additional weights, and A3) couch movement and additional weights. The QA Check Phantom is a rigid object with a mass of ~ 2 kg. The additional weights that are placed on the couch are exactly the same weights (80 kg) that are used to measure the couch calibration profile. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Fig. 5. Noise and drift measurements of the Sentinel system: (top row) A1 stationary couch, no additional weights; A2 couch movement, no additional weights; A3 couch movement, additional calibration weights; between A2 and A3 the calibration weights are placed on the couch; (b) zoomed and offset corrected noise measurement of setting A1; (c) zoomed and drift corrected noise measurement of setting A3; (d) signal drift during couch movement without additional weights; and (e) with additional calibration weights corresponding to settings A2 and A3. Fig. 5. Noise and drift measurements of the Sentinel system: (top row) A1 stationary couch, no additional weights; A2 couch movement, no additional weights; A3 couch movement, additional calibration weights; between A2 and A3 the calibration weights are placed on the couch; (b) zoomed and offset corrected noise measurement of setting A1; (c) zoomed and drift corrected noise measurement of setting A3; (d) signal drift during couch movement without additional weights; and (e) with additional calibration weights corresponding to settings A2 and A3. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 341 341 Heinz et al.: Evaluation of different respiratory monitoring systems 341 Heinz et al.: Evaluation of different respiratory monitoring systems E.4  Signal-to-noise ratio The signal-to-noise ratio for each system is calculated by using Eq. (1) and the root mean square (RMS) values of the estimated input and noise signals. (1) SNRdB = 20 ⋅ log10 RMSsignal RMSnoise ( ) (1) To facilitate calculations, it is assumed that both input signals are sinusoidal. Actually breathing signals are rarely sinusoidal. However, the assumption is applied to both respiratory systems and the results are comparable in that way. For sinusoidal signals with an amplitude, a, the RMS value can be formulated as: RMSsignal = a ⋅ 1 √2 (2) RMSsignal = a ⋅ 1 √2 (2) Thereby the amplitude a is half the peak-to-peak amplitude. The noise observed in the Anzai measurements is characterized by an alternating value in the least significant bit. Therefore, the RMS value is estimated by a pulse wave signals (Eq. (3)) with a pulse height a and the ratio t/T of pulse duration t and period time T. The ratio t/T is set to 0.5 since the error might occur with that probability. (3) RMSnoise = a ⋅ t T (3) RMSnoise = a ⋅ t T RMSnoise = a ⋅ t T (3) Thereby the amplitude a equals the peak-to-peak amplitude. Thereby the amplitude a equals the peak-to-peak amplitude. Thereby the amplitude a equals the peak-to-peak amplitude. For the calculation of the Sentinel’s RMSnoise value, the more general form (see Eq. (4)) is used in combination with the three settings A1), A2), and A3). Whereas n is the number of measured points and xi represents the noise value at point i in the measurement. (4) RMSnoise = xi 2 1 n 2 ∑ n i=1 RMSnoise = xi 2 1 n 2 ∑ n i=1 (4) RMSnoise = xi 2 1 n 2 ∑ n i=1 (4) Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 E.7  Clinical examples In order to discuss the performance of both respiratory systems on clinical data, three exemplary patients from the patient Set B are selected. The breathing signal of each respiratory monitoring system, as well as the patient setup, is visualized and the examples are discussed. E.6  Trigger compatibility A major requirement to all respiratory monitoring systems used in combination with the Toshiba CT scanner is a high reliability of the online delivered cycle-based trigger events. Thereby the compatibility of trigger events includes the requirement that all triggers are released at the same respiratory phase, as well as the correct number of released triggers. The Anzai system is able to deliver triggers at the same respiratory phase, by triggering on the inspiration peak. The Sentinel system delivers amplitude-based triggers only. For periodic and piecewise monotonic signals like a sinusoidal signal, the signal’s phase is correlated with the signal’s absolute value. In such a case, the amplitude-based triggers delivered by the Sentinel system are correlated to the same respiratory phase. However, respiratory signals are not strictly periodic, but quasi-periodic, signals that vary in amplitude as well as in cycle length. Therefore, the triggers delivered by the Sentinel system are not necessarily correlated to the same respiration phase (Fig. 1(b)) as it is expected by the Toshiba CT scanner and phase shifts are introduced. As the user is interested in the most extreme tumor positions during the respiration cycle, the amplitude threshold of the Sentinel system should be set as close as possible to the value of the inspiration peak to minimize phase shift errors for the maximum inspiration phase. To test both respiratory systems with respect to the correct number of released triggers for each respiratory curve in patient Set B, a ground truth trigger signal is created by setting trig­ ger events manually at the beginning of each respiratory cycle (inspiration peak). The Anzai system is set up to trigger on the inspiration peak, too. Since the trigger events from the Sentinel system cannot be saved in a data file, the triggers are extracted from the respiratory curve using a threshold value. Therefore, all breathing curves of the Sentinel system are imported into MATLAB (MathWorks, Natick, MA) and rescaled to an arbitrary scale. To account for instabilities in signal amplitudes, two different threshold values are used (80% and 60% of the signal maximum) to extract triggers as they would be delivered by the Sentinel system. The absolute number of trigger events delivered by each respiratory system is compared to the number of ground truth trigger events expected for each patient. E.5  Signal linearity g y Each of the respiratory monitoring system is expected to have a linear dependency between its input and output signal to avoid the introduction of additional distortion on the correlation of tumor motion and the monitored respiratory signal. To test both systems, two breathing patterns are applied to a dynamic thorax phantom (Model 008A; Computerized Imaging Reference Systems, Inc., Norfolk, VA) and measured simultaneously with the Anzai and the Sentinel system. Pattern A is a sinusoidal signal with a cycle length of 4 sec and peak-to-peak amplitude of 1 cm. Pattern B is a breathing curve from patient Set A rescaled to a maximal excursion of 1 cm between the global maximum and minimum of the pattern. Both patterns are chosen to reproduce a patient-like breathing signal for both respiratory monitoring systems. 342 342 Heinz et al.: Evaluation of different respiratory monitoring systems A. Time resolution h l i f b A. Time resolution The sample time for both respiratory systems should match ≤ 100 msec. Both trigger systems meet that requirement. The Anzai system is the fastest system with a constant sample acquisi­ tion every 25 msec. The sample times of the Sentinel system are not constant. A mean sample time of 42.2 ± 0.9 msec can be observed in the data. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Heinz et al.: Evaluation of different respiratory monitoring systems Evaluation results of Patient Set B with respect to the Sentinel’s input signal amplitude (surface motion) and the absolute number of expected triggers (ground truth) and the delivered triggers for each setup. Table 2. Evaluation results of Patient Set B with respect to the Sentinel’s input signal amplitude (surface motion) and the absolute number of expected triggers (ground truth) and the delivered triggers for each setup. Peak-To-Peak Patient Signal Amplitude Ground Sentinel Sentinel from [Avg.±Std.] Truth Anzai Triggers Triggers Set B = mm Triggers Triggers (thr=80%) (thr=60%) 1 4.2±0.7 9 9 6 10 2 7.0±1.9 13 9 3 9 3 3.7±0.4 14 14 5 15 4 11.5±2.7 12 10 7 13 5 4.9±1.0 11 11 5 8 6a 0.8±0.2 15 15 27 - 7 6.6±0.7 14 14 5 14 8 7.1±0.5 11 10 11 11 9 6.4 ±0.7 10 9 8 10 10 26.4±2.0 8 8 8 8 11 5.3±0.5 16 14 12 16 12 13.4±2.0 12 11 6 12 13 11.8 ±2.2 10 10 4 9 14 5.0±0.5 13 13 13 13 15 5.0±0.7 10 10 11 15 16a 2.3±0.2 13 13 29 - 17 7.6±2.5 15 11 4 11 18 6.9±1.0 8 8 3 8 19 5.7±0.3 9 9 9 10 Mean 7.7±4.8 a Patient is removed from the calculation of the overall mean peak-to-peak signal amplitude due to an improper place­ ment of the region observed by the Sentinel system. a Patient is removed from the calculation of the overall mean peak-to-peak signal amplitude due to an improper place­ ment of the region observed by the Sentinel system. RMSsignal value is calculated from the mean signal amplitude by the use of Eq. (2). The result­ ing value for the Sentinel system is RMSsignal = 2.7 mm. RMSsignal value is calculated from the mean signal amplitude by the use of Eq. (2). The result­ ing value for the Sentinel system is RMSsignal = 2.7 mm. signal Due to different principles in measurement and different units of the signals, a direct com­ parison of the signal amplitudes or the RMS values is not possible. Heinz et al.: Evaluation of different respiratory monitoring systems Heinz et al.: Evaluation of different respiratory monitoring systems 343 343 Table 1. Overview of the Anzai pressure transducers and the calculation base of the corresponding SNR values. Input Range / Peak-To-Peak Signal Resolution Signal Amplitude Pressure Amplification (measured) (coverage 1/3) RMSsignal RMSnoise Transducer (gain) [mass] = g [mass] = g [mass] = g [mass] = g Low 1.0 170 / 1.13 56.7 20.0 0.80 10.0 28 / 0.18 9.3 3.3 0.13 High 1.0 280 / 1.85 93.3 33.0 1.31 10.0 50 / 0.33 16.7 5.9 0.23 Table 2. Evaluation results of Patient Set B with respect to the Sentinel’s input signal amplitude (surface motion) and the absolute number of expected triggers (ground truth) and the delivered triggers for each setup. Peak-To-Peak Patient Signal Amplitude Ground Sentinel Sentinel from [Avg.±Std.] Truth Anzai Triggers Triggers Set B = mm Triggers Triggers (thr=80%) (thr=60%) 1 4.2±0.7 9 9 6 10 2 7.0±1.9 13 9 3 9 3 3.7±0.4 14 14 5 15 4 11.5±2.7 12 10 7 13 5 4.9±1.0 11 11 5 8 6a 0.8±0.2 15 15 27 - 7 6.6±0.7 14 14 5 14 8 7.1±0.5 11 10 11 11 9 6.4 ±0.7 10 9 8 10 10 26.4±2.0 8 8 8 8 11 5.3±0.5 16 14 12 16 12 13.4±2.0 12 11 6 12 13 11.8 ±2.2 10 10 4 9 14 5.0±0.5 13 13 13 13 15 5.0±0.7 10 10 11 15 16a 2.3±0.2 13 13 29 - 17 7.6±2.5 15 11 4 11 18 6.9±1.0 8 8 3 8 19 5.7±0.3 9 9 9 10 Mean 7.7±4.8 a Patient is removed from the calculation of the overall mean peak-to-peak signal amplitude due to an improper place­ ment of the region observed by the Sentinel system. Table 1. Overview of the Anzai pressure transducers and the calculation base of the corresponding SNR values. Input Range / Peak-To-Peak Signal Resolution Signal Amplitude Pressure Amplification (measured) (coverage 1/3) RMSsignal RMSnoise Transducer (gain) [mass] = g [mass] = g [mass] = g [mass] = g Low 1.0 170 / 1.13 56.7 20.0 0.80 10.0 28 / 0.18 9.3 3.3 0.13 High 1.0 280 / 1.85 93.3 33.0 1.31 10.0 50 / 0.33 16.7 5.9 0.23 Table 1. Overview of the Anzai pressure transducers and the calculation base of the corresponding SNR values. Table 2. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 B. Signal amplitude The measurements on the input ranges and estimations about the signal amplitudes of the Anzai system are summarized in Table 1. For instance, the peak-to-peak amplitude for the pressure transducer “low” and a gain of 10 results in 9.3 g (input range of 28 g multiplied by the estimated input range coverage of 1/3). The corresponding RMSsignal values are calculated using Eq. (2). The measurements on the input ranges and estimations about the signal amplitudes of the Anzai system are summarized in Table 1. For instance, the peak-to-peak amplitude for the pressure transducer “low” and a gain of 10 results in 9.3 g (input range of 28 g multiplied by the estimated input range coverage of 1/3). The corresponding RMSsignal values are calculated using Eq. (2). The signal amplitude for the Sentinel system is calculated from the respiratory signals in patient Set B. The mean peak-to-peak signal amplitude over all patients in Set B is 7.7 ± 4.8 mm (Table 2). Two patients (#6, #16) are excluded from the calculation due to extremely small sig­ nal amplitudes. For these patients, the region observed by the Sentinel system was placed next to the Anzai belt but not at the abdomen, but rather at the patient’s chest. The ­corresponding signal The signal amplitude for the Sentinel system is calculated from the respiratory signals in patient Set B. The mean peak-to-peak signal amplitude over all patients in Set B is 7.7 ± 4.8 mm (Table 2). Two patients (#6, #16) are excluded from the calculation due to extremely small sig­ nal amplitudes. For these patients, the region observed by the Sentinel system was placed next to the Anzai belt but not at the abdomen, but rather at the patient’s chest. The ­corresponding Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 C. Noise The noise observed in the Anzai measurements is characterized by an alternating value in the least significant bit. The resulting amplitude, a, of that alternating least significant bit equals the resolution of the Anzai system. Values for the resolution are given in Table 1 and range from 0.18 g up to 1.85 g. By the use of Eq. (3) the corresponding RMSnoise values are calculated, which are also given in Table 1. To examine different error contributions on the Sentinel signal, noise was measured for three different settings. Figure 5 shows the complete measurement, where the settings (top row of figure) A1, A2, and A3 correspond to the sections of the same name. Figure 5(b) shows a zoomed and offset corrected image of setting A1. Depending on the load of the scanner couch, differ­ ent signal drifts can be observed in Fig. 5(d) (weightless) and 5(e) (with calibration weights). Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Heinz et al.: Evaluation of different respiratory monitoring systems 344 344 The drift signals are the smoothed signals of the settings A2 and A3. Beside the drift, there is an increase in noise when the couch is moved, compared to the stationary noise (Fig. 5(b) and 5(c)). The measurement is exposed to noise from the electronic of the wire ruler and the laser projection. On the other hand, external errors are introduced by the deflection of the CT couch and its movement. A couch deflection depending on the actual load affects the measurement and appears as an additional signal drift during couch movement. Typical noise amplitudes results in calculation errors for the Z position of 0.1 millimeter, whereas drift errors are an order of magnitude greater than the noise. The overall RMSnoise, including noise and drift, results in RMSnoise = 1.07 mm for weightless and RMSnoise = 0.41 mm for the loaded couch. Noise and drift errors depend on the actual deviation of the patient’s weight from the calibration weight (80 kg). A conservative estimation on a deviation of ~ 11 kg (actual patient weight between 69 kg and 91 kg) results in an estimation for the Sentinel’s noise by RMSnoise = 0.5 mm. D. Signal-to-noise ratio The SNR values for both respiratory monitoring systems are calculated by the use of Eq. (1). The Anzai system shows an SNR value of ~ 28 dB for all measured combinations of pressure transducers and selected signal amplifications. According to the figures from Table 1, the SNR values for the pressure transducer “low” result in 28.0 dB using a signal amplification of 1 and 28.3 dB for a signal amplification of 10. By analogy, the SNR value for the pressure transducer “high” result in 28.0 dB for both selected signal amplifications. The SNR value for the Sentinel system is 14.6 dB. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 E. Signal linearity The comparison of two test signals applied to both respiratory monitoring systems shows a nonlinearity (Fig. 6) introduced by the Anzai system. Although the fixation belt was double- checked and the quality indicator LEDs reported a perfect signal adaption, it seemed that the pressure sensor reached saturation or that the fixation belt introduced additional forces to the pressure sensor. Further analysis showed that both sensors (low/high) generate a nearly linear response (Fig. 7). Hence the introduced nonlinearity is generated by the rubber-like elasticity of the fixation belt. The magnitude of the nonlinearity depends on the size of the belt and the applied forces. With decreasing belt size and increasing signal amplitude the nonlinear effects Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Fig. 6. Different motion patterns applied to the motion phantom and resulting signals detected by both respiratory moni­ toring systems. Patterns: (a) sinusoidal motion with a cycle length tcycle = 4 sec and a peak-to-peak amplitude spp = 1 cm; (b) breathing pattern of a patient rescaled to an amplitude smax-min = 1 cm. Fig. 6. Different motion patterns applied to the motion phantom and resulting signals detected by both respiratory moni­ toring systems. Patterns: (a) sinusoidal motion with a cycle length tcycle = 4 sec and a peak-to-peak amplitude spp = 1 cm; (b) breathing pattern of a patient rescaled to an amplitude smax-min = 1 cm. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Heinz et al.: Evaluation of different respiratory monitoring systems 345 345 Fig. 7. Sensor response on applied pressures for (a) the pressure transducer “low” and (b) the pressure transducer “high”. The linear regression fit is shown as blue line for both transducers. Fig. 7. Sensor response on applied pressures for (a) the pressure transducer “low” and (b) the pressure transducer “high”. The linear regression fit is shown as blue line for both transducers. increases. In the signal linearity measurements, the smallest belt size was chosen to mount the pressure transducer on the motion phantom. Therefore, the observed magnitude of nonlinearity is higher than in clinical data. G. Clinical examples To visualize the effects on the clinical data, three exemplary patients were selected from Set B. For each of them, the measured Anzai signal, as well as the Sentinel signal, is plotted in one diagram followed by a small picture of the corresponding patient setup (Fig. 9). To visualize the effects on the clinical data, three exemplary patients were selected from Set B. For each of them, the measured Anzai signal, as well as the Sentinel signal, is plotted in one diagram followed by a small picture of the corresponding patient setup (Fig. 9). For Patient A, a well-adapted signal can be observed for both respiratory monitoring systems, caused by a regular respiration with good intrafractional amplitude stability and large signal amplitudes. The signal from the Anzai system shows steeper signal edges and overshooting of the signal at inspiration peaks. Supposedly, both effects result mainly from the nonlinear­ ity introduced by the fixation belt. Although the respiration curve is close to an ideal signal, the generation of triggers by the use of an amplitude threshold (Sentinel) will introduce phase shifts into reconstruction data. Due to amplitude variability, the selected threshold value does not correlate with the same respiration phase. Patient B shows a characteristic drift artifact of the Sentinel system, caused by the use of inappropriate values for the correction of couch deflection. The absolute peak-to-peak ampli­ tudes of the Sentinel system range between 5 to 7 mm and show good agreement to the figures presented in Table 2. Although the signal drift is only about 1 mm, amplitude based-triggers would introduce a phase shift and thereby reconstruction artifacts in the 4D CT. Cycle-based triggers relying on the detection of the inspiration peak (Anzai) would provide better results in reconstruction. The effect of different SNR values for both respiratory monitoring systems is demonstrated by Patient C, who exhibits an extreme shallow respiration. The absolute signal from the Sentinel system ranges only between 0.5 to 1 mm. The two error contributions — noise and signal drift — are added onto the respiration signal and make it impossible to estimate the respira­ tory cycle based on an amplitude threshold as it is used by the Sentinel system. Besides, it is obvious that the selected region to extract the surrogate signal for the Sentinel system is not a good choice in this example. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 F. Trigger compatibility gg p y Both systems show errors in the trigger release (Fig 8(a)). However, the Anzai system deliv­ ers the more accurate trigger signal with respect to the cycle-based triggers expected by the Toshiba CT. In 12 of 19 patient curves, the Anzai system releases a correct number of triggers by the automatic detection of the inspiration peak. The fact that the triggers are released at the inspiration peak assures that the length of a breathing cycle can be calculated from the Toshiba CT correctly. y The absolute number of trigger events delivered by the Sentinel system is less accurate. For a threshold level of 80%, only 4 of 19 patients show a correct number of trigger events. By a decreased threshold level of 60%, the number of patients for whom a correct number of triggers is released increases to 8 of 19 patients. In addition to missed breathing cycles due to amplitudes lower than the threshold value, noise on the Sentinel’s signal leads to trigger events that are not correlated with a breathing cycle. That is why for some patients more triggers are released than expected from the ground truth trigger signal (compare Table 2 and Fig. 8(b)). Heinz et al.: Evaluation of different respiratory monitoring systems 346 346 Fig. 8. Evaluation of trigger reliability for both respiratory monitoring systems: (a) respiratory signals for both systems and the corresponding trigger events for a Patient #1; (b) comparison of delivered trigger events and ground truth triggers for all patients in Patient Set B. Fig. 8. Evaluation of trigger reliability for both respiratory monitoring systems: (a) respiratory signals for both systems and the corresponding trigger events for a Patient #1; (b) comparison of delivered trigger events and ground truth triggers for all patients in Patient Set B. G. Clinical examples The recommended region to extract surrogate signals is the right upper quadrant of the human abdomen. Heinz et al.: Evaluation of different respiratory monitoring systems 347 347 p y g y Fig. 9. Three exemplary patients with respiratory signals recorded simultaneously with the Anzai and the Sentinel sys­ tem. The right-hand pictures show the patient setup with the Anzai belt (blue) and the Sentinel region (red). Patterns: (a) regular breathing patient with large signal amplitudes; (b) drift error of the Sentinel system resulting from inappropri­ ate correction of couch deflection; (c) small surrogate signal due to shallow breathing and an inappropriate selection of the Sentinel’s measurement area. Fig. 9. Three exemplary patients with respiratory signals recorded simultaneously with the Anzai and the Sentinel sys­ tem. The right-hand pictures show the patient setup with the Anzai belt (blue) and the Sentinel region (red). Patterns: (a) regular breathing patient with large signal amplitudes; (b) drift error of the Sentinel system resulting from inappropri­ ate correction of couch deflection; (c) small surrogate signal due to shallow breathing and an inappropriate selection of the Sentinel’s measurement area. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 IV. DISCUSSION In this study, the trigger events delivered by the Sentinel system are less accurate with respect to the absolute number of delivered triggers and the possibility to extract the correct length of a breathing cycle from the delivered triggers. Besides, the selection of the proper threshold value has to be done before the acquisition of the 4D CT, since the Toshiba CT scanner expects online triggers. Therefore, it is unknown what the respiratory signal will exactly look like during the 4D CT acquisition and whether the selected threshold value is a good choice.i To fulfill the requirement of reliable, cycle-base delivered online triggers, the Sentinel system requires a high stability in respiratory amplitudes, since only then amplitude-based triggers are comparable to cycle-based triggers. Amplitude stability can be improved by visual feedback of the respiratory signal to the patient (see Kini et al.(12)). One option to establish a visual feedback would be the application of the video googles available for the Sentinel system. Regardless of the chosen respiratory monitoring system, every deviation in respiration amplitude will cause motion artifacts in 4D CT reconstructions, since the reconstructed 4D CT contains multiple respiration cycles that are combined to one single respiration cycle. Therefore, amplitude stability is a general requirement for 4D CT acquisition. However, amplitude stabil­ ity is also an essential requirement of the Sentinel system to be able to deliver triggers that are comparable to cycle-based triggers, whereas the Anzai system does not necessarily require highly stable amplitudes of the respiratory signal to release correct cycle-based trigger events. Referring to requirements given by the input signal of each respiratory monitoring system, a time resolution of 100 msec is recommended, which is met by both respiratory monitoring systems. The SNR value for the Anzai system is clearly better (≥ 28 dB) than the Sentinel’s SNR (≥14.6 dB). In order to improve the correct number of delivered triggers delivered by the Sentinel system, it is desirable that the Sentinel’s SNR is improved by the manufacturer. This can be achieved, for example, by the use of low pass filters and by fine-tuning the correction for the weight and position dependent couch deflection, which is the major error contribution. The software should be adapted such that multiple couch calibration profiles are acquired and used depending on the actual weight of a patient. IV. DISCUSSION Toshiba’s 4D CT mode makes use of a cycle-based image sorting algorithm and therefore expects cycle-based trigger pulses. Erroneous triggers have to be corrected retrospectively on the Toshiba console, otherwise they will lead to reconstruction artifacts. A retrospective cor­ rection of the trigger signal on the Toshiba console is impractical, since the software does not show a respiratory curve, but solely the received triggers. That is why any respiratory monitor­ ing system used in combination with the Toshiba CT is required to deliver online, cycle-based triggers with high reliability. The Anzai system is able to generate correct cycle-based trigger events for a majority of patients (12/19). The Sentinel system supports amplitude-based triggering only. That causes two problems. First, the signal amplitude is sensitive to noise and drift errors, which directly implies that trigger events are sensitive to such errors. Due to the noise on the respiratory sig­ nal of the Sentinel system, the number of released triggers may exceed the number of triggers expected from the respiratory signal (e.g., Fig 8(b)). Second, the choice of a proper threshold value is a critical decision. As shown in Fig. 8(b), the threshold value will influence the number of released triggers. In cases where the inspiration peak is below the threshold value, no trigger will be released. A decrease of the threshold value from 80% to 60% will increase the number of patients with a correct detected number of triggers from 4 to 8 out of 19 patients (compare Fig. 8(b)). However, by decreasing the threshold value from 80% to 60%, the uncertainty about the trigger assigned respiratory phase will grow. This effect can be observed in Fig. 8(a) at time points between 20 sec and 27 sec. By using a threshold value of 80% the inspiration peak at 26 sec is not detected. The lower threshold of 60% assures the release of two trigger events; nevertheless, the first trigger is released in a midinspiration phase and the second trigger is released just under the inspiration peak. Therefore, the two triggers do not represent a breathing Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 348 Heinz et al.: Evaluation of different respiratory monitoring systems 348 cycle as it is expected from the Toshiba CT scanner. IV. DISCUSSION Another way to improve the compatibility between the Sentinel system and the Toshiba CT scanner would be the implementation of a cycle-based online triggering option (inspiration peak detection) in the c4D software. The Anzai system introduces a nonlinear dependency between the actual motion and the measured signal, mainly caused by rubber elasticity of the fixation belt. The magnitude of this additional error is hardly predictable and depends on the fixation belt and the applied forces. For triggering on the inspiration peak, the introduced nonlinearity does not matter. However, in gating applications, that nonlinearity of the respiratory signal compromises the gating window. That is why the Sentinel system is the better choice for gating applications in radiotherapy, beside the fact that the Sentinel system uses an absolute respiratory signal in comparison to the relative signal of the Anzai system. Another advantage of the Sentinel system is the contactless measurement of the respiratory signal. In contrast to that, the fixation belt of the Anzai system may constrain the free breathing of a patient and thereby introduce an unintentional interference between the measuring device and the patient’s breathing. This is a potential error in cases where the fixation belt is used during 4D CT acquisition but not during treatment, because estimations on tumor movement might be incorrect. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 V. CONCLUSIONS In clinical practice the combination of a Toshiba CT scanner and the Anzai system will provide better results in 4D CT reconstruction due to the compatibility of methods in image sorting (CT) and trigger release (respiratory monitoring system). To improve the performance of the Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 Heinz et al.: Evaluation of different respiratory monitoring systems 349 349 Sentinel system in combination with a Toshiba CT scanner, a cycle-based trigger option should be added to the c4D software by the vendor. In the 4D CT acquisition using the Toshiba CT scanner and the current version of the Sentinel system, a visual feedback of the respiratory signal is strongly recommended. By visual feedback of the respiratory signal to the patient, it is possible to improve amplitude stability, which is a prerequisite to deliver triggers comparable to cycle-based triggers using the amplitude triggering method of the Sentinel system. Another benefit of the amplitude stability accomplished by visual feedback would be to address the intrinsic problems caused by irregular breathing in the cycle-based 4D CT reconstructions. Journal of Applied Clinical Medical Physics, Vol. 16, No. 2, 2015 tion. MHRA Evaluation Report 04045. St. London: Medicines & Healthcare Products Regulatory Agency; 2004. 12. Kini VR, Vedam SS, Keall PJ, Patil S, Chen C, Mohan R. Patient training in respiratory-gated radiotherapy. Med Dosim. 2003;28(1):7–11. REFERENCES 1. Ford EC, Mageras GS, Yorke E, Ling CC. Respiration-correlated spiral CT: a method of measuring respiratory- induced anatomic motion for radiation treatment planning. Med Phys. 2003;30(1): 88–97. 2. Low D, Nystrom M, Kalinin E, et al. A method for the reconstruction of four-dimensional synchronized CT scans acquired during free breathing. Med Phys. 2003;30(6):1254–63. q g g y ( ) 3. Vedam SS, Keall PJ, Kini VR, Mostafavi H, Shukla HP, Mohan R. Acquiring a four-dimensional computed tomography dataset using an external respiratory signal. Phys Med Biol. 2003;48(1):45–62. 4 ll d SS G illi i l i d 4 C i i i d g y g y g y 4. Keall PJ, Vedam SS, George R, Williamson JF. Respiratory regularity gated 4D-CT acquisition: concepts and proof of principle. Australas Phys Eng Sci Med. 2007;30(3):211–20.l 4. Keall PJ, Vedam SS, George R, Williamson JF. Respiratory regulari proof of principle. Australas Phys Eng Sci Med. 2007;30(3):211–20. 5. D’Souza WD, Kwok Y, Deyoung C, et al. Gated CT imaging using a free-breathing respiration signal from flow- volume spirometry. Med Phys. 2005;32(12):3641–49. p y y ( ) 6. Coolens C, Bracken J, Driscoll B, Hope A, Jaffray D. Dynamic volume vs. respiratory correlated 4DCT for motion assessment in radiation therapy simulation. Med Phys. 2012;39(5):2669–81. 7. Endo M, Tsunoo T, Kandatsu S, Tanada S, Aradate H, Saito Y. Four-dimensional computed tomography (4D CT) — concepts and preliminary development. Radiat Med. 2003;21(1):17–22.l 7. Endo M, Tsunoo T, Kandatsu S, Tanada S, Aradate H, Saito Y. Four-dimensiona CT) — concepts and preliminary development. Radiat Med. 2003;21(1):17–22.l ) p p y p ; ( ) 8. Guckenberger M, Weininger M, Wilbert J, et al. Influence of retrospective sorting on image quality in respiratory correlated computed tomography. Radiother Oncol. 2007;85(2):223–31. p g p y ( ) 9. Glide-Hurst CK, Schwenker Smith M, Ajlouni M, Chetty IJ. Evaluation of two synchronized external surrogates for 4D CT sorting. J Appl Clin Med Phys. 2013;14(6):117–32.i 9. Glide-Hurst CK, Schwenker Smith M, Ajlouni M, Chetty IJ. Evaluation of tw for 4D CT sorting. J Appl Clin Med Phys. 2013;14(6):117–32.i 10. Centre for Evidence-based Purchasing (CEP). Comparative specifications wide bore ct scanners. CEP08029. London: Department of Health; 2009. p 11. Imaging Performance Assessment of CT Scanners (ImPACT). Toshiba Aquilion 16 CT scanner technical evalua­ tion. MHRA Evaluation Report 04045. St. London: Medicines & Healthcare Products Regulatory Agency; 2004. 11. REFERENCES Imaging Performance Assessment of CT Scanners (ImPACT). Toshiba Aquilion 16 CT scanner technical evalua­ ti MHRAE l ti R t 04045 St L d M di i & H lth P d t R l t A 2004 11. Imaging Performance Assessment of CT Scanners (ImPACT). Toshiba Aquilion 16 CT scanner technical evalua­ tion. MHRA Evaluation Report 04045. St. London: Medicines & Healthcare Products Regulatory Agency; 2004. 12. Kini VR, Vedam SS, Keall PJ, Patil S, Chen C, Mohan R. Patient training in respiratory-gated radiotherapy. Med Dosim. 2003;28(1):7–11.
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IV. <i>On surface concentration, and the formation of liquid films</i>
˜The œLondon, Edinburgh and Dublin philosophical magazine and journal of science
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Philosophical Magazine Series 6 ISSN: 1941-5982 (Print) 1941-5990 (Online) Journal homepage: http://www.tandfonline.com/loi/tphm17 Philosophical Magazine Series 6 Date: 22 June 2016, At: 09:39 + Communicated by the Author. "f l'5"de R~yleig'h, Proc. Royal Soc. xlvii, p, 281 (1890). Loc. c#. IV. On surface concentration, and the formation of liquid films S.R. Milner D.Sc. (Lond.) To cite this article: S.R. Milner D.Sc. (Lond.) (1907) IV. On surface concentration, and the formation of liquid films , Philosophical Magazine Series 6, 13:73, 96-110, DOI: 10.1080/14786440709463586 To link to this article: http://dx.doi.org/10.1080/14786440709463586 Published online: 16 Apr 2009. Submit your article to this journal Article views: 12 View related articles Citing articles: 34 View citing articles Download by: [University of California, San Diego] Download by: [University of California, San Diego] IV. On SuT/'~ce Conce.tration, and the l"ormation of Liquid ]+'ilm~ ~. ~j S. R. M~L~'Ea, D.Se. (Lond.) ~. T HE nature of the mechanism by which solutions of soap and :~ few other substances are able to form durable fihns has long been a problem of great interest. It is easy to understaml why in pure liquids, where the surface tension is rigorously uniform, lasting fihns should not exist ; for the slightest local disturb~mce of equilibrium, such as that due to the weight of the fihns themselves, would make them collapse. A capability of local variation in the tension of its surface is thus seen to be essential fbr the stability of a film. Marangonit in 1871 suggested that this capability is due to the presence on the surface of the fihn of a pelIicle, com- posed of matter having a smaller capillary tension than that of water. By wtriafions in the thickness of this pellicle which immediately ensue as it is pulled over the surface, the tensions will everywhere automatically take up the values necessary to balance all the other forces, and a stable equilibrimn will result throughout the fihn. In 1890 Lord Rayleigh + con- sidered the question, and gave a strong support to the theory by showing that the tension of a soap solution, measured le.~s than 1 second after the formation of the surface, approxi- To6 mates to that of pure water--tbr the formation of a pellicle may be reasonably expected to be a matter of thne. The existence of a pellicle (or something equiwdent to it) may be looked on as substantiated by this work, but little or nothing is known as regards its nature or method of formation. In the following paper some observations are recorded which, I think, throw a further light on {tlese points and on the mechanism by which the stability of the fihn is maintained. T H Downloaded by [University of California, San Diego] at 09:39 22 June 2016 y y VMuable information on the conditions of the surface of solutions may be gained by studying their surfi~ce-tension curves in the light of the thermodynamic relations which exist between the surface tension and the concentration of the dissolved substance in the surface. Consider a solution of which the surface and the volume are capable of independent reversible alterations, the latter by means of a semi-permeable partition which separates the solution from pure water. Let s be the area of the surface, v the volume, T the surface tension, p the osmotic pressure of the solute. The work done on th.e system by increasin~ the area by ds at constant volume is rds, flint on increasing the volume by dv the ,5'a~fa6e C, mcentratloJ,, and l'ormatton ~f Liquid 1 dins. 97 surface being kept constant is -pdv. If these two rever- sible operations be performed successively, the final result is independent of the order in which they ~tre performed, and by the second law of thermodynamics l~he same must be the case of the work done in reaching it. Consequently [University of California, San Diego] at 09:39 22 June 2016 Downloaded by [University of California, San Diego] at 09:39 22 June 2016 and therefore ,l'r __ dp . . . . . . (1) dv ds ~ (1) (1) shows that the surface tension will vary with the volmne of the solution (i. e., with the concentration) only when the osmotic pressure depends on the surface. In order that this should be the case, we must suppose that in the thin surface-film which is the seat of the capillary forces, the con- ten,ration of the solute is different fl-om what it is in the interior of the solution, in the case of an excess of con- centration in the surface, any increase in the area will result in drawing away from the interior a certain amount of solute which was previously operative in prod acing osmotic pressure; with a defect the opposite will be the ease. The actual con- centration in the surface-film is indeterminate, in the absence of' any kno~vledge as to how it varies with the distance away from the surface, but the whole excess of' solute associated with the surface m~y readily be calculated from equation (1). Let a be the " surface excess," i. e. the nmnber of gram-molecules of the solute associated with each sq. cm. of the surface which are drawn out from the interior and made ineffective on the osmotic pressure. I[ N is the number of gram-molecules of the solute originally dissolved, the concentration in the interior, on which variable alone both the surface tension and the osmotic pressure depend, will be c . . . . . . . (2) V c . . . . . . . (2) V (2) Changing to the concentration as the variable by (2), we have d~" d'r dc c d'r dv de dv v dc d/~= dp de =_ ~ d_p. ds dc ds v de and and Phil. Mag. S. 6' Vol. i3. No. 73. Jan. 1907 H Phil. Mag. S. 6' Vol. i3. No. 73. Jan. 1907 H Phil. Mag. S. 6' Vol. i3. No. 73. Jan. 1907 H Phil. Mag. S. 6' Vol. i3. No. 73. Jan. 1907 H Dr. S. R. MUner (,n S~<t'~we (?oJweJd,atio~, 98 Hence the surface excess is given by the equation dr ~ dl, de (~ dc . . . . . . . (3) dr ~ dl, de (~ dc . . . . . . . 9 l%rch, Ann. tier Physik, xvii. p. 744 (1905). t Zeit.fib" Phys. Chem. xxxix, p. 166 (1902). 9 l%rch, Ann. tier Physik, xvii. p. 744 (1905). , y , p ( ) t Zeit.fib" Phys. Chem. xxxix, p. 166 (1902). Downloaded by [University of California, San Diego] at 09:39 22 June 2016 X 5 3 z 5( I tk5 0 4( Curve ]~, ,, II. , III. I , 9 i x, I I 7 I 2. 4. 5 <3 q "Z "3 "~. .5 ,6 "7 "8~{~./RYL'~ Surface Tension (dynes/cm.) with concentration (gm.-mol./]itre). ,, . . logarithm of concentration. Surface Excess (gin. mol./sq, cm.) with concentration. B ~ CU/i'yE~ Fig. 1. 10--1o X 5 3 z 5( I tk5 0 4( Curve ]~, ,, II. III I , 9 i x, I I 7 I 2. 4. 5 <3 q "Z "3 "~. .5 ,6 "7 "8~{~./RYL'~ Surface Tension (dynes/cm.) with concentration (gm.-mol./]itre). ,, . . logarithm of concentration. Surface Excess (gin mol /sq cm ) with concentration B ~ CU/i'yE~ Fig. 1. by [University of California, San Diego] at 09:39 22 June 2016 Downloaded by [University of California, San Diego] at 09:39 22 June 2016 Downloaded by [University of California, San Diego] at 09:39 Curve ]~, ,, II. , III. q , { Surface Tension (dynes/cm.) with concentration (gm.-mol./]itre). ,, . . logarithm of concentration. Surface Excess (gin. mol./sq, cm.) with concentration. is very approximately logarithmic, as is better seen in curve II,, where the same results are plotted with the logarithms of the concentrations. In acetic acid the molecular depression of the freezing-point, throughout the range of concentration included in the curves, is very approximately constant and has the value 2 ~ per gm.-mol, per litre. The factor i is thus 1"08~ and consequently is very approximately logarithmic, as is better seen in curve II,, where the same results are plotted with the logarithms of the concentrations. In acetic acid the molecular depression of the freezing-point, throughout the range of concentration included in the curves, is very approximately constant and has the value 2 ~ per gm.-mol, per litre. The factor i is thus 1"08~ and consequently 1 d~" 1 dr o'----- -- I"=-O--~RT c dc 1"08 RT d loge c 1 d~" 1 dr o'----- -- I"=-O--~RT c dc 1"08 RT d loge c The second form of this equation shows that if the surface tension vary linearly with the logarithm of the concentration, the surface excess will be constant. Curve II. shows that this must be very nearly the case in acetic acid. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 (3) (3) By this equation o" may always be calculated when the curve of the surface tension with the concentration is known. @ @ If the osmotic pressure obeys the ordinary gas law, ~tc =RT; but whether this is the case or not, it may always be obtained from fl.eezing-point measurements. If i stands for the ratio of the molecular lowering of the freezing-point (lowering per gram-tool, per litre) to the normal lowering 1"86, p=iRTc, and consequently dp RTc di. 3(; = iRT + d,. When, as is usually the case, the variation of i with the concentration is small, the second ~erm of this equation may be neglected in comparison with the first, and we have approxi- mately from (3) ,t"r = _ iRT o- (4). dc c ,t"r = _ iRT o- (4). dc c (4). In all inorganic solutions so far measured the surface tension dr increases linearly with the concentration, that is Tcc is positive and constant; hence ~ is negative, and 7 is constant, i. e., c there is a defect of salt in the surface-fihn of an amount proportional to the concentration. As regards the actual magnitude of the defect, sodium chloride may be cited as an average case. The increase of surf:ace tension * is 1"72 dynes per ch~. for an increase in concentration of 1 gm.-mol[per litre. Also i= 1"74, taking an average value of the fi~t.tor, R= 8"32 x 10 z ergs per degree C., T=290 ~ Substituting these nmnbers in (4) we obtain for a normal solution, o-= --4'09 • 10 -~L gm.-mol, per sq. era. This is equivalent to a defect of "024 mgm. per sq. metre. This is equivalent to a defect of "024 mgm. per sq. metre On the other hand, in several organic solutions the surface tension is less than that of water, and there is consequently an excess of solute in the surface. Acetic acid, the surthce tension of which has been measured by Whatmough ?, forms 99 and the Formation of Liquid Films, an instructive example. Whatmough's results, plotted with the concentration, are shown in curve ]:. (fig. 1). The curve Fig. 1. an instructive example. Whatmough's results, plotted with the concentration, are shown in curve ]:. (fig. 1). The curve Fig. 1. an instructive example. Whatmough s results, plotted with the concentration, are shown in curve ]:. (fig. 1). The curve Fig. 1. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 y y g It is interesting to observe that very dilute solutions of acetic acid will give a lasting foam, but that strong solutions will hardly foam at all. We may infer that the question is one of the relation of the surface excess to the concentration, and the consequent difference in its rate of formation by diffusion of the solute into the surface in strong and weak solutions. In strong solutions, on the surface excess being pulled away from any portion of a fihn by a local inequality of the forces, it may be formed again so quickly that the surface tension remains practically unchanged, and the film thins rapidly and collapses as in a pure liquid. In dilute solutions the excess must take an appreciable thne to form, and the life of the film is correspondingly increased. p g y Sodium Oleate Solutions.--Here it is more difficult to deter- mine the sur~hee excess from the tension curve. Marangoni has shown that the surface tension, while verv much less than that of pure water, is practicaly independent of the concen- tration, and indeed based on that~ fact an argumen~ for the existence of the pelliele. For the calculation of the excess a knowledge is required of the rate of diminution of the tension with the concentration. I coh~equently made a series of measurements, and continued them to as low con- centrations as possible, to see it' there were any portion of the d~ hi h d/ i h b b i d Th l i Sodium Oleate Solutions.--Here it is more difficult to deter- mine the sur~hee excess from the tension curve. Marangoni has shown that the surface tension, while verv much less than that of pure water, is practicaly independent of the concen- tration, and indeed based on that~ fact an argumen~ for the existence of the pelliele. For the calculation of the excess a knowledge is required of the rate of diminution of the tension with the concentration. I coh~equently made a series of measurements, and continued them to as low con- centrations as possible, to see it' there were any portion of the d~ curve at which d/c might be obtained. The solutions were contained in a U-tube, having one large and one capillary limb, the difference in the heights of the liquid in the two limbs being measured with a cathetometer. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 There is perhaps a slight curvature in the direction which implies that r increases a little with the concentration, as shown in curve III., which represents its magnitudes at the differen~ concentrations as determined from the differences of the H2 100 Dr. S. R. Milner ore Sttrface Concentration, successive experimental nmnbers. The value is 3"3 • 10 -l~ gm.-mol, per sq. cm., or "20 mgm. per sq. metre in a norma] solution, which is nearly ten times as much as the corre- sponding defect in salt solutions; and it alters by less than 15 per cent. even when tile concentration is increased eight-fold. At concentrations lower than normal the curve becomes uncertain ; bu~ it is evident that, as the solution becomes more dilute, the ratio of the excess to the concentration must ulti- mately become very large. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 By forcing a little air into the wide limb, the liquid could be expelled over the top of the capillary and a perfectly fresh surface formed at any instant. It was found that the surface tension, after a fresh surface was formed in this way, always varied with the time, falling rapidly at first and afterwards more slowly until it reached a final value independent of the time. Fig. 2 shows two specimen curves of the variation in solutions of different concentrations. In moderately strong solutions the fall was initially very rapid, the tension reaching within 101 and the Formation of Liquid ffilms. 1 per cent. of its final value in one or two minutes, but in the dilutest solutions the fall extended over many hours. The Fig. '2. 4-0 ~3"5 1• /~-Co#, "NrR~rlO~ .O02ZSAI | "O ,a- 8 t~ I6 eo s e~ ~a final value, when once obtained, was quite definite (so long as the surfaee was not disturbed); but on repeating the time- curves by making a fresh surface in the capillary without altering the other conditions, erratic differences of as much as 5 per cent. in the ultimate values obtained were often met with. Before reaching its final value the tension almost invariably passed through a slight minimum,--this is only just perceptible in the curves, but was very obvious in the measurements with the eathetometer. Fig. '2. 4-0 ~3"5 1• /~-Co#, "NrR~rlO~ .O02ZSAI | "O ,a- 8 t~ I6 eo s e~ ~a Fig. '2. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 by [University of California, San Diego] at 09:39 22 June 2016 final value, when once obtained, was quite definite (so long as the surfaee was not disturbed); but on repeating the time- curves by making a fresh surface in the capillary without altering the other conditions, erratic differences of as much as 5 per cent. in the ultimate values obtained were often met with. Before reaching its final value the tension almost invariably passed through a slight minimum,--this is only just perceptible in the curves, but was very obvious in the measurements with the eathetometer. final value, when once obtained, was quite definite (so long as the surfaee was not disturbed); but on repeating the time- curves by making a fresh surface in the capillary without altering the other conditions, erratic differences of as much as 5 per cent. ~'he curves rou_~hlv aoree with those calculated on this supposition, but the effects are so much comphcated by the a teratlons of the surface as the liquid moves in the tube~ that I have not been able to make an accurate calculation. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 in the ultimate values obtained were often met with. Before reaching its final value the tension almost invariably passed through a slight minimum,--this is only just perceptible in the curves, but was very obvious in the measurements with the eathetometer. The following numbers show the relative values of the surface tension ultimately reached in the different concen- trations obtained in one series of measurements. They represent the differences of the heights of the liquid in the two tubes in eros. multiplied by the densities of the ~olutions. Concentration. Ilelative Surface Tension. 0'2 normal '066 i "00824 I -00225 "002(~; Water. 2'330 2'300 2"275 2"305 2'240 6'15 102 Dr. S. R. Milner on Surface Concentration, These nun ibers show no trace of any finite value for d These nun ibers show no trace of any finite value for dc down to a concentration of "002 normal, and would in them- selves seem to negative the idea of a surface excess. On the other hand, the very large negative, and probably infinite dT dT value which ~c must possess in the neighbourhood of zero Downloaded by [University of California, San Diego] at 09:39 22 June 2016 concentration, renders it probable that an excess of consi- derable magnitude exists even in the dilutest solutions. This conclusion is supported by the way in which in the different solutions the tension ~aries with the time. The tlme-curves suggest that the surface excess is so large, that in dilute solutions the diffusion of sufficient oleate into the surface to form it takes an appreciable tim% and that the fall in the tension is proportional to the amount of oleate which has diffused into the surface in the interval *. The theoretical method for the determination of the surf.me excess thus fails to give a result; but I have verified its existence experimentally~ and nmde a rough estimate of its value, in the following way. A current of air, saturated by previously bubbling through water, was passed through a number of fine holes into a beaker about two-thirds full of a solution of sodium oleate. The foam rising to the top of the beaker was periodically removed, and the resistance of the solution was measured in s~tu at intervals during the experiment. In dilute solutions the resistance was always found to perceptibly increase after bubbling for about an hour's time--indicating that the oleate is removed from the interior by the continual re-formation of the surface. By measuring the rate of increase of the volmne of the foam and the average size of the bubbles, and by assuming that each bubble as it tbrms removes a portion of'the surface equal to its sectional area, the total area of the surface removed in a given time can be readily, if only roughly, estimated. Also, from the alterations in the concentration determined from the electrical measurements, the amount of oleate removed from the interior of the solution by the foam is known. Thus the surface excess, which is the amount removed per sq. cm. of fresh surface formed, is simply calculated. The following table shows the results obtained :-- a,d the _Fo~'matio~t o~ Liq.id .~Tlms. 103 I I Dim n etei of bubbles. "5 era. "15 till. Volume of tbam (lit.res). 29"8 42"6 570 11"16 17"24 Surface !Resistance, removed ! corrected (square for Iempe- metres ) . future. 1374~ '~:~ 1456 12"8 1467 17"1 1509 ...... t17i 4"5 120a 11"2 1237 17"2 1273 Collcell- tration (gm.-mol. per litre). '002056 "l}01940 "001925 "001872 "002220 '002156 "002102 "002042 Oleate Surface removed :Excess (gram-moI.). (gm.-mol. per sq. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 era.). 1.1ix'ib -~ 1-adx'i5-'o 1'34 ,, 1"05 ,, 1"86 ,, 1'09 ,, 0'64• 10 -5 1"42• -l~ 1"18 ,, 1"06 ,, 1"78 ,, 1"03 ,, ~fean volume of solution, 100 c.cs. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 The mean result--l'2x 10 -1~ gm.-mol, per sq. era., or 9 ~ mgm. per sq. metre--is doubtless a moderately good estimate for the surface excess at the moment of formation el the bubbles, but it must necessarily be very much less them the ultimate value of the excess. The curves of fig. 2' show that at the concentration of these experiments the excess is not fully refined until after the lapse of { hr., while in the bubbling the whole surface of the solution in the beaker was renewed in about 2 seconds,--so that only a very small fraction of the excess can have been formed. The existence of a surh:ce excess having been confirmed experimentally, a di~culty arises with regard to the theoretical interpretation of the surface-tension curve. According to the thermodynamieal equation (~) an excess of the magnitude found should cause a very considerable dinfinution of the tension with the concentration; but, as a, matter of fact, the ultim~lte values of the tension are practically independent of the concentration. Since the application of the thermo- dynamical reasoning is limited only by the reversibility of the processes concerned, we are driven to the conclusion that the formation of the sm'face excess in oleate solutions is not a reversible process. This idea is in con%rmity with the con- clusion already arrived at, that the excess tends to be formed however dilut~' tbe solution ; for this means that the reverse action (passage of the excess from the surface into the interior) is very small) if not actually zero. This leads us to expect that, since the surface cannot take up an indefinite amount of the oleate, it will ultimately in finite concentrations become saturated, when irreversibility will be produced by the con- tinuous precipitation of the oleate into another phase. Several observations go to support this inference :--(1) It is well known that an insolnble scum is fbrmed on the surface of 10~ Dr. S. R. Miiner on b'~t~j~ce Concentrations, sodimn-oleate solutions to an cxten~ which increases with the rime : this may well be the solid phase which causes the irre- versibility. O) ]if tile surface of a soap-bubble, which has thinned so as to exhibiL the reds and greens, is carefully watched as the bubble is allowed to decrease in size, it will be found to suddenly beeome covered with a large nmnber of minute white flakes. -~ The minimum value of the surface tension and the variability of its ultinaate vahe in the same solution described on p. 100 may be easily accounted for as due to a slight supersaturation of the surface. g p t Phil. Trans elxxxiv, p.505 (1898). Downloaded by [University of California, San Diego] at 09:39 22 June 2016 These flakes are not formed in ~ film of constant size, except at places towards which the surface is being pulled by local want of balance of the tensions. On the above view, we should expect the precipitate to be formed in such cases, where, but for its occurrence, super- satm'ation ~" of the oleate in the surface would be produced. (3) In the experiments on the surface tension, in dilute solutions, on sucking the liquid down in ~he capillary it is brought back sharply to its previous position ; but after pressing it up, it comes back only very slowly in accordance with the time-curve of fig. 2. This shows that while the tension increases when the surthce is inere',sed, it does not decrease, but even tends to increase, when the surface is diminished. Thus, whatever the cause of the want of re- versibility may be, there can l:e little doubt as to the fact of its existence. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 [n the rest of tiffs paper the above conceptions are applied to some results on the conductivity of soap-fihns obtained by Messrs. tleinold and [Riicker, and to tim explanation of the manner of thinning of fihns, and of the formation of the black spot. p ('omlucli'city of d,'ilms.~Reinold and Riickert measured the true, and what they called the " electrical" thickness (i. e., the thickness calculated fi'om the conductance on tim assumption that the conductivity is the same as that of the liquid in mass) of a series of fihns tbrmed from a solution containing 1part,of sodimn oleate to 60 parts of water. They found that the electrical thickness was always greater than the true thickness lnea~ured optically; and from their results we may determine approximately the magnitude of the surface excess at different thicknesses of the film. On the assumption that the conducting-power per molecule of the surface excess is the same as that of the rest of tho solution, the conductance of a film of' thickness t and con- eentration ~ and possessing an extra number ~ of' gram- molecules per sq. cm. of each surface, will be proportional to 105 and the _~brmation of Liquid Films. ct + 2a. The electrical thickness, t,, was calculated by Reinotd and Rficker on the assumption that the conductance was proportional to cte. Consequently, ct+ 2o'----- cte; and if r= ~ the ratio of the electrical to the actual thickness, o-=Xct(r--1). o-=Xct(r--1). o-=Xct(r--1). [University of California, San Diego] at 09:39 22 June 2016 Downloaded by [University of California, San Diego] at 09:39 22 June 2016 Downloaded by [University of California, San Diego] at 09:39 22 June 2016 In the experiments c was "0164 gin. per c.c.; thus, t being measured in micromillimetres~ c~='0082 x 10-Tt(r--1) gin. per sq. era., ='0082 t (r--l) milligrams per sq. metre. c~='0082 x 10-Tt(r--1) gin. per sq. era., ='0082 t (r--l) milligrams per sq. metre. The following table shows the values of the excess at different thicknesses, ealculated from this equation. The experimental numbers are taken from tables xiv., xv., and xvi. of their paper, and arranged in order of decreasing thickness of fihn. different thicknesses, ealculated from this equation. The experimental numbers are taken from tables xiv., xv., and xvi. of their paper, and arranged in order of decreasing thickness of fihn. 4 Optical [ thickness_.'. 641 484 388 332 320 314 314 307 301 =)96 97 97 27'7 Ratio of Electrical to Optical thickness. 1"66 1"69 1"60 1"81 1'78 2"07 190 1 "84 1"84 1 '98 4"47 4'19 Surface :Excess (mgm. per sq. metre). 3"46 2"73 1"91 2"21 2"05 2'75 2"32 2"12 2"08 2"38 2"75 2"53 Mean 2"4 5"8 1'09 Ratio recalculated. 1 "46 1"6l 1"75 t "87 !'9t 1 "93 1 '93 1 "95 1"97 1 "99 4"02 4'02 These results show that the surface excess remains prac- tically constant down to a thickness of 97tqz. The first three numbers, it is true, show fairly large differences among them~ selves, but this is due to the experimental error being here a good deal magnified in the calculation ; and an examination of the fourth column, which contains the theoretical ratios calculated using the mean value, 2"4 mgm. per sq. m., of tim excess, shows that the differences between them and the expe- rimental ratios nowhere exceed the possible errors of th~te 106 Dr. S. R. Milner on Surface Conce~trat~on, very dimcult measurements. The mean value is several times larger than that obtained from the experiments on bubbling. This might have been anticipated, since, as was there pointed out, only a small fraction of the final excess can have been formed in those experiments. p The table shows further that in the black film of 27"7/q~ thickness, a reduction to less than half its value, in thick films has occurred in the excess. This is, I think, a point of con- siderable importance in the explanation of the formation of the black film, and is considered more fully on p. 107. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 , y p Thinning oJ F(lms.--Accordlng to the theory of the for- marion of a pellicle, the surface of a vertical soap-fihn is in a state of statical equilibrium, and the tliinning is due to the gradual draining down of the layer between the two surfaces impeded by the ordinary fluid viscosity of the liquid. In view of the continual re-formation of the surface excess when the surfiJee is renewed, another eause will be seen to be operative in the thinning. Consider a horizontal fihn suddenly turned into a vertical position. In-rJrder for the fihn to he in equi- librium, the tensions at different levels must be different by the weight of the intervening fihn. This not being initially the ease, the surface, and with it a portion of the excess which it contains, will be pulled down by the tension from higher to lower levels. The surface tensions of the higher levels will be thus increased until equilibrium results. This increase in the tension is, however, only temporary, since the excess removed from any portion of the surface is continually being replaced by diffusion of the oleate into the surface from the interior. But this will he similarly removed, and thus a con- tinuM circu'lation over the surface will be produced, the rate of which, other things being equal, will depend on the rate at which the sm'faee excess is capable of being formed. Both circulation and draining will assist in thinning the fihn, and it is difficult to say a priori which of the two will be the more potent cause. We may, however, infer that, since the rate of tbrmation of the surface excess is decreased by a decrease in the concentration of the solution, the same will be the case for the rate of thinning by circulation over the surface; so that the extent to which this cause is oper:ttive may be gauged by the extent to which the rate of thinning of films depends on the concentration I made some experiments on this point by measuring the time required by fihns, formed in the same way front sodium oleate solutions of different strengths, to undergo a given.degree of thinning. The films were formed on a rectangular frame of glass fibre, 2 eros. wide by 4 eros. high, having the handle attached at the bottom, and the whole aml the ~bbrmation of Liquid Films. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 107 aml the ~bbrmation of Liquid Films. 107 being so arranged that the framework could be raised out of the solution without removing a glass shade which covered the whole apparatus. Concentration. II. IlL 9 20 no~maI, '07 "02 '007 '002 iS"l sees. ] 8"3 19",~ 20.~,) 2L'0 12"7 sees. 173 19'8 Downloaded by [University of California, San Diego] at 09:39 22 June 2016 In the above table the numbers in column i[. are the times after the formation of the film at which the green of the 6th order (thickness ll00/~br had reached a point 2 cms. from the top of the frame ; those in column III. the times at which the blue of the 2nd order (250 m*) had reached a point "3 cm. from the top ; they are each the mean of about ten measure- ments, which throughout nowhere differed among themselves by more than 1 second. The table shows that the time required for a given degree of thinning increases in a marked way with tile dilution of the solution; and we may conse- quently infer that circulation over the surface is an appreciable factor in producing the thinning. p g g The circulation must, however, cease when the whole of the oleate has been removed from the interior of the film. The surface excess will then everywhere take up a steady value of such a magnitude as will enable the tensions to remain in statical equilibrium throughout the film ; this point, however, does not appear to be reached--unless perhaps in very dilute solutions--before the black spot becomes formed. Black 1,~ilms.--Many observers have endeavoured to trace the effect on the surfhce tension of the approach to molecular magnitudes which films make as they thin ; but the results have always shown that there is no appreciable difference in the tensions of thick and of the thinnest black films. Any other result would indeed be difl3cult to nnderstand from the point of view of the equilibrium of the fihn introduced by Marangoni; but it is to be observed that it by no means indicates that a thickness of twice the radius of molecular action has not been reached and passed. If a fihn of pure water could be obtained with a thickness less than twice the radius of molecular action, its tension would doubtless be less than that of a thick water-flhn ; but the tension of a soap-film 108 Dr. S. It. Milner on &~face Conce~tratlon, of the same thickness will depend olt both the thickness and the magnitude of the surface excess. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 While a diminution of the thickness will tend to decrease the tension, a diminution of the excess will tend to increase it ; and down to ~ certain limiting thickness there will always he a possible wdue of the excess which will enable the tension to keep uniform. excess which will enable the tension to keep uniform. Now from the results on p. 105 fbr the value of the surface excess ealeulated from the eondnetivity of fihns, we must con- elude that it is considerably less in black fihns than in thiek ones. Another argmnent for the same thing may be adduced in the fact that when the black spot is formed its edge iv (in my experience) always surrounded by ~ great many of' the little white flakes, the, formation of which as a precipitate formed from a supersaturated solution has already been adverted to. On this view they form tt visible piece of evidence of oleate removed from the black spot. We must consequently conclude that the tension so far as it depends on the thickness has been diminished, and that in black fihns it thiekness less than the range of molecular action has been reached. It would be interesting to know, even if only roughly, what the tension of a pure water-fihn of the same thickness would be. If'we assume, as is sua'gested by the approximate "~greement of' the time-curves ~or the surface tension with diffusion curves, that the diminution of the surface tension of oleate solutions below that of pure water is proportional to the actual amount of the surface excess, an estimate of the value may be made from the results of tleinold and t/tieker's conductivity experiments. On this assumption, if r is the surface tension of a water-film, that of a soap-fihn will be r(1--lc~), where ~ is the magnitude of the surface excess; and since both in thick fihns and thin ones the tension must have the same value, 27"5 dynes per cln., we must have, for a thick fihn, where ~" is 74 dynes per era., and r is 2"4, 27"5 = 7t(1-- k x 2"4), and for a hlack fihn of 27"7#t* thickness~ where r 27"5 = r k x 1"09). This makes -r----38"5, only about half as great as the m)rmal value. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 Although no great stress can be laid on the actual number, it is evident froln the calculation that a thickness of 27"7t*t* is well within the limits of the range of molecular action. This makes -r----38"5, only about half as great as the m)rmal value. Although no great stress can be laid on the actual number, it is evident froln the calculation that a thickness of 27"7t*t* is well within the limits of the range of molecular action. There remain outstanding the sudden formation, and the discontinuous thickness at the edge, of the })lack spot,--phe- nomena which have long" been recognized as an indication a~d the Fo,,'mation of Liq_~dd _bTlms. 109 that tile thickness is approaching molecular dimensions, but ~he actual cause of which has remained completely obscure. I have only been able to come across two suggested expla- nations of the discontinuity. Reinold and Riicker ~ suggest that it is due to the forces between the molecules becoming repulsive within certain limited regions, whereby the surface tension of the film alternately decreases and increases as the ~hickness is diminished. On the other band, Bakkert connects it with the region of instability in the isothermal which represents the passage of liquid into vapour. An objection to both of these explanations is that they neglect to givo a part to be played by the soap, although it has an obvious and essential role in the experimental production of the spectacle. It seems to me that an explanation on the following lines is more satisfactory. g y Let, us suppose (simply to make the argument more definite) that the tension of a thin soap-fihn of thickness t and surface excess a is actually represented by the formj(t){1--ka}. It is true that from a mathematical point of view an infinite nmnber of corresponding values of t and o- may exist ychich will make this function have the same value as in a thick film. We have seen that if in any place the tension is accidentally less than this v~lue, thinning occurs by the extreme surfbce layer, and with it the excess of oleate which it contains, being drugged away from the place by the surrounding tensions ; but ~ -'Shen we come to molecular magnitudes tho alterations of the thickness and the excess produced in this way cannot be perfectly continuous, but must occur in finite, if very small, steps. * Phil. Trans. vol. clxxvii, p. 6"27 (1886). Ann. der Physik, Bd. xx. p. 35 (1906). Proe. Royal Soc. March 1906, vol. lxxvii, p. 314. + Phil. M~g. June 190(;, p. 746. :~ Communicated by the Author. w Rutherford, Phil. Mag. Oct. ]906. Rutherford ard Hahn, Phil. Mag. Oct. 1906. Proe. Royal Soc. March 1906, vol. lxxvii, p. 314. y + Phil. M~g. June 190(;, p. 746. g ( p :~ Communicated by the Author. y w Rutherford, Phil. Mag. Oct. ]906. Rutherford ard Hahn, Phil. Mag. Oct. 1906. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 Thus we may imagine that the thickness cannot alter by less than that of a layer i molecule thick, and similarly the surface excess by the amount of oleate contained in such a layer. But unless the increase in the tension produced by the second oI~ these alterations is exactly of the right amount to neutralize the decrease produced by the first, the portion of the film thus thinned cannot remain in equilibrimn with the thicker parts. Hence either the thinning will not take plac% or the next layer will be removed simultaneot~sly with the first, when the same considerations will again be applicable. It nmy not be until after a considerable number of layers of molecules are considered removed, that a point is reached a~ which the sums o[ the decreases of the thickness and of the surfac~e e~cess which correspond to the removal of each layer exactly neutralize eaqh other in their effects on the 110 Pro[. E. 1Rutherford on the Velocity eo,d t~ension, and enable the thinned fihn to form in stable equi- librium with the thicker parts. If this be the case, the practical process of thinning will take place by the sudden removal of a finite thickness from a limited area, forming a spot with a sharply defined edge. From the point of view of this explanation there is no necessity to limit to a single case the number of' films, having thicknesses finitely different from each other, which might be capable ef being formed in this way. It is well known indeed that.bh~ck films occur in two different thicknesses ; and Stansfield ~ and Johonnottt have recently drawn attention to the existence of a series of at least three grey films of |hicknesses intermediate between those of tha white and the black, g (;,p :~ Communicated by the Author. Downloaded by [University of California, San Diego] at 09:39 22 June 2016 The University, Sheffield. August 1906. The University, Sheffield. August 1906. The University, Sheffield. August 1906. u The Velocity and _ENergy of the a particles from Radio.- active Substances. ~y E. RE'mE,FORD, F.R.S., Macdonald ProJessor of .Physics, Me Gill University, Montreal ~. I N I N two previous papers w an account has been given of measurements of the velocity and mass of the a particle expelled from selected products of radium, ~ctinium, and thorium. ]t has been shown that, within the limit of experi- mental error, the a particles expelled from each of the substances examined have the same mass but differ in the initial velocity of their projection. y p j l_~nder the normal conditions of experiment, the a particle carries a positive charge and behaves as if it were a charged atom o! matter projected from the radioactive matter at very great velocity. The value of e/m~the ratio of the charge of the ~ particle to its mass--was found to be 507 X 10 ~. The p~ obability that the ~r particle is an atom of helium is discussed in detail in one of the papels alre:,dy mentioned. p p y The range of ionization in air of the a particles from a thin layer of active matter is a de6nite trod easily measured quantity, which is a characteristic of each individual product. The ranges in air of the radium products have been accurately
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English
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Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei
Molecular and biochemical parasitology
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To cite this version: Andreas Raabe, Oliver Billker, Henri Vial, Kai Wengelnik. Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei. Molecular and Biochemical Parasitology, 2009, 168 (2), pp.172-176. ￿10.1016/j.molbiopara.2009.08.004￿. ￿hal-03246862￿ Distributed under a Creative Commons Attribution 4.0 International License 1. Results Onlysuccessfulinfectionof themosquitoallows the malaria par- asite to reproduce sexually and eventually infect new vertebrate hosts, thereby spreading the disease. Targeting this pivotal step in the Plasmodium life cycle with transmission blocking drugs or vac- cines is still poorly explored even though it is a promising approach in the fight against malaria. The need to investigate the transmis- sion blocking potential of antimalarial drugs is widely recognised as important for the elimination of malaria. The only cells capa- ble of infecting the mosquito are the sexual blood stages, male (micro) and female (macro) gametocytes. Duration of their matu- ration is species dependent and can range from little more than the asexual cycle (26–30 h) for P. berghei to >10 days for P. falciparum [1–3]. Mature gametocytes are developmentally arrested while circulating in the vertebrate blood stream, but become activated in the midgut of the mosquito seconds after ingestion, forming male and female gametes. Macrogametocytes give rise to a single macrogamete while microgametocytes re-enter the cell cycle and replicate their genome to the octoploid level in only 10 min, making this a remarkably rapid process [4]. Three rounds of endomitosis and concomitant assembly of eight axonemes finally give rise to eight flagellated and motile microgametes (exflagellation). Fusion Gametocyte activation is triggered in vitro by simultaneous exposure to two stimuli: a drop in temperature of more than 5 ◦C [5] and xanthurenic acid (XA), the latter of which can be substi- tuted by a pH shift from 7.5 to 8.0 [6–9]. The underlying molecular mechanisms are intensively investigated [10–12] given the criti- cal role of gametocyte activation in transmission of malaria. The standard method to monitor successful gametogenesis is by count- ing exflagellation centres under the microscope. However, this method is labour-intensive, somewhat subjective, cannot be auto- mated, and is thus not suited for medium-throughput applications. DNA replication during microgametogenesis has been investi- gated previously using DNA staining with various fluorescent dyes and subsequent analysis of individual cells by fluorescence microscopy or of cell populations by flow cytometry [4,10,11,13]. These methods gave fundamental insight into the kinetics and gen- eral parameters of microgametocyte DNA synthesis and allowed phenotypic analysis of mutants; however they are not easily adapted to higher throughput analyses. Here we present an assay adapted to the 96-well format to monitor activation of gametocytes based on incorporation of radioactive hypoxanthine into newly synthesised DNA of microgametes. a r t i c l e i n f o Article history: Received 4 August 2009 Received in revised form 17 August 2009 Accepted 19 August 2009 Available online 25 August 2009 Keywords: Malaria Gametocyte Transmission Exflagellation Article history: Received 4 August 2009 Received in revised form 17 August 2009 Accepted 19 August 2009 Available online 25 August 2009 Targeting the crucial step of Plasmodium transition from vertebrate host to mosquito vector is a promis- ing approach to eliminate malaria. Uptake by the mosquito activates gametocytes within seconds, and in the case of male (micro) gametocytes leads to rapid DNA replication and the release of eight flagel- lated gametes. We developed a sensitive assay to monitor P. berghei microgametocyte activation based on [3H]hypoxanthine incorporation into DNA. Optimal pH range and xanthurenic acid concentrations for gametocyte activation were established and the kinetics of DNA replication investigated. Signifi- cance of the method was confirmed using P. berghei mutants and the assay was applied to analyse the effect of protease inhibitors, which revealed differences regarding their inhibitory action. The developed method thus appears suitable for reproducible determination of microgametocyte activation, medium- throughput drug screenings and deeper investigation of early blocks in gametogenesis and will facilitate the analysis of compounds for transmission blocking activities. Keywords: Malaria Gametocyte Transmission Exflagellation © 2009 Elsevier B.V. Open access under CC BY license. © 2009 Elsevier B.V. Open access under CC BY license. of micro- and macrogametes leads to formation of a diploid zygote that itself develops into the motile ookinete which escapes from the blood meal by penetrating the mosquito gut wall. Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei Andreas C. Raabe a,b, Oliver Billker b,c, Henri J. Vial a, Kai Wengelnik a,∗ a CNRS UMR5235, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France b Imperial College London, Division of Cell and Molecular Biology, London SW7 2AZ, UK c The Wellcome Trust Sanger Institute, Cambridge CB10 SA1, UK Andreas C. Raabe a,b, Oliver Billker b,c, Henri J. Vial a, Kai Wengelnik a,∗ a CNRS UMR5235, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France b Imperial College London, Division of Cell and Molecular Biology, London SW7 2AZ, UK c The Wellcome Trust Sanger Institute, Cambridge CB10 SA1, UK HAL Id: hal-03246862 https://hal.science/hal-03246862v1 Submitted on 16 Jun 2022 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 Molecular & Biochemical Parasitology 168 (2009) 172–176 ∗Corresponding author. Tel.: +33 467144898; fax: +33 467144286. E-mail address: kaiweng@univ-montp2.fr (K. Wengelnik). 0166-6851 © 2009 Elsevier B.V. doi:10.1016/j.molbiopara.2009.08.004 Open access under CC BY license. 1. Results Values are expressed as cpm per 106 cells and are averages of duplicates in one experiment representative of five. (B) Radioactive labelling of nucleic acids over time with fixed amount of radioactive [3H]hypoxanthine (0.5 Ci/well, 260 nM) supplemented with the indicated concentrations of cold hypoxanthine. (C) Total hypoxanthine incorporation during gametogenesis calculated from data shown in (B). (D) Effect of pre-incubation of gametocytes with 260 nM [3H]hypoxanthine before activation. Activation was performed at the indicate time and cells incubated for 20 min. Results are shown with S.E.M, n = 3. All graphs and analyses were generated using GraphPad Prism software. A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 173 A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 17 hi i i i DNA d i i i (A) I i f [3H]h hi i f i G ifi Fig. 1. [3H]hypoxanthine incorporation into DNA during microgametogenesis. (A) Incorporation of [3H]hypoxanthine over time of gametogenesis. Gametocytes were purified as described previously [10] with minor modifications. Female NMRI mice (Charles River) were pre-treated with 0.1 ml phenylhydrazine (25 mg/ml in PBS) and infected two days later with 0.5–2 × 107 P. berghei ANKA clone 2.34 parasites from frozen blood stocks. On day 4 p.i. 20 mg/ml sulfadiazine in drinking water was applied to kill asexual stages. On day 6 p.i., mice were bled by cardiac puncture, the blood washed in gametocyte maintenance buffer (GMB: 4 mM sodium bicarbonate, 20 mM glucose, 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 20 mM Hepes pH 7.24–7.29, 0.1% BSA) and purified on a 48% Nycodenz/GMB gradient (Nycodenz stock solution: 27.6% (w/v) Nycodenz in 3 mM KCl, 0.3 mM EDTA, 5 mM Tris–HCl pH 7.2). Gametocytes were resuspended in GMB and kept at 20 ◦C. As determined by Giemsa stained blood film, gametocytes were enriched to approximately 95% with contaminants being late stage trophozoites (≈4%), few red blood cells and occasionally some very few white blood cells. Gametocytes were activated at room temperature (22–26 ◦C) by transferring them to gametocyte activation medium (GAM; RPMI 1640 with 20 mM Hepes, 4 mM sodium bicarbonate, adjusted to pH 8.0 and supplemented with 100 M XA) while the medium for controls was adjusted to pH 7.0. 1. Results The radioactive purine precursor [3H]hypoxanthine is readily metabolised into nucleotides by Plasmodium and routinely used to label DNA in asexual stages [14]. Given the considerable need for A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 173 Fig. 1. [3H]hypoxanthine incorporation into DNA during microgametogenesis. (A) Incorporation of [3H]hypoxanthine over time of gametogenesis. Gametocytes were purified as described previously [10] with minor modifications. Female NMRI mice (Charles River) were pre-treated with 0.1 ml phenylhydrazine (25 mg/ml in PBS) and infected two days later with 0.5–2 × 107 P. berghei ANKA clone 2.34 parasites from frozen blood stocks. On day 4 p.i. 20 mg/ml sulfadiazine in drinking water was applied to kill asexual stages. On day 6 p.i., mice were bled by cardiac puncture, the blood washed in gametocyte maintenance buffer (GMB: 4 mM sodium bicarbonate, 20 mM glucose, 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 20 mM Hepes pH 7.24–7.29, 0.1% BSA) and purified on a 48% Nycodenz/GMB gradient (Nycodenz stock solution: 27.6% (w/v) Nycodenz in 3 mM KCl, 0.3 mM EDTA, 5 mM Tris–HCl pH 7.2). Gametocytes were resuspended in GMB and kept at 20 ◦C. As determined by Giemsa stained blood film, gametocytes were enriched to approximately 95% with contaminants being late stage trophozoites (≈4%), few red blood cells and occasionally some very few white blood cells. Gametocytes were activated at room temperature (22–26 ◦C) by transferring them to gametocyte activation medium (GAM; RPMI 1640 with 20 mM Hepes, 4 mM sodium bicarbonate, adjusted to pH 8.0 and supplemented with 100 M XA) while the medium for controls was adjusted to pH 7.0. GAM was supplemented with 0.5 M [3H]hypoxanthine (previously evaporated from 52 M in water/ethanol 1:1, specific activity of 1 mCi/ml, GE Healthcare). 100 l aliquots containing (3–15) × 106 gametocytes were removed at the indicated time points and shock frozen in 96-well microtiter plates on liquid N2. Subsequently, macromolecules including DNA were recovered by filtering the lysates onto glass-fiber filter 96 plates (Perkin Elmer UniFilter 96 GF/C and Packard Filtermate Harvester). The membrane plate was washed 5 times by perfusion with H2O, bleached with 10% H2O2, dried at 50 ◦C for 30 min, loaded with 30 l/well PerkinElmer Microscint 0, and the radioactivity determined in a TopCount NXT microplate scintillation counter (Packard Instruments). 1. Results GAM was supplemented with 0.5 M [3H]hypoxanthine (previously evaporated from 52 M in water/ethanol 1:1, specific activity of 1 mCi/ml, GE Healthcare). 100 l aliquots containing (3–15) × 106 gametocytes were removed at the indicated time points and shock frozen in 96-well microtiter plates on liquid N2. Subsequently, macromolecules including DNA were recovered by filtering the lysates onto glass-fiber filter 96 plates (Perkin Elmer UniFilter 96 GF/C and Packard Filtermate Harvester). The membrane plate was washed 5 times by perfusion with H2O, bleached with 10% H2O2, dried at 50 ◦C for 30 min, loaded with 30 l/well PerkinElmer Microscint 0, and the radioactivity determined in a TopCount NXT microplate scintillation counter (Packard Instruments). Values are expressed as cpm per 106 cells and are averages of duplicates in one experiment representative of five. (B) Radioactive labelling of nucleic acids over time with fixed amount of radioactive [3H]hypoxanthine (0.5 Ci/well, 260 nM) supplemented with the indicated concentrations of cold hypoxanthine. (C) Total hypoxanthine incorporation during gametogenesis calculated from data shown in (B). (D) Effect of pre-incubation of gametocytes with 260 nM [3H]hypoxanthine before activation. Activation was performed at the indicate time and cells incubated for 20 min. Results are shown with S.E.M, n = 3. All graphs and analyses were generated using GraphPad Prism software. per 100 l solution at 37 ◦C for 2 h completely removed RNA from non-radioactive control samples as detected by ethidium bromide staining of agarose gel separated nucleic acids. When lysates of [3H]hypoxanthine labelled parasites were digested with RNaseA before the macromolecules were filtered and analysed, we found no difference to untreated samples, showing that [3H]hypoxanthine was essentially incorporated into DNA (data not shown). nucleotides during gametogenesis it seemed likely that gameto- cytes too would scavenge external hypoxanthine for incorporation into their DNA. Purified gametocytes were activated by trans- fer to gametocyte activation medium (GAM) in the presence of [3H]hypoxanthine and the incorporation of the radioactive label into macromolecules analysed. Strong radiolabel incorporation into nucleic acids was observed in gametocytes activated by a shift to pH 8.0 but not under non-activating conditions at pH 7.0 (Fig. 1A). At pH 8.0, label incorporation commenced after a short lag phase reaching an intermediate step after 6 min and continued to rise until a plateau was reached after 10 min. This pattern was repeatedly observed in independent experiments (data not shown). 1. Results To date, it is not clear how these two stimuli are recognised by the parasite, but it has previously been shown by counting exflagellation events that addition of XA can extend the permissive pH range for exflagellation, and it was therefore suggested that the two stimuli synergise in vivo [16]. To validate our assay, we asked whether the hypoxanthine incorporation would reflect this synergy of pH and XA. Purified gametocytes were resuspended in GAM spanning a pH range of 6.8–8.2 either with or without 100 M XA added. In the absence of XA, incorporation of radioactive hypoxanthine commenced at pH 7.5 and steadily rose until a maximum was reached at pH 7.8 (Fig. 2A). Addition of XA shifted the curve by 0.3 pH units to the acidic range. Gametocytes were not activated below pH 7.2 even in the presence of XA. We also identified the EC50, (effec- tive concentration 50%) of XA, the concentration at which half the cells become activated. Purified gametocytes were resuspended in XA free GAM at pH 7.4 to avoid unintentional activation by alkaline pH alone, and cells were then activated by the addition of different concentrations of XA. The resulting activation pat- tern followed a concentration dependent curve with an EC50 of 1.1 M (95% confidence interval 0.8–1.4 M) (Fig. 2B). These results show that hypoxanthine incorporation was intimately linked with the response to activation by pH and XA, thus validating our assay as an appropriate method to quantify gametocyte activa- tion. After having established the basic parameters of gametocyte activation, we sought to subject parasites with a known exflag- ellation deficiency to this assay to validate the system as a tool for phenotypic analysis. The two different P. berghei parasite lines that seemed suitable were a knock out of the calcium-dependent kinase 4 (CDPK4, clone 3.0.7) and a knock out of the Map-2 kinase (Map-2, clone 2.4.9), respectively. Neither of the parasite lines exflagellates. CDPK4 parasites have been described to have a block in the signalling pathway upstream of DNA replication, i.e. game- tocytes become activated but fail to initiate DNA replication [10]. Map-2 parasites have a later block at the stage of cytokinesis [11], i.e. the parasites can replicate their DNA but fail to segregate into gametes. Consistent with this we found that at pH 7.0 neither of the three strains replicated their DNA. 1. Results As we were using only trace con- centrations of [3H]hypoxanthine (i. e. 260 nM), we first tested whether addition of non-radioactive (cold) hypoxanthine rang- ing from 3.1 to 200 M would increase the overall incorporation of external hypoxanthine (Fig. 1B). As calculated on the basis of the specific activity, total hypoxanthine incorporation indeed increased with the concentration of cold hypoxanthine from 1 to 48 pmol/107 cells (Fig. 1C). However, dilution of label by cold hypoxanthine outweighed this assimilation surplus, resulting in an overall decrease in DNA labelling. Importantly however, the kinetics of label incorporation were identical and independent of the specific activity. The curves showed the same characteristic incorporation pattern described above with an intermediate step at around 6 min and a plateau reached at 12 min post-activation (Fig. 1B). Further experiments were thus performed in media with [3H]hypoxanthine as the only source of hypoxanthine. Secondly, we tested whether pre-incubation with radioactive hypoxanthine prior to activation would increase label incorporation. Purified gametocytes were pre-incubated for up to 90 min in the presence of radioactive hypoxanthine before they were activated by transfer to GAM containing the same concentration of [3H]hypoxanthine. The pre-incubation did not modify the level of DNA labelling when com- pared to control cells without pre-incubation (Fig. 1D). At the same time, washing of pre-incubated gametocytes to remove the label, followed by activation in the absence of [3H]hypoxanthine did not lead to detectable labelling of DNA (data not shown). These some- what surprising results indicated that gametocytes did not take up and incorporate [3H]hypoxanthine into nucleotides in the rest- ing state (at least during the 90 min before activation). However, upon activation, and perhaps depending upon their emergence from the host erythrocyte, microgametocytes replenished their nucleotide pool at a rate that was high enough to allow rapid detec- tion of [3H]hypoxanthine incorporation into DNA from 2 min on, although the precursor has first to be taken up by the parasite and metabolised into nucleotides, before it can be incorporated into DNA. The efficiency of [3H]hypoxanthine incorporation only upon gametocyte activation facilitated the assay as cells did not require pre-incubation and could be used directly after purifica- tion. p g Gametocytes can be activated in vitro by either a pH shift to the alkaline range or by XA. 1. Results For one batch of purified gametocytes, we counted exflagellation events under the microscope and observed that at pH 8.0 exflagel- lation peaked at 14 min while no exflagellation was observed at pH 7.0 (data not shown). Thus, hypoxanthine incorporation correlated well with successful exflagellation. nucleotides during gametogenesis it seemed likely that gameto- cytes too would scavenge external hypoxanthine for incorporation into their DNA. Purified gametocytes were activated by trans- fer to gametocyte activation medium (GAM) in the presence of [3H]hypoxanthine and the incorporation of the radioactive label into macromolecules analysed. Strong radiolabel incorporation into nucleic acids was observed in gametocytes activated by a shift to pH 8.0 but not under non-activating conditions at pH 7.0 (Fig. 1A). At pH 8.0, label incorporation commenced after a short lag phase reaching an intermediate step after 6 min and continued to rise until a plateau was reached after 10 min. This pattern was repeatedly observed in independent experiments (data not shown). For one batch of purified gametocytes, we counted exflagellation events under the microscope and observed that at pH 8.0 exflagel- lation peaked at 14 min while no exflagellation was observed at pH 7.0 (data not shown). Thus, hypoxanthine incorporation correlated well with successful exflagellation. We also excluded the possibility that asexual stages, essen- tially late trophozoites, which are minor contaminants (<4%) in our purified gametocyte preparations might contribute to the total radioactive DNA measured. For this, identical parasite numbers of asexual stage parasites from gametocyte deficient strain 2.33, and purified gametocytes from wild type strain 2.34 were acti- vated in GAM in the presence of [3H]hypoxanthine. After 20 min, label incorporation was observed in gametocytes but not asex- ual stages (4300 cpm versus 440 cpm). From this we estimate that contaminating asexual stages in our enriched gametocyte prepa- rations only account for approximately 0.4% of the total label Purines deriving from hypoxanthine can be incorporated into both DNA or RNA. Treatment of lysates with 10 g RNaseA A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 174 incorporation we observed, which is negligible for most applica- tions. could be observed in a population of cells indicates that under our experimental conditions, gametocytes were highly synchronised until the point of exflagellation. We next tested whether [3H]hypoxanthine incorporation into nucleic acids could be improved with the aim to reduce the radioactivity used per assay. 1. Results When activated at pH 8.0, Map-2 and wild type parasites showed similar levels of DNA repli- cation, while CDPK4 parasites did not replicate their DNA (Fig. 2C). Our assay was thus suitable for phenotypic analysis of P. berghei mutants. Our experiments revealed interesting insight into the kinetics of DNA replication in the course of gamete formation. Previous studies based on total DNA measurements using Feulgen-stained cells showed a steady increase of DNA over the time of gameto- genesis [13]. On the contrary, we observed three distinct stages in most experiments (Fig. 1A and B and data not shown). The first occurrence of radioactive label was generally very weak but could be observed from 2 min after activation. After 4 min, a stronger increase lasted until 5 min when a brief plateau was reached. After another minute, a further surge in radioactive label incorporation lasted until the final plateau was reached after 10 min. We hypothe- size that each of the three phases of label incorporation corresponds to an S-phase, when DNA is synthesised, while each intermediate step in the curve likely represents an M-phase, coinciding with a mitotic division. Earlier studies described the formation of mitotic spindles during microgametogenesis with the second mitotic spin- dle being observed after about 5 min post-activation [15]. This time point corresponds to the brief pause in label incorporation we observed (Fig. 1A), which suggests that the actual M-phase might only take 1 min during which DNA synthesis is stopped. In some experiments, this time point was shifted and occurred between 6 and 7 min but remained clearly distinguishable as a period of reduced DNA synthesis (Fig. 1B). The fact that these distinct stages Our assay is suited for medium-throughput screenings of compounds that interfere with gametocyte activation or DNA repli- cation and thus might have transmission blocking potential. As an example of such an investigation, we chose to analyse the inhibitory effects of three protease inhibitors on DNA replication. The serine/cysteine protease inhibitors TLCK and TPCK as well as the metalloprotease inhibitor 1,10-phenanthroline have been previously shown to block exflagellation centre formation in P. berghei [17] and similar effects had been described in P. falci- parum [18]. However, it is not clear from these studies at which point during gametogenesis these compounds act. 1. Results We first con- firmed that these compounds completely abolished exflagellation under our assay conditions when used at the reported concentra- tions (100 M TLCK or TPCK, 1 mM 1,10-phenanthroline) [17] (data not shown). When we applied these compounds in the hypoxan- thine assay, we observed potent inhibition of DNA synthesis for 1,10-phenanthroline with an IC50 of 200 M and TPCK with an IC50 of 25 M while TLCK had no effect (Fig. 2D). These results indicate that TPCK and 1,10-phenanthroline inhibit gametogen- A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 175 Fig. 2. Applications of the [3H]hypoxanthine assay. (A) Synergy of XA and pH in gametocyte activation. Purified gametocytes were resuspended in GAM of different pH con- taining 0.5 M [3H]hypoxanthine either with or without 100 M XA added. Reactions were stopped after 20 min. The pH of all solutions was adjusted prior to the experiment and confirmed after the experiment. Values are presented as percentage of control at pH 8.0. Shown is one representative experiment of four. (B) Purified gametocytes were activated in GAM at pH 7.4 with varying concentrations of XA. Values are presented as percentage of control at 100 M XA. Shown is one representative experiment of five. (C) DNA replication of Map-2 and CDPK4 parasite strains compared to wild type (WT). Gametocytes were purified, activated in GAM and [3H]hypoxanthine incorporation quantified 20 min after activation. Shown is the average and S.E.M. of three independent experiments. The difference in [3H]hypoxanthine incorporation in CDPK4 parasites at pH 7 and pH 8 was not statistically significant. (D) Effect of protease inhibitors on DNA synthesis during gametogenesis. Stock solutions were prepared immediately before the experiment at the following concentrations: 10 mM TLCK (N-a-tosyl-l-lysine chloromethyl ketone) in water pH 3, 10 mM TPCK (N-tosyl-l-phenylalanine chloromethyl ketone) in DMSO, 100 mM 1,10-phenanthroline in DMSO (all from Sigma–Aldrich). Purified gametocytes were activated in the presence of the inhibitors and [3H]hypoxanthine incorporation was quantified 20 min after activation. Values are the mean ± SD and are presented as percentage of control without inhibitor (n = 3). plications of the [3H]hypoxanthine assay. (A) Synergy of XA and pH in gametocyte activation. References esis upstream of DNA synthesis, i.e. within the first 2 min after gametocyte activation, while TLCK exerted an inhibitory effect on exflagellation only later, after DNA replication was concluded. The mechanisms through which the different protease inhibitors act on microgametogenesis remain to be identified in more detailed analyses. [1] Mons B, Janse CJ, Boorsma EG, van der Kaay HJ. Synchronized erythrocytic schizogony and gametocytogenesis of Plasmodium berghei in vivo and in vitro. Parasitology 1985;91(Pt 3):423–30. [2] Hawking F, Wilson ME, Gammage K. Evidence for cyclic development and short- lived maturity in the gametocytes of Plasmodium falciparum. Trans R Soc Trop Med Hyg 1971;65:549–59. In summary, the presented assay that is adapted to the 96- well plate format allows rapid and easy analysis of multiple conditions and provides reproducible information about male gametocyte activation. The assay can be readily used with the same reagents and equipment that are commonly employed for antimalarial growth assays [14] and will thus be useful in drug screenings for transmission blocking studies as well as closer phenotypical analysis of developmental blocks in gametogene- sis. [3] Dixon MW, Thompson J, Gardiner DL, Trenholme KR. Sex in Plasmodium: a sign of commitment. Trends Parasitol 2008;24:168–75. [4] Janse CJ, van der Klooster PF, van der Kaay HJ, Van der PM, Overdulve JP. Rapid repeated DNA replication during microgametogenesis and DNA syn- thesis in young zygotes of Plasmodium berghei. Trans R Soc Trop Med Hyg 1986;80:154–7. [5] Sinden RE. Sexual development of malarial parasites. Adv Parasitol 1983;22:153–216. [6] Nijhout MM. Plasmodium gallinaceum: exflagellation stimulated by a mosquito factor. Exp Parasitol 1979;48:75–80. [7] Billker O, Lindo V, Panico M, et al. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature 1998;392:289–92. 1. Results Purified gametocytes were resuspended in GAM of different pH con- M [3H]h po anthine either ith or ithout 100 M XA added Reactions ere stopped after 20 min The pH of all solutions as adjusted prior to the e periment Fig. 2. Applications of the [3H]hypoxanthine assay. (A) Synergy of XA and pH in gametocyte activation. Purified gametocytes were resuspended in GAM of different pH con- taining 0.5 M [3H]hypoxanthine either with or without 100 M XA added. Reactions were stopped after 20 min. The pH of all solutions was adjusted prior to the experiment and confirmed after the experiment. Values are presented as percentage of control at pH 8.0. Shown is one representative experiment of four. (B) Purified gametocytes were activated in GAM at pH 7.4 with varying concentrations of XA. Values are presented as percentage of control at 100 M XA. Shown is one representative experiment of five. (C) DNA replication of Map-2 and CDPK4 parasite strains compared to wild type (WT). Gametocytes were purified, activated in GAM and [3H]hypoxanthine incorporation quantified 20 min after activation. Shown is the average and S.E.M. of three independent experiments. The difference in [3H]hypoxanthine incorporation in CDPK4 parasites at pH 7 and pH 8 was not statistically significant. (D) Effect of protease inhibitors on DNA synthesis during gametogenesis. Stock solutions were prepared immediately before the experiment at the following concentrations: 10 mM TLCK (N-a-tosyl-l-lysine chloromethyl ketone) in water pH 3, 10 mM TPCK (N-tosyl-l-phenylalanine chloromethyl ketone) in DMSO, 100 mM 1,10-phenanthroline in DMSO (all from Sigma–Aldrich). Purified gametocytes were activated in the presence of the inhibitors and [3H]hypoxanthine incorporation was quantified 20 min after activation. Values are the mean ± SD and are presented as percentage of control without inhibitor (n = 3). [16] Billker O, Miller AJ, Sinden RE. Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis. Parasitology 2000;120(Pt 6):547–51. [17] Torres JA, Rodriguez MH, Rodriguez MC. Hernandez-Hernandez dlC, Plasmod- ium berghei: effect of protease inhibitors during gametogenesis and early zygote development. Exp Parasitol 2005;111:255–9. Acknowledgements [8] Nijhout MM, Carter R. Gamete development in malaria parasites: bicarbonate- dependent stimulation by pH in vitro. Parasitology 1978;76:39–53. We would like to thank Shilpa Nagaraju-Shastri for critical read- ing of the manuscript. This work is part of the activities of the BioMalPar European Network of Excellence (LSHP-CT-2004-258 503578), and also received support from the Wellcome Trust (grant number 089085/Z/09/Z). This research adhered to the Principles of Laboratory Animal Care [19]. Animal studies were approved by the local animal use committee. [9] Garcia GE, Wirtz RA, Barr JR, Woolfitt A, Rosenberg R. Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite. J Biol Chem 1998;273:12003–5. [10] Billker O, Dechamps S, Tewari R, Wenig G, Franke-Fayard B, Brinkmann V. Cal- cium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite. Cell 2004;117:503–14. [11] Tewari R, Dorin D, Moon R, Doerig C, Billker O. An atypical mitogen-activated protein kinase controls cytokinesis and flagellar motility during male gamete formation in a malaria parasite. Mol Microbiol 2005;58:1253–63. [19] U.S. National Institutes of Health. Laboratory animal welfare; proposed U.S. government principles for the utilization and care of vertebrate animals used in testing, research and training. Fed Regist 1984;49:29350–01. yg p p [18] Rupp I, Bosse R, Schirmeister T, Pradel G. Effect of protease inhibitors on exflag- ellation in Plasmodium falciparum. Mol Biochem Parasitol 2008;158:208–12. [18] Rupp I, Bosse R, Schirmeister T, Pradel G. Effect of protease inhibitors on exflag- ellation in Plasmodium falciparum. Mol Biochem Parasitol 2008;158:208–12. [19] U.S. National Institutes of Health. Laboratory animal welfare; proposed U.S. government principles for the utilization and care of vertebrate animals used in testing, research and training. Fed Regist 1984;49:29350–01. [12] McRobert L, Taylor CJ, Deng W, et al. Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS Biol 2008;6:e139. [16] Billker O, Miller AJ, Sinden RE. Determination of mosquito bloodmeal pH in situ by ion-selective microelectrode measurement: implications for the regulation of malarial gametogenesis. Parasitology 2000;120(Pt 6):547–51. [17] Torres JA, Rodriguez MH, Rodriguez MC. Hernandez-Hernandez dlC, Plasmod- ium berghei: effect of protease inhibitors during gametogenesis and early zygote development. Exp Parasitol 2005;111:255–9. [18] Rupp I, Bosse R, Schirmeister T, Pradel G. Effect of protease inhibitors on exflag- ellation in Plasmodium falciparum. Mol Biochem Parasitol 2008;158:208–12. [19] U.S. National Institutes of Health. Laboratory animal welfare; proposed U.S. government principles for the utilization and care of vertebrate animals used in testing, research and training. Fed Regist 1984;49:29350–01. A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 A.C. Raabe et al. / Molecular & Biochemical Parasitology 168 (2009) 172–176 176 [12] McRobert L, Taylor CJ, Deng W, et al. Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS Biol 2008;6:e139. [13] Janse CJ, van der Klooster PF, van der Kaay HJ, Van der PM, Overdulve JP. DNA synthesis in Plasmodium berghei during asexual and sexual development. Mol Biochem Parasitol 1986;20:173–82. [17] Torres JA, Rodriguez MH, Rodriguez MC. Hernandez-Hernandez dlC, Plasmod- ium berghei: effect of protease inhibitors during gametogenesis and early zygote development. Exp Parasitol 2005;111:255–9. [14] Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979;16:710–8. [18] Rupp I, Bosse R, Schirmeister T, Pradel G. Effect of protease inhibitors on exflag- ellation in Plasmodium falciparum. Mol Biochem Parasitol 2008;158:208–12. [15] Billker O, Shaw MK, Jones IW, Ley SV, Mordue AJ, Sinden RE. Azadirachtin dis- rupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei. J Eukaryot Microbiol 2002;49:489–97.
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https://journals.iucr.org/e/issues/2011/03/00/hb5794/hb5794.pdf
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(Dimethyl sulfoxide-κ<i>O</i>){4,4′,6,6′-tetra-<i>tert</i>-butyl-2,2′-[1,2-dicyanoethene-1,2-diylbis(nitrilomethylidyne)]diphenolato-κ<sup>4</sup><i>O</i>,<i>N</i>,<i>N</i>′,<i>O</i>′}zinc(II) acetonitrile monosolvate
Acta crystallographica. Section E
2,011
cc-by
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E. S. Aazam,a‡ Seik Weng Ngb and Edward R. T. Tiekinkb* Experimental Crystal data [Zn(C34H42N4O2)(C2H6OS)]- C2H3N Mr = 723.27 Monoclinic, P21=n a = 12.3288 (7) A˚ b = 17.7043 (9) A˚ c = 17.3932 (9) A˚  = 92.4391 (8) V = 3793.0 (3) A˚ 3 Z = 4 Mo K radiation  = 0.74 mm1 T = 100 K 0.45  0.30  0.10 mm Data collection Bruker SMART APEX CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.603, Tmax = 0.746 35700 measured reflections 8699 independent reflections 7157 reflections with I > 2(I) Rint = 0.041 Refinement R[F 2 > 2(F 2)] = 0.032 wR(F 2) = 0.080 S = 1.02 8699 reflections 448 parameters H-atom parameters constrained max = 0.56 e A˚ 3 min = 0.29 e A˚ 3 Experimental Experimental Crystal data [Zn(C34H42N4O2)(C2H6OS)]- C2H3N Mr = 723.27 Monoclinic, P21=n a = 12.3288 (7) A˚ b = 17.7043 (9) A˚ c = 17.3932 (9) A˚  = 92.4391 (8) V = 3793.0 (3) A˚ 3 Z = 4 Mo K radiation  = 0.74 mm1 T = 100 K 0.45  0.30  0.10 mm Data collection Bruker SMART APEX CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.603, Tmax = 0.746 35700 measured reflections 8699 independent reflections 7157 reflections with I > 2(I) Rint = 0.041 Refinement R[F 2 > 2(F 2)] = 0.032 wR(F 2) = 0.080 S = 1.02 8699 reflections 448 parameters H-atom parameters constrained max = 0.56 e A˚ 3 min = 0.29 e A˚ 3 aDepartment of Chemistry, Girls Campus, King Abdulaziz University, PO Box 6171, Jeddah 21442, Saudi Arabia, and bDepartment of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia Correspondence e-mail: edward.tiekink@gmail.com  = 92.4391 (8) V = 3793.0 (3) A˚ 3 Z = 4 Mo K radiation  = 0.74 mm1 T = 100 K 0.45  0.30  0.10 mm Received 27 January 2011; accepted 27 January 2011 Key indicators: single-crystal X-ray study; T = 100 K; mean (C–C) = 0.002 A˚; R factor = 0.032; wR factor = 0.080; data-to-parameter ratio = 19.4. The Zn atom in the title acetonitrile solvate, [Zn(C34H42N4O2)(C2H6OS)]CH3CN, exists in a distorted square-pyramidal geometry with the basal plane defined by the N2O2 atoms of the tetradentate Schiff base and with the dimethyl sulfoxide O atom in the apical position. metal-organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Table 1 Selected bond lengths (A). Zn—O1 1.9470 (11) Zn—O2 1.9390 (11) Zn—O3 2.0467 (12) Zn—N1 2.0939 (14) Zn—N2 2.1001 (13) metal-organic compounds Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HB5794). E. S. Aazam,a‡ Seik Weng Ngb and Edward R. T. Tiekinkb* The tetradentate mode of coordination of the Schiff base ligand leads to a five-membered ZnN2C2 chelate ring which adopts an envelope conformation with the Zn atom at the flap, and two six-membered ZnOC4N chelate rings, one of which is approximately planar (r.m.s. deviation = 0.054 A˚ ) but the other has significant puckering (r.m.s. deviation = 0.203 A˚ ). 448 parameters H-atom parameters constrained max = 0.56 e A˚ 3 min = 0.29 e A˚ 3 Table 1 Selected bond lengths (A˚ ). Zn—O1 1.9470 (11) Zn—O2 1.9390 (11) Zn—O3 2.0467 (12) Zn—N1 2.0939 (14) Zn—N2 2.1001 (13) Related literature For background to metal salicylaldiminato complexes as optoelectronic materials, see: Liuzzo et al. (2010); Shirai et al., (2000). For background to zinc complexes as organic light- emitting diodes, see: Chen et al. (2009). For related structures, see: MacLachlan et al. (1996). For geometrical analysis, see: Addison et al. (1984). Data collection: APEX2 (Bruker, 2008); cell refinement: SAINT (Bruker, 2008); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 (Farrugia, 1997), DIAMOND (Brandenburg, 2006); soft- ware used to prepare material for publication: publCIF (Westrip, 2010) and PLATON (Spek, 2009). The authors acknowledge King Abdulaziz University for financial support (grant No. 17–013/430). The authors also thank the University of Malaya for support of the crystal- lographic facility. ‡ Additional correspondence author, e-mail: wayfield8@yahoo.com. ‡ Additional correspondence author, e-mail: wayfield8@yahoo.com. m314 Aazam et al. Acta Cryst. (2011). E67, m314–m315 doi:10.1107/S160053681100359X Addison, A. W., Rao, T. N., Reedijk, J., van Rijn, J. & Verschoor, G. C. (1984). J. Chem. Soc. Dalton Trans. pp. 1349–1356. Brandenburg, K. (2006). DIAMOND. Crystal Impact GbR, Bonn, Germany. Bruker (2008). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Chen, L., Qiao, J., Xie, J., Duan, L., Zhang, D., Wang, L. & Qiu, Y. (2009). Inorg. Chim. Acta, 362, 2327–2333. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Liuzzo, V., Oberhauser, W. & Pucci, A. (2010). Inorg. Chem. Commun. 13, 686–688. MacLachlan, M. J., Park, M. K. & Thompson, L. K. (1996). Inorg. Chem. 35, 5492–5499. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Shirai, K., Matsuoka, M. & Fukunishi, K. (2000). Dyes Pigments, 47, 107–115. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. MacLachlan, M. J., Park, M. K. & Thompson, L. K. (1996). Inorg. Chem. 35, 5492–5499. Liuzzo, V., Oberhauser, W. & Pucci, A. (2010). Inorg. Chem. Commun. 13, 686–688. Acta Cryst. (2011). E67, m314–m315 Aazam et al.  [Zn(C34H42N4O2)(C2H6OS)]C2H3N m315 Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Inorg. Chim. Acta, 362, 2327 2333. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Chen, L., Qiao, J., Xie, J., Duan, L., Zhang, D., Wang, L. & Qiu, Y. (2009). Inorg Chim Acta 362 2327–2333 Shirai, K., Matsuoka, M. & Fukunishi, K. (2000). Dyes Pigments, 47, 107–115. S k A L (2009) A C D65 148 155 Shirai, K., Matsuoka, M. & Fukunishi, K. (2000). Dyes Pigments, 47, 107–115. Spek, A. L. (2009). Acta Cryst. D65, 148–155. p ( ) y Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. supporting information Acta Cryst. (2011). E67, m314–m315 [doi:10.1107/S160053681100359X] Acta Cryst. (2011). E67, m314–m315 [doi:10.1107/S160053681100359X] Acta Cryst. (2011). E67, m314–m315 [doi:10.1107/S160053681100359X] S1. Comment Metal complexes with salicylaldiminato ligands are promising materials for optoelectronic applications due to their outstanding photo- and electro-luminescent properties (Liuzzo et al., 2010; Shirai et al., 2000). One of the main appeals of this class of coordination complexes is that molecular engineering permits systematically the optimizing of spectroscopic and chemical properties. This chemical flexibility allows for the design of systems that respond to specific environmental variables. Recently, zinc complexes have been introduced to OLED's (organic light-emitting diodes) and recognized as useful electron transport materials (Chen et al., 2009). The above motivated the synthesis and structural characterization of the title complex, (I). The Zn atom in (I), Fig. 1, is tetracoordinated by the N2O2 donor atoms of the tetradentate Schiff-base ligand and the O atom derived from the dimethyl sulfoxide ligand, Table 1; the asymmetric unit is completed by a non-coordinating aceto- nitrile molecule. The resulting N2O3 donor set is based on a square pyramidal arrangement with the dimethyl sulfoxide- O3 atom occupying an axial site. The value of τ = 0.16 compares with τ = 0 and 1.0 for ideal square pyramidal and trigonal bipyramidal geometries, respectively (Addison et al., 1984). The r.m.s. deviation of the O1, O2, N1 and N2 atoms from their least-squares plane is 0.0836 Å and the Zn atom lies 0.3976 (7) Å out of the plane towards the O3 atom. The tetradentate mode of coordination of the Schiff-base leads to the formation of a five- and two six-membered rings. The former has a conformation based on an envelope on Zn (Spek, 2009). While the chelate ring involving the O1 atom is approximately planar (r.m.s. = 0.054 Å), there is significantly more distortion in the O2-containing chelate ring (r.m.s. = 0.203 Å). Schiff-base ligands derived from diaminomaleonitrile have been documented and shown to adopt comparable coordination modes towards transition metals (MacLachlan et al., 1996). References Liuzzo, V., Oberhauser, W. & Pucci, A. (2010). Inorg. Chem. Commun. 13, 686–688. MacLachlan, M. J., Park, M. K. & Thompson, L. K. (1996). Inorg. Chem. 35, 5492–5499. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. , ( ) y , Shirai, K., Matsuoka, M. & Fukunishi, K. (2000). Dyes Pigments, 47, 107–115. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Aazam et al.  [Zn(C34H42N4O2)(C2H6OS)]C2H3N m315 Acta Cryst. (2011). E67, m314–m315 supporting information S2. Experimental A mixture of diaminemaleonitrile (0.1 g, 0.93 mmol), 3,5-di-tert-butyl-2-hydroxybenzaldehyde (0.1 g, 1.86 mmol), zinc(II) acetate dihydrate (0.2 g, 0.93 mmol) and ethanol (5 ml) were placed in a glass Petri dish and capped with a glass cover. The dish was placed in a microwave oven (700 W) and irradiated for 1 min. The reaction mixture was cooled and washed with 15 ml of ethanol. The purple solid was filtered off and washed with ethanol. Re-crystallization was by slow evaporation of an acetonitrile/dimethyl sulfoxide (90/10 v/v) solution which yielded purple blocks of (I). Yield: 70%. M.pt. > 623 K (dec.). 1H NMR (DMSO-d6, 500 MHz): δ = 1.25 (s, 18H, C(CH3)3), 1.47 (s, 18H, C(CH3)3), 2.06 (MeCN), 2.50 (DMSO), 7.24 (s, 2H, Ar—H), 7.43 (s, 2H, Ar—H), 8.58 (s, 2H, N═CH) p.p.m.. 13C NMR (DMSO-d6, 500 MHz): δ = 28.19, 29.80 (C(CH3)3), 32.61, 34.13 (C(CH3)3), 110.48, 116.85, 120.31, 128.10, 130.17, 134.20, 141.11, 161.68 and 172.33 p.p.m. IR: 2952, 2212 (C≡N), 1616 (C═N), 1569, 1519, 1433, 1372, 1170, 1154, 1119, 1032, 795, 656 cm-1. λmax M.pt. > 623 K (dec.). 1H NMR (DMSO-d6, 500 MHz): δ = 1.25 (s, 18H, C(CH3)3), 1.47 (s, 18H, C(CH3)3), 2.06 (MeCN), 2.50 (DMSO), 7.24 (s, 2H, Ar—H), 7.43 (s, 2H, Ar—H), 8.58 (s, 2H, N═CH) p.p.m.. 13C NMR (DMSO-d6, 500 MHz): δ = 28.19, 29.80 (C(CH3)3), 32.61, 34.13 (C(CH3)3), 110.48, 116.85, 120.31, 128.10, 130.17, 134.20, 141.11, 161.68 and 172.33 p.p.m. IR: 2952, 2212 (C≡N), 1616 (C═N), 1569, 1519, 1433, 1372, 1170, 1154, 1119, 1032, 795, 656 cm-1. λmax Acta Cryst. (2011). E67, m314–m315 sup-1 supporting information (DMSO, 10 -5 mol L-1): 574, 501, 380, 375, 318, 245 nm. (DMSO, 10 -5 mol L-1): 574, 501, 380, 375, 318, 245 nm. S3. Refinement Carbon-bound H-atoms were placed in calculated positions (C–H = 0.95 to 0.98 Å) and were included in the refinement in the riding model approximation, with Uiso(H) set to 1.2–1.5Uequiv(C). In the final refinement three low angle reflections evidently effected by the beam stop were omitted, i.e. (011), (101) and (110). Figure 1 The molecular structure of (I) showing displacement ellipsoids at the 50% probability level supporting information Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.032 wR(F2) = 0.080 S = 1.02 8699 reflections 448 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.036P)2 + 1.6768P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.002 Δρmax = 0.56 e Å−3 Δρmin = −0.29 e Å−3 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.032 wR(F2) = 0.080 S = 1.02 8699 reflections 448 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.036P)2 + 1.6768P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.002 Δρmax = 0.56 e Å−3 Δρmin = −0.29 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.036P)2 + 1.6768P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.002 Δρmax = 0.56 e Å−3 Δρmin = −0.29 e Å−3 Figure 1 gu e The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. g The molecular structure of (I) showing displacement ellipsoids at the 50% probability level. (Dimethyl sulfoxide-κO){4,4′,6,6′-tetra-tert-butyl-2,2′-[1,2- dicyanoethene-1,2- diylbis(nitrilomethylidyne)]diphenolato- κ4O,N,N′,O′}zinc(II) acetonitrile monosolvate Crystal data [Zn(C34H42N4O2)(C2H6OS)]·C2H3N Mr = 723.27 Monoclinic, P21/n Hall symbol: -P 2yn a = 12.3288 (7) Å b = 17.7043 (9) Å c = 17.3932 (9) Å β = 92.4391 (8)° V = 3793.0 (3) Å3 Z = 4 F(000) = 1536 Dx = 1.267 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 9906 reflections θ = 2.3–28.3° µ = 0.74 mm−1 T = 100 K Block, purple 0.45 × 0.30 × 0.10 mm Data collection Bruker SMART APEX CCD diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.603, Tmax = 0.746 35700 measured reflections 8699 independent reflections 7157 reflections with I > 2σ(I) Rint = 0.041 θmax = 27.5°, θmin = 2.3° h = −16→16 k = −23→23 l = −22→22 sup-2 Acta Cryst. (2011). E67, m314–m315 supporting information Special details Geometry. All s.u.'s (except the s.u. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell s.u.'s are taken into account individually in the estimation of s.u.'s in distances, angles and torsion angles; correlations between s.u.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell s.u.'s is used for estimating s.u.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > 2σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Acta Cryst. (2011). E67, m314–m315 Special details Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq Zn 0.516854 (14) 0.654898 (11) 0.558637 (10) 0.01242 (6) S1 0.41362 (3) 0.82176 (2) 0.50384 (2) 0.01635 (9) O1 0.63893 (9) 0.70123 (7) 0.61554 (7) 0.0184 (3) O2 0.44436 (9) 0.61428 (7) 0.64636 (6) 0.0167 (2) O3 0.41323 (9) 0.73722 (7) 0.51757 (7) 0.0198 (3) N1 0.61797 (11) 0.64054 (7) 0.46602 (8) 0.0129 (3) N2 0.42744 (10) 0.57668 (7) 0.49091 (7) 0.0121 (3) N3 0.64563 (12) 0.60956 (9) 0.27021 (9) 0.0224 (3) N4 0.34261 (12) 0.51244 (9) 0.30973 (9) 0.0221 (3) N5 0.63439 (16) 1.00678 (11) 0.54854 (14) 0.0500 (6) C1 0.73798 (13) 0.71491 (9) 0.59846 (9) 0.0133 (3) C2 0.81352 (12) 0.74304 (9) 0.65753 (9) 0.0127 (3) C3 0.91590 (13) 0.76392 (9) 0.63645 (9) 0.0136 (3) H3 0.9635 0.7851 0.6751 0.016* C4 0.95562 (12) 0.75609 (9) 0.56135 (9) 0.0128 (3) C5 0.88612 (12) 0.72505 (9) 0.50676 (9) 0.0129 (3) H5 0.9107 0.7177 0.4563 0.015* C6 0.77787 (12) 0.70320 (9) 0.52280 (9) 0.0130 (3) C7 0.78027 (12) 0.74618 (9) 0.74153 (9) 0.0138 (3) C8 0.87537 (14) 0.77112 (11) 0.79528 (10) 0.0212 (4) H8A 0.9355 0.7353 0.7914 0.032* H8B 0.8520 0.7724 0.8484 0.032* H8C 0.8994 0.8216 0.7804 0.032* C9 0.68745 (14) 0.80283 (10) 0.75108 (10) 0.0206 (4) H9A 0.6683 0.8044 0.8052 0.031* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2011). E67, m314–m315 supporting information supporting information H9B 0.6240 0.7871 0.7191 0.031* H9C 0.7108 0.8531 0.7350 0.031* C10 0.74430 (15) 0.66745 (10) 0.76792 (10) 0.0211 (4) H10A 0.8033 0.6312 0.7613 0.032* H10B 0.6801 0.6515 0.7370 0.032* H10C 0.7266 0.6695 0.8223 0.032* C11 1.07286 (13) 0.77942 (9) 0.54726 (9) 0.0148 (3) C12 1.08302 (14) 0.86593 (10) 0.55278 (11) 0.0221 (4) H12A 1.0340 0.8894 0.5140 0.033* H12B 1.1580 0.8809 0.5437 0.033* H12C 1.0636 0.8826 0.6042 0.033* C13 1.10819 (14) 0.75406 (11) 0.46817 (10) 0.0226 (4) H13A 1.0609 0.7773 0.4281 0.034* H13B 1.1029 0.6989 0.4643 0.034* H13C 1.1834 0.7697 0.4614 0.034* C14 1.15020 (13) 0.74290 (10) 0.60857 (10) 0.0186 (4) H14A 1.1375 0.6883 0.6098 0.028* H14B 1.1367 0.7645 0.6591 0.028* H14C 1.2256 0.7527 0.5958 0.028* C15 0.71711 (12) 0.66741 (9) 0.46187 (9) 0.0131 (3) H15 0.7509 0.6624 0.4141 0.016* C16 0.56431 (13) 0.60475 (9) 0.40459 (9) 0.0122 (3) C17 0.60834 (13) 0.60514 (9) 0.32927 (9) 0.0140 (3) C18 0.46527 (12) 0.57224 (9) 0.41737 (9) 0.0119 (3) C19 0.40078 (13) 0.53784 (9) 0.35600 (9) 0.0141 (3) C20 0.33269 (12) 0.55019 (9) 0.50908 (9) 0.0130 (3) H20 0.2899 0.5258 0.4697 0.016* C21 0.28904 (12) 0.55523 (9) 0.58319 (9) 0.0122 (3) C22 0.18261 (13) 0.52592 (9) 0.59065 (9) 0.0144 (3) H22 0.1463 0.5038 0.5469 0.017* C23 0.13077 (13) 0.52844 (9) 0.65864 (9) 0.0139 (3) C24 0.18870 (13) 0.56085 (9) 0.72222 (10) 0.0153 (3) H24 0.1538 0.5625 0.7699 0.018* C25 0.29211 (13) 0.59022 (9) 0.72021 (9) 0.0143 (3) C26 0.34663 (13) 0.58787 (9) 0.64890 (9) 0.0137 (3) C27 0.01364 (13) 0.50025 (10) 0.66406 (10) 0.0170 (3) C28 0.00085 (18) 0.42155 (12) 0.63012 (14) 0.0377 (5) H28A 0.0470 0.3861 0.6599 0.056* H28B 0.0225 0.4220 0.5766 0.056* H28C −0.0752 0.4057 0.6320 0.056* C29 −0.02449 (15) 0.49953 (13) 0.74654 (11) 0.0312 (5) H29A 0.0230 0.4666 0.7783 0.047* H29B −0.0992 0.4806 0.7468 0.047* H29C −0.0217 0.5509 0.7675 0.047* C30 −0.06285 (15) 0.55409 (12) 0.61742 (12) 0.0287 (4) H30A −0.1381 0.5372 0.6214 0.043* H30B −0.0437 0.5538 0.5633 0.043* H30C −0.0552 0.6054 0.6380 0.043* sup-4 Acta Cryst. (2011). E67, m314–m315 supporting information supporting information sup-5 Acta Cryst. (2011). supporting information E67, m314–m315 C31 0.34962 (14) 0.62514 (10) 0.79195 (10) 0.0175 (3) C32 0.27723 (16) 0.62444 (13) 0.86125 (11) 0.0293 (4) H32A 0.2104 0.6527 0.8487 0.044* H32B 0.3159 0.6480 0.9054 0.044* H32C 0.2590 0.5722 0.8740 0.044* C33 0.45222 (14) 0.57956 (10) 0.81404 (10) 0.0213 (4) H33A 0.4881 0.6017 0.8601 0.032* H33B 0.5018 0.5808 0.7715 0.032* H33C 0.4322 0.5271 0.8246 0.032* C34 0.37892 (15) 0.70810 (10) 0.77625 (11) 0.0227 (4) H34A 0.3126 0.7366 0.7629 0.034* H34B 0.4280 0.7105 0.7335 0.034* H34C 0.4147 0.7300 0.8224 0.034* C35 0.44163 (17) 0.86449 (11) 0.59519 (11) 0.0272 (4) H35A 0.3795 0.8573 0.6277 0.041* H35B 0.4546 0.9186 0.5883 0.041* H35C 0.5062 0.8411 0.6198 0.041* C36 0.53942 (15) 0.84257 (11) 0.46161 (12) 0.0260 (4) H36A 0.5411 0.8186 0.4109 0.039* H36B 0.5994 0.8232 0.4947 0.039* H36C 0.5469 0.8974 0.4560 0.039* C37 0.71611 (16) 0.99001 (11) 0.57521 (12) 0.0284 (4) C38 0.82087 (17) 0.96856 (14) 0.60957 (14) 0.0398 (5) H38A 0.8254 0.9844 0.6636 0.060* H38B 0.8786 0.9932 0.5818 0.060* H38C 0.8294 0.9136 0.6066 0.060* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Zn 0.01068 (9) 0.01551 (10) 0.01115 (10) −0.00142 (7) 0.00141 (7) −0.00244 (7) S1 0.01486 (19) 0.0156 (2) 0.0185 (2) −0.00009 (15) −0.00009 (15) −0.00080 (16) O1 0.0123 (6) 0.0290 (7) 0.0141 (6) −0.0051 (5) 0.0029 (4) −0.0070 (5) O2 0.0122 (5) 0.0260 (7) 0.0120 (6) −0.0043 (5) 0.0008 (4) 0.0004 (5) O3 0.0160 (6) 0.0143 (6) 0.0291 (7) 0.0003 (5) −0.0007 (5) −0.0002 (5) N1 0.0125 (6) 0.0148 (7) 0.0112 (7) 0.0002 (5) −0.0010 (5) −0.0006 (5) N2 0.0133 (6) 0.0126 (7) 0.0105 (7) 0.0012 (5) 0.0014 (5) 0.0003 (5) N3 0.0248 (8) 0.0260 (8) 0.0167 (8) −0.0025 (6) 0.0036 (6) −0.0005 (6) N4 0.0203 (8) 0.0251 (8) 0.0207 (8) −0.0033 (6) −0.0005 (6) −0.0046 (6) N5 0.0405 (11) 0.0296 (11) 0.0778 (16) −0.0031 (9) −0.0226 (11) 0.0054 (10) C1 0.0124 (7) 0.0145 (8) 0.0130 (8) 0.0003 (6) 0.0007 (6) −0.0011 (6) C2 0.0135 (7) 0.0121 (8) 0.0125 (8) 0.0012 (6) 0.0002 (6) −0.0018 (6) C3 0.0139 (7) 0.0134 (8) 0.0132 (8) −0.0004 (6) −0.0015 (6) −0.0020 (6) C4 0.0120 (7) 0.0132 (8) 0.0131 (8) 0.0007 (6) 0.0014 (6) 0.0019 (6) C5 0.0129 (7) 0.0150 (8) 0.0109 (8) 0.0023 (6) 0.0018 (6) 0.0013 (6) C6 0.0126 (7) 0.0141 (8) 0.0122 (8) 0.0004 (6) 0.0002 (6) 0.0006 (6) C7 0.0135 (8) 0.0169 (8) 0.0113 (8) 0.0004 (6) 0.0013 (6) −0.0024 (6) C8 0.0187 (9) 0.0323 (10) 0.0125 (8) −0.0032 (7) −0.0005 (7) −0.0052 (7) sup-5 Acta Cryst. Acta Cryst. (2011). E67, m314–m315 supporting information (2011). Acta Cryst. (2011). E67, m314–m315 supporting information E67, m314–m315 supporting information supporting informatio sup- Acta Cryst (2011) E67 m314–m315 C9 0.0215 (9) 0.0236 (10) 0.0169 (9) 0.0038 (7) 0.0037 (7) −0.0046 (7) C10 0.0271 (9) 0.0213 (9) 0.0154 (9) −0.0026 (7) 0.0060 (7) 0.0006 (7) C11 0.0118 (7) 0.0191 (8) 0.0135 (8) −0.0022 (6) 0.0020 (6) 0.0004 (7) C12 0.0181 (8) 0.0208 (9) 0.0274 (10) −0.0044 (7) 0.0011 (7) 0.0040 (8) C13 0.0136 (8) 0.0375 (11) 0.0171 (9) −0.0064 (7) 0.0040 (7) −0.0040 (8) C14 0.0124 (8) 0.0231 (9) 0.0203 (9) 0.0004 (7) 0.0017 (7) 0.0024 (7) C15 0.0135 (7) 0.0147 (8) 0.0112 (8) 0.0020 (6) 0.0017 (6) 0.0010 (6) C16 0.0144 (7) 0.0121 (8) 0.0100 (8) 0.0020 (6) 0.0001 (6) −0.0003 (6) C17 0.0127 (7) 0.0145 (8) 0.0146 (8) −0.0008 (6) −0.0014 (6) −0.0007 (6) C18 0.0138 (7) 0.0110 (8) 0.0108 (7) 0.0013 (6) 0.0000 (6) 0.0005 (6) C19 0.0139 (7) 0.0149 (8) 0.0138 (8) 0.0007 (6) 0.0033 (6) 0.0002 (6) C20 0.0144 (7) 0.0108 (8) 0.0138 (8) 0.0004 (6) −0.0010 (6) −0.0010 (6) C21 0.0125 (7) 0.0118 (8) 0.0125 (8) 0.0010 (6) 0.0009 (6) 0.0011 (6) C22 0.0150 (8) 0.0135 (8) 0.0145 (8) −0.0008 (6) −0.0003 (6) 0.0001 (6) C23 0.0131 (7) 0.0126 (8) 0.0162 (8) 0.0002 (6) 0.0018 (6) 0.0029 (6) C24 0.0167 (8) 0.0166 (8) 0.0129 (8) 0.0007 (6) 0.0038 (6) −0.0003 (6) C25 0.0160 (8) 0.0149 (8) 0.0118 (8) 0.0017 (6) 0.0005 (6) 0.0001 (6) C26 0.0136 (7) 0.0134 (8) 0.0140 (8) 0.0013 (6) −0.0006 (6) 0.0022 (6) C27 0.0166 (8) 0.0175 (9) 0.0172 (8) −0.0054 (6) 0.0055 (6) −0.0020 (7) C28 0.0358 (12) 0.0261 (11) 0.0527 (14) −0.0109 (9) 0.0218 (10) −0.0110 (10) C29 0.0212 (9) 0.0500 (13) 0.0228 (10) −0.0085 (9) 0.0055 (8) 0.0026 (9) C30 0.0181 (9) 0.0363 (12) 0.0316 (11) −0.0033 (8) −0.0012 (8) 0.0057 (9) C31 0.0181 (8) 0.0221 (9) 0.0125 (8) −0.0027 (7) 0.0018 (6) −0.0027 (7) C32 0.0260 (10) 0.0454 (12) 0.0168 (9) −0.0096 (9) 0.0045 (8) −0.0105 (9) C33 0.0236 (9) 0.0263 (10) 0.0136 (8) −0.0007 (7) −0.0037 (7) 0.0002 (7) C34 0.0257 (9) 0.0225 (9) 0.0196 (9) −0.0002 (7) −0.0022 (7) −0.0049 (7) C35 0.0344 (11) 0.0256 (10) 0.0215 (10) −0.0044 (8) 0.0019 (8) −0.0061 (8) C36 0.0213 (9) 0.0274 (10) 0.0300 (10) −0.0052 (8) 0.0092 (8) −0.0012 (8) C37 0.0312 (11) 0.0192 (10) 0.0345 (11) −0.0027 (8) −0.0034 (9) 0.0023 (8) C38 0.0298 (11) 0.0479 (14) 0.0412 (13) 0.0011 (10) −0.0056 (10) 0.0119 (11) Geometric parameters (Å, º) Zn—O1 1.9470 (11) C14—H14C 0.9800 Zn—O2 1.9390 (11) C15—H15 0.9500 Zn—O3 2.0467 (12) C16—C18 1.377 (2) Zn—N1 2.0939 (14) C16—C17 1.439 (2) Zn—N2 2.1001 (13) C18—C19 1.439 (2) S1—O3 1.5155 (12) C20—C21 1.421 (2) S1—C35 1.7803 (19) C20—H20 0.9500 S1—C36 1.7826 (18) C21—C22 1.422 (2) O1—C1 1.2919 (19) C21—C26 1.440 (2) O2—C26 1.2950 (19) C22—C23 1.369 (2) N1—C15 1.317 (2) C22—H22 0.9500 N1—C16 1.386 (2) C23—C24 1.412 (2) N2—C20 1.310 (2) C23—C27 1.535 (2) N2—C18 1.382 (2) C24—C25 1.379 (2) N3—C17 1.146 (2) C24—H24 0.9500 C9 0.0215 (9) 0.0236 (10) 0.0169 (9) 0.0038 (7) 0.0037 (7) −0.0046 (7) C10 0.0271 (9) 0.0213 (9) 0.0154 (9) −0.0026 (7) 0.0060 (7) 0.0006 (7) C11 0.0118 (7) 0.0191 (8) 0.0135 (8) −0.0022 (6) 0.0020 (6) 0.0004 (7) C12 0.0181 (8) 0.0208 (9) 0.0274 (10) −0.0044 (7) 0.0011 (7) 0.0040 (8) C13 0.0136 (8) 0.0375 (11) 0.0171 (9) −0.0064 (7) 0.0040 (7) −0.0040 (8) C14 0.0124 (8) 0.0231 (9) 0.0203 (9) 0.0004 (7) 0.0017 (7) 0.0024 (7) C15 0.0135 (7) 0.0147 (8) 0.0112 (8) 0.0020 (6) 0.0017 (6) 0.0010 (6) C16 0.0144 (7) 0.0121 (8) 0.0100 (8) 0.0020 (6) 0.0001 (6) −0.0003 (6) C17 0.0127 (7) 0.0145 (8) 0.0146 (8) −0.0008 (6) −0.0014 (6) −0.0007 (6) C18 0.0138 (7) 0.0110 (8) 0.0108 (7) 0.0013 (6) 0.0000 (6) 0.0005 (6) C19 0.0139 (7) 0.0149 (8) 0.0138 (8) 0.0007 (6) 0.0033 (6) 0.0002 (6) C20 0.0144 (7) 0.0108 (8) 0.0138 (8) 0.0004 (6) −0.0010 (6) −0.0010 (6) C21 0.0125 (7) 0.0118 (8) 0.0125 (8) 0.0010 (6) 0.0009 (6) 0.0011 (6) C22 0.0150 (8) 0.0135 (8) 0.0145 (8) −0.0008 (6) −0.0003 (6) 0.0001 (6) C23 0.0131 (7) 0.0126 (8) 0.0162 (8) 0.0002 (6) 0.0018 (6) 0.0029 (6) C24 0.0167 (8) 0.0166 (8) 0.0129 (8) 0.0007 (6) 0.0038 (6) −0.0003 (6) C25 0.0160 (8) 0.0149 (8) 0.0118 (8) 0.0017 (6) 0.0005 (6) 0.0001 (6) C26 0.0136 (7) 0.0134 (8) 0.0140 (8) 0.0013 (6) −0.0006 (6) 0.0022 (6) C27 0.0166 (8) 0.0175 (9) 0.0172 (8) −0.0054 (6) 0.0055 (6) −0.0020 (7) C28 0.0358 (12) 0.0261 (11) 0.0527 (14) −0.0109 (9) 0.0218 (10) −0.0110 (10) C29 0.0212 (9) 0.0500 (13) 0.0228 (10) −0.0085 (9) 0.0055 (8) 0.0026 (9) C30 0.0181 (9) 0.0363 (12) 0.0316 (11) −0.0033 (8) −0.0012 (8) 0.0057 (9) C31 0.0181 (8) 0.0221 (9) 0.0125 (8) −0.0027 (7) 0.0018 (6) −0.0027 (7) C32 0.0260 (10) 0.0454 (12) 0.0168 (9) −0.0096 (9) 0.0045 (8) −0.0105 (9) C33 0.0236 (9) 0.0263 (10) 0.0136 (8) −0.0007 (7) −0.0037 (7) 0.0002 (7) C34 0.0257 (9) 0.0225 (9) 0.0196 (9) −0.0002 (7) −0.0022 (7) −0.0049 (7) C35 0.0344 (11) 0.0256 (10) 0.0215 (10) −0.0044 (8) 0.0019 (8) −0.0061 (8) C36 0.0213 (9) 0.0274 (10) 0.0300 (10) −0.0052 (8) 0.0092 (8) −0.0012 (8) C37 0.0312 (11) 0.0192 (10) 0.0345 (11) −0.0027 (8) −0.0034 (9) 0.0023 (8) C38 0.0298 (11) 0.0479 (14) 0.0412 (13) 0.0011 (10) −0.0056 (10) 0.0119 (11) sup-6 supporting information N4—C19 1.147 (2) C25—C26 1.436 (2) N5—C37 1.131 (3) C25—C31 1.538 (2) C1—C6 1.439 (2) C27—C28 1.519 (3) C1—C2 1.446 (2) C27—C29 1.529 (2) C2—C3 1.380 (2) C27—C30 1.546 (3) C2—C7 1.535 (2) C28—H28A 0.9800 C3—C4 1.421 (2) C28—H28B 0.9800 C3—H3 0.9500 C28—H28C 0.9800 C4—C5 1.367 (2) C29—H29A 0.9800 C4—C11 1.533 (2) C29—H29B 0.9800 C5—C6 1.428 (2) C29—H29C 0.9800 C5—H5 0.9500 C30—H30A 0.9800 C6—C15 1.421 (2) C30—H30B 0.9800 C7—C8 1.533 (2) C30—H30C 0.9800 C7—C9 1.536 (2) C31—C32 1.530 (2) C7—C10 1.539 (2) C31—C33 1.535 (2) C8—H8A 0.9800 C31—C34 1.540 (3) C8—H8B 0.9800 C32—H32A 0.9800 C8—H8C 0.9800 C32—H32B 0.9800 C9—H9A 0.9800 C32—H32C 0.9800 C9—H9B 0.9800 C33—H33A 0.9800 C9—H9C 0.9800 C33—H33B 0.9800 C10—H10A 0.9800 C33—H33C 0.9800 C10—H10B 0.9800 C34—H34A 0.9800 C10—H10C 0.9800 C34—H34B 0.9800 C11—C13 1.528 (2) C34—H34C 0.9800 C11—C12 1.540 (2) C35—H35A 0.9800 C11—C14 1.542 (2) C35—H35B 0.9800 C12—H12A 0.9800 C35—H35C 0.9800 C12—H12B 0.9800 C36—H36A 0.9800 C12—H12C 0.9800 C36—H36B 0.9800 C13—H13A 0.9800 C36—H36C 0.9800 C13—H13B 0.9800 C37—C38 1.450 (3) C13—H13C 0.9800 C38—H38A 0.9800 C14—H14A 0.9800 C38—H38B 0.9800 C14—H14B 0.9800 C38—H38C 0.9800 O2—Zn—O1 97.38 (5) N3—C17—C16 176.04 (18) O2—Zn—O3 103.71 (5) C16—C18—N2 117.53 (14) O1—Zn—O3 109.57 (5) C16—C18—C19 121.55 (14) O2—Zn—N1 150.43 (5) N2—C18—C19 120.86 (14) O1—Zn—N1 88.26 (5) N4—C19—C18 174.84 (17) O3—Zn—N1 101.60 (5) N2—C20—C21 124.94 (14) O2—Zn—N2 87.04 (5) N2—C20—H20 117.5 O1—Zn—N2 159.88 (5) C21—C20—H20 117.5 O3—Zn—N2 88.21 (5) C20—C21—C22 116.52 (14) N1—Zn—N2 78.69 (5) C20—C21—C26 123.51 (14) O3—S1—C35 106.31 (8) C22—C21—C26 119.97 (14) sup-7 Acta Cryst. supporting information O3—S1—C36 106.12 (8) C23—C22—C21 122.24 (15) C35—S1—C36 98.09 (9) C23—C22—H22 118.9 C1—O1—Zn 132.84 (10) C21—C22—H22 118.9 C26—O2—Zn 128.42 (10) C22—C23—C24 116.77 (15) S1—O3—Zn 138.77 (7) C22—C23—C27 121.18 (15) C15—N1—C16 122.47 (14) C24—C23—C27 122.00 (14) C15—N1—Zn 125.65 (11) C25—C24—C23 124.81 (15) C16—N1—Zn 111.62 (10) C25—C24—H24 117.6 C20—N2—C18 122.86 (14) C23—C24—H24 117.6 C20—N2—Zn 123.57 (11) C24—C25—C26 118.56 (15) C18—N2—Zn 111.52 (10) C24—C25—C31 121.73 (14) O1—C1—C6 123.11 (14) C26—C25—C31 119.71 (14) O1—C1—C2 119.20 (14) O2—C26—C25 119.29 (14) C6—C1—C2 117.68 (14) O2—C26—C21 123.08 (14) C3—C2—C1 118.19 (14) C25—C26—C21 117.63 (14) C3—C2—C7 121.84 (14) C28—C27—C29 109.03 (16) C1—C2—C7 119.93 (14) C28—C27—C23 110.91 (15) C2—C3—C4 124.96 (15) C29—C27—C23 112.83 (14) C2—C3—H3 117.5 C28—C27—C30 108.11 (16) C4—C3—H3 117.5 C29—C27—C30 107.01 (15) C5—C4—C3 116.58 (14) C23—C27—C30 108.77 (14) C5—C4—C11 124.38 (14) C27—C28—H28A 109.5 C3—C4—C11 119.00 (14) C27—C28—H28B 109.5 C4—C5—C6 122.44 (15) H28A—C28—H28B 109.5 C4—C5—H5 118.8 C27—C28—H28C 109.5 C6—C5—H5 118.8 H28A—C28—H28C 109.5 C15—C6—C5 116.26 (14) H28B—C28—H28C 109.5 C15—C6—C1 123.81 (14) C27—C29—H29A 109.5 C5—C6—C1 119.87 (14) C27—C29—H29B 109.5 C8—C7—C2 111.26 (13) H29A—C29—H29B 109.5 C8—C7—C9 107.46 (14) C27—C29—H29C 109.5 C2—C7—C9 110.91 (13) H29A—C29—H29C 109.5 C8—C7—C10 107.55 (14) H29B—C29—H29C 109.5 C2—C7—C10 110.07 (13) C27—C30—H30A 109.5 C9—C7—C10 109.50 (14) C27—C30—H30B 109.5 C7—C8—H8A 109.5 H30A—C30—H30B 109.5 C7—C8—H8B 109.5 C27—C30—H30C 109.5 H8A—C8—H8B 109.5 H30A—C30—H30C 109.5 C7—C8—H8C 109.5 H30B—C30—H30C 109.5 H8A—C8—H8C 109.5 C32—C31—C33 107.50 (15) H8B—C8—H8C 109.5 C32—C31—C25 111.81 (14) C7—C9—H9A 109.5 C33—C31—C25 109.75 (14) C7—C9—H9B 109.5 C32—C31—C34 107.25 (15) H9A—C9—H9B 109.5 C33—C31—C34 110.41 (14) C7—C9—H9C 109.5 C25—C31—C34 110.06 (14) H9A—C9—H9C 109.5 C31—C32—H32A 109.5 H9B—C9—H9C 109.5 C31—C32—H32B 109.5 C7—C10—H10A 109.5 H32A—C32—H32B 109.5 106.12 (8) C23—C22—C21 122.24 (15) 98.09 (9) C23—C22—H22 118.9 132.84 (10) C21—C22—H22 118.9 128.42 (10) C22—C23—C24 116.77 (15) 138.77 (7) C22—C23—C27 121.18 (15) 122.47 (14) C24—C23—C27 122.00 (14) 125.65 (11) C25—C24—C23 124.81 (15) 111.62 (10) C25—C24—H24 117.6 122.86 (14) C23—C24—H24 117.6 123.57 (11) C24—C25—C26 118.56 (15) 111.52 (10) C24—C25—C31 121.73 (14) 123.11 (14) C26—C25—C31 119.71 (14) 119.20 (14) O2—C26—C25 119.29 (14) 117.68 (14) O2—C26—C21 123.08 (14) 118.19 (14) C25—C26—C21 117.63 (14) 121.84 (14) C28—C27—C29 109.03 (16) 119.93 (14) C28—C27—C23 110.91 (15) 124.96 (15) C29—C27—C23 112.83 (14) 117.5 C28—C27—C30 108.11 (16) 117.5 C29—C27—C30 107.01 (15) 116.58 (14) C23—C27—C30 108.77 (14) 124.38 (14) C27—C28—H28A 109.5 119.00 (14) C27—C28—H28B 109.5 122.44 (15) H28A—C28—H28B 109.5 118.8 C27—C28—H28C 109.5 118.8 H28A—C28—H28C 109.5 116.26 (14) H28B—C28—H28C 109.5 123.81 (14) C27—C29—H29A 109.5 119.87 (14) C27—C29—H29B 109.5 111.26 (13) H29A—C29—H29B 109.5 107.46 (14) C27—C29—H29C 109.5 110.91 (13) H29A—C29—H29C 109.5 107.55 (14) H29B—C29—H29C 109.5 110.07 (13) C27—C30—H30A 109.5 109.50 (14) C27—C30—H30B 109.5 109.5 H30A—C30—H30B 109.5 109.5 C27—C30—H30C 109.5 109.5 H30A—C30—H30C 109.5 109.5 H30B—C30—H30C 109.5 109.5 C32—C31—C33 107.50 (15) 109.5 C32—C31—C25 111.81 (14) 109.5 C33—C31—C25 109.75 (14) 109.5 C32—C31—C34 107.25 (15) 109.5 C33—C31—C34 110.41 (14) 109.5 C25—C31—C34 110.06 (14) 109.5 C31—C32—H32A 109.5 109.5 C31—C32—H32B 109.5 109.5 H32A—C32—H32B 109.5 sup-8 Acta Cryst. supporting information (2011). E67, m314–m315 supporting information supporting information (2011). E67, m314–m315 supporting information C7—C10—H10B 109.5 C31—C32—H32C 109.5 H10A—C10—H10B 109.5 H32A—C32—H32C 109.5 C7—C10—H10C 109.5 H32B—C32—H32C 109.5 H10A—C10—H10C 109.5 C31—C33—H33A 109.5 H10B—C10—H10C 109.5 C31—C33—H33B 109.5 C13—C11—C4 111.82 (13) H33A—C33—H33B 109.5 C13—C11—C12 108.83 (14) C31—C33—H33C 109.5 C4—C11—C12 109.44 (13) H33A—C33—H33C 109.5 C13—C11—C14 107.92 (14) H33B—C33—H33C 109.5 C4—C11—C14 109.64 (13) C31—C34—H34A 109.5 C12—C11—C14 109.13 (14) C31—C34—H34B 109.5 C11—C12—H12A 109.5 H34A—C34—H34B 109.5 C11—C12—H12B 109.5 C31—C34—H34C 109.5 H12A—C12—H12B 109.5 H34A—C34—H34C 109.5 C11—C12—H12C 109.5 H34B—C34—H34C 109.5 H12A—C12—H12C 109.5 S1—C35—H35A 109.5 H12B—C12—H12C 109.5 S1—C35—H35B 109.5 C11—C13—H13A 109.5 H35A—C35—H35B 109.5 C11—C13—H13B 109.5 S1—C35—H35C 109.5 H13A—C13—H13B 109.5 H35A—C35—H35C 109.5 C11—C13—H13C 109.5 H35B—C35—H35C 109.5 H13A—C13—H13C 109.5 S1—C36—H36A 109.5 H13B—C13—H13C 109.5 S1—C36—H36B 109.5 C11—C14—H14A 109.5 H36A—C36—H36B 109.5 C11—C14—H14B 109.5 S1—C36—H36C 109.5 H14A—C14—H14B 109.5 H36A—C36—H36C 109.5 C11—C14—H14C 109.5 H36B—C36—H36C 109.5 H14A—C14—H14C 109.5 N5—C37—C38 179.9 (3) H14B—C14—H14C 109.5 C37—C38—H38A 109.5 N1—C15—C6 125.57 (15) C37—C38—H38B 109.5 N1—C15—H15 117.2 H38A—C38—H38B 109.5 C6—C15—H15 117.2 C37—C38—H38C 109.5 C18—C16—N1 117.67 (14) H38A—C38—H38C 109.5 C18—C16—C17 121.36 (14) H38B—C38—H38C 109.5 N1—C16—C17 120.90 (14) O2—Zn—O1—C1 150.82 (15) C3—C4—C11—C12 −69.76 (19) O3—Zn—O1—C1 −101.74 (15) C5—C4—C11—C14 −127.48 (17) N1—Zn—O1—C1 −0.04 (15) C3—C4—C11—C14 49.91 (19) N2—Zn—O1—C1 49.2 (2) C16—N1—C15—C6 −178.57 (15) O1—Zn—O2—C26 166.41 (14) Zn—N1—C15—C6 7.9 (2) O3—Zn—O2—C26 54.13 (14) C5—C6—C15—N1 177.20 (15) N1—Zn—O2—C26 −94.02 (16) C1—C6—C15—N1 0.0 (3) N2—Zn—O2—C26 −33.31 (14) C15—N1—C16—C18 173.74 (15) C35—S1—O3—Zn −60.18 (14) Zn—N1—C16—C18 −11.89 (17) C36—S1—O3—Zn 43.54 (14) C15—N1—C16—C17 −9.4 (2) O2—Zn—O3—S1 119.64 (12) Zn—N1—C16—C17 164.96 (12) O1—Zn—O3—S1 16.51 (13) C18—C16—C17—N3 142 (2) sup-9 Acta Cryst. (2011). E67, m314–m315 supporting information supporting information −75.78 (12) N1—C16—C17—N3 −34 (3) −153.85 (12) N1—C16—C18—N2 −1.0 (2) −108.97 (15) C17—C16—C18—N2 −177.83 (14) −7.03 (13) N1—C16—C18—C19 176.23 (14) 102.59 (13) C17—C16—C18—C19 −0.6 (2) −171.63 (14) C20—N2—C18—C16 177.49 (14) 76.88 (14) Zn—N2—C18—C16 13.30 (17) 178.82 (11) C20—N2—C18—C19 0.2 (2) −71.56 (11) Zn—N2—C18—C19 −163.95 (12) 14.22 (10) C16—C18—C19—N4 −144 (2) 27.25 (13) N2—C18—C19—N4 33 (2) 130.72 (15) C18—N2—C20—C21 −178.19 (15) −76.57 (13) Zn—N2—C20—C21 −15.9 (2) −178.78 (13) N2—C20—C21—C22 177.32 (15) −168.69 (11) N2—C20—C21—C26 −2.7 (3) −65.23 (19) C20—C21—C22—C23 −178.94 (15) 87.48 (11) C26—C21—C22—C23 1.0 (2) −14.73 (10) C21—C22—C23—C24 −0.9 (2) 6.6 (2) C21—C22—C23—C27 176.69 (15) −173.09 (11) C22—C23—C24—C25 0.5 (2) −174.06 (15) C27—C23—C24—C25 −176.99 (16) 6.2 (2) C23—C24—C25—C26 −0.4 (3) 8.2 (2) C23—C24—C25—C31 179.57 (15) −171.50 (14) Zn—O2—C26—C25 −153.79 (12) −3.7 (2) Zn—O2—C26—C21 26.7 (2) 173.99 (15) C24—C25—C26—O2 −179.06 (15) −0.4 (2) C31—C25—C26—O2 1.0 (2) −177.95 (15) C24—C25—C26—C21 0.5 (2) 1.7 (2) C31—C25—C26—C21 −179.45 (14) 179.12 (15) C20—C21—C26—O2 −1.3 (2) −176.22 (15) C22—C21—C26—O2 178.71 (15) 1.1 (2) C20—C21—C26—C25 179.17 (15) −7.7 (3) C22—C21—C26—C25 −0.8 (2) 172.05 (15) C22—C23—C27—C28 50.7 (2) 175.20 (15) C24—C23—C27—C28 −131.85 (18) −5.1 (2) C22—C23—C27—C29 173.39 (16) −2.6 (2) C24—C23—C27—C29 −9.2 (2) 175.02 (15) C22—C23—C27—C30 −68.0 (2) 116.96 (17) C24—C23—C27—C30 109.38 (18) −65.43 (19) C24—C25—C31—C32 −1.6 (2) −121.71 (17) C26—C25—C31—C32 178.31 (16) 55.90 (19) C24—C25—C31—C33 117.58 (17) −7.8 (2) C26—C25—C31—C33 −62.5 (2) 169.57 (15) C24—C25—C31—C34 −120.71 (17) 112.85 (18) C26—C25—C31—C34 59.2 (2) sup-10 Acta Cryst. (2011). E67, m314–m315
https://openalex.org/W3133958590
https://jds-online.org/journal/JDS/article/165/file/pdf
English
null
Comparison of estimation methods for unit-Gamma distribution
Journal of data science
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cc-by
13,981
Sanku Dey1, Andre F. B. Menezes2 and Josmar Mazucheli2 1Department of Statistics, St. Anthony's College, Shillong, Meghalaya, India 2Department of Statistics, Universidade Estadual de Maring Maringa, PR, Brazil Sanku Dey1, Andre F. B. Menezes2 and Josmar Mazucheli2 1Department of Statistics, St. Anthony's College, Shillong, Meghalaya, India 2Department of Statistics, Universidade Estadual de Maring Maringa, PR, Brazil Journal of Data Science,17(4). P. 768–801,2019 DOI:10.6339/JDS.201910_17(4).0009 Journal of Data Science,17(4). P. 768–801,2019 DOI:10.6339/JDS.201910_17(4).0009 Abstract In this study we have considered different methods of estimation of the unknown parameters of a two-parameter unit-Gamma (UG) distribution from the frequentists point of view. First, we briefly describe different frequentists approaches: maximum likelihood estimators, moments estimators, least squares estimators, maximum product of spacings estimators, method of Cramer-von-Mises, methods of Anderson- Darling and four variants of Anderson-Darling test and compare them using extensive numerical simulations. Monte Carlo simulations are performed to compare the performances of the proposed methods of estimation for both small and large samples. The performances of the estimators have been compared in terms of their bias and root mean squared error using simulated samples. Also, for each method of estimation, we consider the interval estimation using the bootstrap method and calculate the coverage probability and the average width of the bootstrap confidence intervals. The study reveals that the maximum product of spacing estimators and Anderson-Darling 2 (AD2) estimators are highly competitive with the maximum likelihood estimators in small and large samples. Finally, two real data sets have been analyzed for illustrative purposes. Keywords: Unit-Gamma distribution, Monte Carlo simulations, Estimation methods, Parametric bootstrap methods. Keywords: Unit-Gamma distribution, Monte Carlo simulations, Estimation methods, Parametric bootstrap methods. Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 769 1 Introduction Grassia (1977) introduced a new probability distribution which was later called by Ratnaparkhl and Mosimann (1990) as unit-Gamma (UG) distribution, since its support is on the unit interval (0, 1). A random variable X follows unit-Gamma distribution if its probability density function is given by: 1 1 , log ) ( ) ( | ) ( f x x x            (1) (1) where 1 0 ( ) u u e u u d       is the complete gamma function, 0  and 0  are the shape parameters. Its corresponding cumulative distribution function (c.d.f.) is written as: 𝐹(𝑥|𝛼, 𝛽) = 𝐹𝑦(−log(𝑥)|𝛼, 𝛽) = 𝛾(𝛼,𝛽(−log 𝑥)) 𝛤(𝛼) (2) (2) where 𝐹𝑦(⋅) denotes the c.d.f. of Gamma distribution with shape (𝛼> 0) and scale (𝛽> 0)parameters and 𝛾(⋅,⋅) is the lower incomplete gamma function, define as 𝛾(a, x) = ∫ta−1 x 0 e−tdt. Comparison of estimation methods for unit-Gamma distribution 770 Figure 1: The unit-Gamma probability density function with different values of α and β. Figure 1: The unit-Gamma probability density function with different values of α and β. Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 771 Figure 2: The unit-Gamma hazard rate function with di erent values of and . Figure 2: The unit-Gamma hazard rate function with di erent values of and . The p.d.f (1) can have increasing, decreasing, constant and unimodal shapes, and the hazard rate function exhibits decreasing and bathtub shapes. Grassia (1977) gave a detailed account of UG distribution and its variants. Ratnaparkhl and Mosimann (1990) used this distribution for deriving some new distributions taking UG as a conditional distribution. Although, UG distribution has not been studied widely, but possesses some properties similar to that of the beta distribution. The applicability of the UG distribution has been found in areas like estimation of bacteria or virus density in dilution assays with host variability to infection using inoculation approach and for deriving other statistical distributions (see Grassia, 1977; Ratnaparkhl and Mosimann, 1990). Tadikamalla (1981) in his discussion paper pointed out that this distribution can be used as an alternative for Beta and Johnson SB distributions. He also investigated some of its properties. Ratnaparkhl and Mosimann (1990) studied the logarithmic and Tukey's lambda-type transformation on the unit-Gamma distribution. More recently, Mousa et al. 1 Introduction (2016) formulated the UG regression model while Mazucheli et al. (2018) derived second order bias corrections for the parameters Comparison of estimation methods for unit-Gamma distribution 772 of UG distribution. Ho et al. (2019) considered the UG distribution to construct control charts to monitor rates and proportions. It is worth mentioning here that in studying real life situations we may come across distributions with bounded support such as percentages, proportions or fractions (see, Marshall and Olkin (2007)). In this respect, Papke and Wooldridge (1996) observed that variables bounded between zero and one arise naturally in many economic setting such as the fraction of total weekly hours spent on working, the proportion of income spent on non-durable consumption, pension plan participation rates, industry market shares, television rating, fraction of land area allocate to agriculture, etc. Various examples of proportions in the unit interval used in empirical finance are also discussed in Cook et al. (2008). Furthermore, when the reliability is measured as percentage or ratio, it is important to have models de ned on the unit interval (see, Genc (2013)) in order to have plausible results. Parameter estimation is vital in the study of any probability distribution. Maximum likeli-hood estimation (MLE) is generally a starting point when it comes to estimating the parameters of any distribution due to its attractive properties. For example, they are asymptotically unbiased, consistent, and asymptotically normally distributed (Lehmann, 1999). However, there are other estimation methods developed over time for other distributions (see Gupta and Kundu (2001) for generalized Exponential distribution, Kundu and Raqab (2005) for generalized Rayleigh distributions, Teimouri et al. (2013) for Weibull distribution, Mazucheli et al. (2013) for weighted Lindley distribution, do Espirito Santo and Mazucheli (2015) for Marshall-Olkin extended Lindley distribution, Dey et al. (2015) for weighted Exponential distribution, Mazucheli et al. (2016) for Marshall-Olkin extended Exponential distribution and Dey et al. (2018) for Kumaraswamy distribution) which are based on different methodologies, such as method of moments estimation (MOM), method of L-moments estimation (LM), method of probability weighted moment estimation (PWM), method of least-squares estimation (LSE), method of weighted least-square estimation (WLSE), method of maximum product spacing estimation (MPS) and method of minimum distance estimation. Mazucheli and Menezes (2019) investigated the parameter estimation for the complementary Beta distribution considering the L-moments and maximum likelihood methods. 2 Estimation Methods In this section, we describe seven estimation methods along with four variants of AD test for estimating the parameters, 𝛼 and 𝛽, that index the unit-Gamma distribution. For all the methods of estimation, we assume that x = (x1, … , xn)T is a random sample of size n from unit-Gamma distribution, (1), with unknown parameters 𝛼 and 𝛽. Besides, consider that x(1) < ⋯< x(n) denote the corresponding order samples. 1 Introduction Almetwally and Almongy (2019) used the maximum likelihood and maximum product spacing methods for estimating the parameters of generalized power Weibull distribution. In this paper, we provide a comprehensive comparison of different methods of estimation for the unknown parameters for unit-Gamma distribution and to study the behaviour of these estimators for different sample sizes and for di erent parameter Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 773 values. We mainly compare: the maximum likelihood estimators, maximum product of spacings estimators, moments estimators, least-squares estimators, weighted least- squares estimators, Cramer-von-Mises estimators and Anderson-Darling estimators and four of its variants. Since, it is difficult to compare theoretically the performances of the different methods of estimation, we perform extensive simulations to compare the performances of the different estimators based on bias and root mean squared error. Also, for each method of estimation, we consider the interval estimation using the bootstrap con dence interval (Efron, 1982a) and calculate the coverage probability and the average width of the con dence interval. The originality of this study comes from the fact that there has been no previous work comparing all of these estimation methods for the unit-Gamma distribution. The final motivation of the paper is to show how different aforementioned frequentist estimators of this distribution perform for different sample sizes and different parameter values and to develop a guideline for choosing the best estimation method for the unit-Gamma distribution, which we think would be of interest to applied statisticians. The remaining part of the paper is organized as follows: In Section 2 we discuss the eleven estimation methods considered in this paper. The comparison of these methods in terms of bias, root mean-squared error, coverage probability and average width is presented in Section 3. The eleven estimation methods are used for fitting two real data sets in Section 4. Some concluding remarks are presented in Section 5. 2.1 Method of Maximum Likelihood The method of maximum likelihood (MLE) is the most popular estimation method in statistical inference, since its underlying motivation is simple and intuitive. Furthermore, the MLE enjoys several attractive properties (see, e.g, Lehmann and Casella, 1998; Pawitan, 2001; Rohde, 2014). For the unit-Gamma distribution, the log-likelihood function, apart from constant term, can be expressed as: l(𝛼, 𝛽|𝑥) ∝𝑛𝛼log𝛽−𝑛log𝛤(𝛼) + 𝛽∑ 𝑥𝑖 𝑛 𝑖=1 + 𝛼∑ log(−log𝑥𝑖) 𝑛 𝑖=1 (3) (3) Comparison of estimation methods for unit-Gamma distribution 774 The maximum likelihood estimators ˆMLE  and ˆ MLE  , of the parameters  and , respectively, can be obtained by maximizing (3), or equivalently solving the following nonlinear equations:   1 1 ( , | ) log ( ) log log ( , | ) log n i i n i i n n x n x                       x x where ( )  denotes the digamma function, define as ( ) log ( ) d x x dx    where ( )  denotes the digamma function, define as ( ) log ( ) d x x dx    2.2 Method of Maximum Product of Spacings The maximum product of spacing (MPS) method was introduced by Cheng and Amin (1979, 1983) as an alternative to MLE for estimating parameters of continuous univariate distributions. Ranneby (1984) independently derived the same method as an approximation to the Kullback-Leibler measure of information. The uniform spacing of a random sample from unit-Gamma distribution is defined as: as:     : 1: ( , ) | , | , i i n i n D F x F x       for   0: 1, , , | , 0 n i n F x    and   1: | , 1 n n F x   .Clearly 1 1 ( , ) 1 n i i D      .     : 1: ( , ) | , | , i i n i n D F x F x       for   0: 1, , , | , 0 n i n F x    and From Cheng and Amin (1979, 1983), the MPSEs, ˆMPS  and ˆ MPS  , are the values of From Cheng and Amin (1979, 1983), the MPSEs, ˆMPS  and ˆ MPS  , are the values of and , which maximize the geometric mean of the spacing: and , which maximize the geometric mean of the spacing: 1 1 1 1 ( , | ) ( , ) n n i i G D             x (6) 1 1 1 ( , | ) log ( , ) 1 n i i H D n       x (7) (6) (7) The estimators ˆMPS  and ˆ MPS  of the parameters  and  can also be obtained by solving the nonlinear equations: Sanku Dey, Andre F. B. 2.2 Method of Maximum Product of Spacings Menezes and Josmar Mazucheli 775         1 1 : 1 1: 1 1 2 : 2 1: 1 1 1 ( , ) | , | , 0 1 ( , ) 1 1 ( , ) | , | , 0 1 ( , ) n i n i n i i n i n i n i i H x x n D H x x n D                                         where     1 : : | , | , i n i n x F x       (8) (8) And     2 : : | , | , i n i n x F x       (9) (9) which must be obtained numerically, ) ( F is defined in Equation (2). It is noteworthy that the MPSE is as efficient as ML estimation and consistent under more general conditions than the ML estimators (Cheng and Amin, 1983) 2.3Method of Moments Another technique fairly simple and commonly used in the parametric estimation is the method of moments (MOM). Grassia (1977) showed that the moment of order r about the origin of (1)is given by:   r r X r            (10) The moment estimators can be obtained by equating the first two moments (10) of unit-Gamma distribution to their counterparts sample moments, that is, 1 1 2 2 1 2 m m                          where 1 1 2 1 2 1 1 and n n i i i i m n x m n x         Comparison of estimation methods for unit-Gamma distribution 776 2.4 Methods of Least Squares The least square methods were originally proposed by Swain et al. (1988) to estimate the parameters of the Beta distributions. Suppose that ( ) ( ) i F X denotes the distribution function of the order statistics from the random sample 1 2 ( , ,... ) n x x x x  . An important result from probability shows that ( ) ( ) ~ ( , 1) i F X Beta i n i  . Therefore, we have     ( ) ( ) 2 ( 1) and Var 1 ( 1) ( 2) i i i i n i F X F X n n n               (11) (11) (11) for further details see Johnson et al. (1995). Using the expectations and variances, we obtain two variants of the least squares methods. 2.4.1 Ordinary Least Squares In case of unit-Gamma distribution, the ordinary least square estimators ˆOLS  and In case of unit-Gamma distribution, the ordinary least square estimators ˆOLS  and ˆ OLS  of the parameters  and  can be obtained by minimizing the function: In case of unit-Gamma distribution, the ordinary least square estimators ˆOLS  and ˆ OLS  of the parameters  and  can be obtained by minimizing the function: ˆ OLS  of the parameters  and  can be obtained by minimizing the function:   2 : 1 ( , | ) | , 1 n i n i i S F x n              x (12) (12) with respect toand . Alternatively, these estimates can also be obtained by solving the following nonlinear equations:         : 1 : 1 : 2 : 1 | , | , 0 1 | , | , 0 1 n i n i n i n i n i n i i F x x n i F x x n                             2.4.2 Weighted Least Squares 2.4.2 Weighted Least Squares For the unit-Gamma distribution, the weighted least square estimators of and . say ˆWLS  and ˆ WLS  , respectively are obtained by minimizing the function: Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 777 777   2 2 : 1 ( 1) ( 2) ( , | ) | , ( 1) 1 n i n i n n i W F x i n i n                 x (13) (13) with respect to and . Equivalently, these estimators are the solution of the following nonlinear equations:         2 : 1 : 1 2 : 2 : 1 ( 1) ( 2) | , | , 0 ( 1) 1 ( 1) ( 2) | , | , 0 ( 1) 1 n i n i n i n i n i n i n n i F x x i n i n n n i F x x i n i n                                   where 1( | , )   and 2( | , )   are defined in Equations (8) and (9), respectively. 2.5 Methods of Minimum Distances Here, we will discuss some methods based on the test statistics of Cramer-von Mises, Anderson-distance between the theoretical and empirical cumulative distribution functions (see for further details e.g., D'Agostino and Stephens, 1986; Luce~no, 2006). The expressions for each method are presented in Table 1. 2.5 Methods of Minimum Distances 2.5.1 Method of Cramer-von-Mises 2.5.1 Method of Cramer-von-Mises In regard to unit-Gamma distribution, the Cramer-von- Mises estimates CvM  and CvM  are obtained by minimizing with respect to  and the function:   2 : 1 1 2 1 ( , | ) | , 12 2 n i n i i C F x n n               x (14) (14) The estimators can also be obtained by solving the following nonlinear equations: The estimators can also be obtained by solving the following nonlinear equations:         : 1 : 1 : 2 : 1 2 1 | , | , 0 2 2 1 | , | , 0 2 n i n i n i n i n i n i i F x x n i F x x n                             where 1( | , )   and 2( | , )   are speci ed in Equations (8) and (9), respectively. 2.5.2 Method of Anderson-Darling 2.5 Methods of Minimum Distances Table 1: Expression for the methods based on the minimum distances Acronym s Expressions CvM 2 2 : 1 1 2 1 12 2 n n i n i i W x n n             AD   2 : ( 1 ) 1 1 (2 1) log log 1 n n i n n i i A n i x x n            ADR   2 : ( 1 ) 1 1 1 2 (2 1)log 1 2 m N n i n n i i i n R x i x n           ADR2   2 : 1 1 ( 1 ) 1 2 1 2 log 1 1 n n n i n i i n i i r x n x           AD2L 2 : 1 1 : 1 2 1 2 log n n n i n i i i n i l x n x        Table 1: Expression for the methods based on the minimum distances Acronym s Expressions CvM 2 2 : 1 1 2 1 12 2 n n i n i i W x n n             AD   2 : ( 1 ) 1 1 (2 1) log log 1 n n i n n i i A n i x x n            ADR   2 : ( 1 ) 1 1 1 2 (2 1)log 1 2 m N n i n n i i i n R x i x n           ADR2   2 : 1 1 ( 1 ) 1 2 1 2 log 1 1 n n n i n i i n i i r x n x           AD2L 2 : 1 1 : 1 2 1 2 log n n n i n i i i n i l x n x        Table 1: Expression for the methods based on the minimum distances Comparison of estimation methods for unit-Gamma distribution 778 AD2   2 : : 1 1 : ( 1 ) 1 2 1 2 1 2 log log 1 1 n n n i n i n i i i n n i i i a x x n x x                          For illustrative purposes, we have presented only the expressions used for the estimation of the parameters for the Cramer-von-Mises and Anderson-Darling methods. 2.5.2 Method of Anderson-Darling Anderson and Darling (1952) developed a test, as an alternative to statistical tests for detecting sample distributions departure from normality. Using these test statistics, we can obtain the Anderson-Darling estimates, ADE  and ADE  , by minimizing the function       : ( 1 ) 1 1 ( , | ) (2 1) log | , log | , n i n n i i A n i F x F x n          x (15) (15) (15) Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 779 with respect to and . Equivalently, these estimators are the solution of the following nonlinear equations:                 1 ( 1 ) 1 : 1 : ( 1 ) 2 ( 1 ) 2 : 1 : ( 1 ) | , | , (2 1) 0 | , | , | , | , (2 1) 0 | , | , n n i i n i i n n i n n i i n i i n n i x x i F x F x x x i F x F x                                           where 1( | , )   and 2( | , )   are specified in Equations (8) and (9), respectively. 2. The bias of ˆdecreases with increasing n for all the methods of estimation. 3 Monte Carlo Simulations In this section, we conduct Monte Carlo simulation studies to compare the performance of the estimators discussed in the previous sections. We evaluate the performance of the estimators based on bias and root mean squared errors (RMSE), for different sample sizes and parameter values. Moreover, we also calculate the parametric bootstrap confidence intervals for each method and evaluate the coverage probability (CP) and the average length (AW) of the simulated confidence intervals. We have taken sample sizes of n = 20; 50; 100 and 200 and the following parameter values: = 0:5; 1:0 and 2:0 and = 0:5; 1:0; 2:0 and 3:0. For each scenario, the number of Monte Carlo simulations is set at 10,000 and the parametric bootstrap replications is fixed at 1000. To generates random samples from the UG distribution, we consider the transformation Y X e  , where ~ Gamma( , ) Y . Simulated bias, RMSE, CP and AW for the estimates are presented in Tables 2{13. Asuperscript indicate the rank of each of the estimators among all the estimators for that metric. For example, Table 2 shows the bias of MLE( ˆ) as 0:1259 for n = 20. This indicates, bias of ˆ obtained using the method of maximum likelihood ranks 9th among all other estimators. Table14 shows the partial and overall rank of the estimators. The Table 14 is used to find the over all performance of estimation techniques. The following observations can be drawn from the Tables 2-13. 1. All the estimators show the property of consistency i.e., the RMSE decreases as sample size increases. 2. The bias of ˆdecreases with increasing n for all the methods of estimation. 2. The bias of ˆdecreases with increasing n for all the methods of estimation. Comparison of estimation methods for unit-Gamma distribution 780 3. The bias of ˆdecreases with increasing n for all the methods of estimation. 3. The bias of ˆdecreases with increasing n for all the methods of estimation. 4. The bias of ˆgenerally increases with increasing  for any given and n and for all methods of estimation ˆ. 5. In terms of RMSE, all the methods of estimation produces smaller RMSE or ˆ compared to that of . 5. In terms of RMSE, all the methods of estimation produces smaller RMSE or ˆ compared to that of . 6. 3 Monte Carlo Simulations In terms of performance of the methods of estimation, we found that maximum product spacing (MPS) estimators is the best as it produces the least biases of the estimates with least RMSE for most of the configurations considered in our studies. The next best method is the AD2, followed by MLE. AD method ranked 4th while WLSE ranked 5th.AD2L ranked 11th among the eleven methods of estimation. The overall positions of the estimators are presented in Table 14, from which we confirm the superiority of MPS and AD2. Table 2: Simulation results for = 0.5 and = 0.5. n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0584 -0.0081 0.24611 0.1118 0.0976 0.15810 0.1259 0.1077 -0.0705 0.0362 0.0463 RMSE( ) 0.1285 0.0121 0.2989 0.51911 0.2888 0.33210 0.2497 0.2166 -0.1033 0.0952 0.1094 Bias( ) 0.3363 0.3022 0.81811 0.49810 0.3946 0.4919 0.3604 0.4168 0.2771 0.3947 0.3775 20 RMSE( ) 0.5913 0.5062 0.95310 1.66511 0.9449 0.9418 0.6444 0.7237 0.4531 0.7236 0.6845 CP( ) 0.9378 0.9429 0.8913 0.9337 0.9215 0.8902 0.8841 0.9216 0.9024 0.94511 0.94410 CP( ) 0.9408 0.95511 0.8772 0.9277 0.9135 0.8843 0.8751 0.9156 0.9044 0.95310 0.9479 AW( ) 0.6703 0.5772 1.60511 0.9699 0.7967 0.99210 0.7405 0.8508 0.4741 0.7456 0.7134 AW ( ) 1.1863 0.9742 1.9028 3.16911 1.9149 1.92010 1.3395 1.4957 0.7611 1.3626 1.2944 Total 374 302 6510 7411 557 629 363 557 201 506 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0204 -0.0325 0.05611 0.0142 0.0336 0.05010 0.0458 0.0357 -0.0509 0.0081 0.0173 RMSE( ) 0.0484 -0.0412 0.0615 0.12911 0.1009 0.11110 0.0918 0.0766 -0.0777 0.0301 0.0443 Bias( ) 0.1864 0.1812 0.32611 0.25410 0.2046 0.2249 0.1863 0.2238 0.1681 0.2067 0.1925 50 RMSE( ) 0.3134 0.2822 0.3868 0.71511 0.43310 0.4109 0.3083 0.3697 0.2651 0.3666 0.3345 CP( ) 0.9488 0.9182 0.94910 0.9395 0.9416 0.9344 0.9283 0.9447 0.8901 0.9499 0.95011 CP( ) 0.9488 0.9324 0.9509 0.95411 0.9355 0.9193 0.9172 0.9366 0.8951 0.95010 0.9477 AW( ) 0.3714 0.3382 0.66311 0.49810 0.4076 0.4549 0.3713 0.4478 0.2971 0.4087 0.3835 AW ( ) 0.6204 0.5322 0.7948 1.38111 0.84810 0.8069 0.6193 0.7307 0.4641 0.7026 0.6525 Total 404 211 7311 7110 588 639 333 567 222 476 445 Table 2: Simulation results for = 0.5 and = 0.5. Sanku Dey, Andre F. B. 3 Monte Carlo Simulations Menezes and Josmar Mazucheli 781 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0094 -0.03210 0.0083 -0.0082 0.0156 0.0239 0.0218 0.0167 -0.03411 0.0021 0.0095 RMSE( ) 0.0203 -0.0479 0.0041 0.0295 0.0458 0.04910 0.0427 0.0336 -0.05611 0.0112 0.0204 Bias( ) 0.1283 0.1315 0.21211 0.17410 0.1376 0.1498 0.1242 0.1539 0.1191 0.1437 0.1314 100 RMSE( ) 0.2114 0.2063 0.2589 0.44111 0.27010 0.2578 0.2012 0.2467 0.1891 0.2436 0.2185 CP( ) 0.9478 0.9001 0.95211 0.9323 0.9447 0.9394 0.9425 0.9446 0.9022 0.94810 0.9489 CP( ) 0.9448 0.8961 0.95311 0.9395 0.9395 0.9344 0.9253 0.9417 0.8972 0.94710 0.9449 AW( ) 0.2514 0.2432 0.42711 0.33910 0.2706 0.2928 0.2443 0.2999 0.2151 0.2777 0.2575 AW ( ) 0.4114 0.3742 0.5149 0.85311 0.53110 0.4998 0.3953 0.4777 0.3331 0.4666 0.4245 Total 384 332 6611 577 588 5910 332 588 301 496 465 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0042 -0.02711 -0.0118 -0.0149 0.0074 0.0117 0.0106 0.0085 -0.02210 0.0011 0.0043 RMSE( ) 0.0092 -0.04211 -0.0176 -0.0124 0.0218 0.0239 0.0207 0.0165 -0.03610 0.0041 0.0093 Bias( ) 0.0893 0.0976 0.15311 0.12510 0.0945 0.1008 0.0852 0.1059 0.0841 0.0987 0.0894 200 RMSE( ) 0.1423 0.1495 0.18610 0.30411 0.1789 0.1668 0.1342 0.1637 0.1311 0.1616 0.1444 CP( ) 0.95011 0.8891 0.9414 0.9263 0.9456 0.9425 0.9467 0.9467 0.9132 0.9479 0.94910 CP( ) 0.95410 0.8901 0.9384 0.9323 0.9518 0.9466 0.9455 0.9497 0.9062 0.95511 0.9539 AW( ) 0.1743 0.1785 0.30211 0.24310 0.1856 0.1988 0.1662 0.2079 0.1551 0.1937 0.1764 AW ( ) 0.2824 0.2733 0.36310 0.58811 0.3559 0.3318 0.2652 0.3267 0.2401 0.3206 0.2875 Total 383 435 6411 6110 557 599 332 568 281 486 424 Overall Total 154 102 4311 3810 307 379 102 307 51 246 195 Table 4: Simulation results for = 2.0 and = 0.5. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0704 -0.0111 0.20910 0.1407 0.1166 0.1959 0.1668 0.25111 -0.0875 0.0242 0.0363 RMSE( ) 0.0854 -0.0141 0.2068 0.2299 0.1656 0.23310 0.1917 0.37611 -0.1035 0.0302 0.0463 Bias( ) 0.3883 0.3442 0.64910 0.5579 0.4617 0.5458 0.4296 0.67311 0.3201 0.4245 0.4024 20 RMSE( ) 0.4503 0.3952 0.6509 0.79910 0.5947 0.6428 0.4926 0.96711 0.3641 0.4905 0.4694 CP( ) 0.9397 0.9418 0.9135 0.9449 0.9306 0.8934 0.8772 0.8671 0.8813 0.95611 0.95110 CP( ) 0.9357 0.9408 0.8965 0.9409 0.9216 0.8894 0.8762 0.8541 0.8833 0.95811 0.95110 AW( ) 2.9983 2.5632 4.73211 4.23210 3.5317 4.0429 3.3236 4.0018 2.0781 3.2175 3.1054 AW( ) 0.8663 0.7322 1.1678 1.56711 1.1407 1.1989 0.9616 1.51810 0.5901 0.9375 0.9024 Total 343 262 6610 7411 527 618 435 649 201 466 424 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0253 -0.0446 0.0698 0.0354 0.0445 0.07110 0.0607 0.07911 -0.0699 0.0031 0.0222 RMSE( ) 0.0303 -0.0534 0.0595 0.0647 0.0636 0.08810 0.0698 0.11411 -0.0799 0.0051 0.0262 Bias( ) 0.2234 0.2032 0.38611 0.3139 0.2447 0.2778 0.2193 0.34910 0.1871 0.2356 0.2275 50 RMSE( ) 0.2544 0.2332 0.3729 0.43810 0.3077 0.3218 0.2483 0.50611 0.2131 0.2716 0.2605 Comparison of estimation methods for unit-Gamma distribution 782 CP( ) 0.9417 0.9052 0.9448 0.95010 0.9396 0.9265 0.9164 0.9153 0.8711 0.95611 0.9489 Table 3: Simulation results for  = 1.0 and  = 0.5. 3 Monte Carlo Simulations B. Menezes and Josmar Mazucheli 783 CP( ) 0.9417 0.9052 0.9478 0.95110 0.9416 0.9195 0.9144 0.9073 0.8731 0.96111 0.9519 AW( ) 1.7073 1.5012 2.98211 2.48110 1.9497 2.1858 1.7124 2.3939 1.3051 1.9046 1.7835 AW( ) 0.4884 0.4262 0.7199 0.87710 0.6077 0.6338 0.4883 0.88211 0.3701 0.5486 0.5115 Total 35 3 221 699 7011 517 628 364 699 242 486 425 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0123 -0.04410 0.0135 0.0022 0.0247 0.0369 0.0318 0.0226 -0.04511 0.0021 0.0124 RMSE( ) 0.0124 -0.05211 0.0092 0.0103 0.0337 0.0459 0.0368 0.0296 -0.05110 0.0021 0.0145 Bias( ) 0.1483 0.1504 0.25811 0.21510 0.1657 0.1778 0.1432 0.2059 0.1341 0.1626 0.1515 100 RMSE( ) 0.1693 0.1724 0.2529 0.29710 0.2027 0.2068 0.1642 0.30511 0.1531 0.1876 0.1735 CP( ) 0.9418 0.8811 0.9459 0.9346 0.9417 0.9335 0.9324 0.9283 0.8842 0.95111 0.94610 CP( ) 0.94610 0.8701 0.9438 0.9386 0.9407 0.9315 0.9304 0.9273 0.8832 0.95211 0.9469 AW( ) 1.1534 1.0812 1.99311 1.65610 1.2836 1.3928 1.1143 1.5559 0.9501 1.2977 1.1855 AW( ) 0.3294 0.3072 0.4859 0.57911 0.3957 0.4028 0.3183 0.57210 0.2701 0.3736 0.3395 Total 394 353 6411 589 557 6010 342 578 291 496 485 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0074 -0.03511 -0.0115 -0.0137 0.0126 0.0189 0.0158 -0.0011 -0.02810 0.0022 0.0063 RMSE( ) 0.0074 -0.04211 -0.0125 -0.0156 0.0167 0.0229 0.0188 -0.0062 -0.03210 0.0021 0.0073 Bias( ) 0.1034 0.1116 0.18011 0.15510 0.1095 0.1209 0.0972 0.1188 0.0941 0.1167 0.1033 200 RMSE( ) 0.1184 0.1275 0.1759 0.21611 0.1347 0.1388 0.1102 0.18110 0.1071 0.1336 0.1173 CP( ) 0.9447 0.8711 0.9364 0.9233 0.94810 0.9416 0.9395 0.9489 0.9052 0.9478 0.94811 CP( ) 0.9478 0.8631 0.9355 0.9223 0.94811 0.9406 0.9406 0.9314 0.9052 0.9479 0.94810 AW( ) 0.7993 0.8004 1.41411 1.16910 0.8736 0.9328 0.7552 1.0049 0.6901 0.9017 0.8105 AW( ) 0.2284 0.2273 0.3449 0.40911 0.2677 0.2688 0.2152 0.37810 0.1961 0.2596 0.2315 Total 383 424 598 6110 598 6311 352 537 281 466 435 Overall Total 133 102 3810 4111 297 379 133 338 51 246 195 Table 5: Simulation results for  = 0.5 and  = 1.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS 20 Bias( ) 0.0694 -0.0001 0.25211 0.1589 0.1206 0.18710 0.1568 0.1417 -0.0795 0.0332 0.0403 RMSE( ) 0.0984 0.0071 0.26110 0.35711 0.2066 0.2609 0.2198 0.2077 -0.1085 0.0512 0.0633 Bias( ) 0.3663 0.3302 0.79611 0.63210 0.4557 0.5579 0.4085 0.4698 0.2931 0.4196 0.4074 RMSE( ) 0.4973 0.4352 0.80910 1.24111 0.7048 0.7579 0.5515 0.6527 0.3811 0.5646 0.5454 CP( ) 0.9399 0.9398 0.8913 0.9257 0.9246 0.8912 0.8751 0.9085 0.8934 0.94811 0.94610 CP( ) 0.9388 0.9449 0.8712 0.9287 0.9206 0.8914 0.8701 0.9135 0.8843 0.95411 0.95310 AW( ) 1.4683 1.2472 3.14211 2.35110 1.8077 2.2279 1.6566 1.9778 1.0001 1.6325 1.5494 AW( ) 0.9743 0.8182 1.60710 2.31911 1.4248 1.5289 1.1106 1.3797 0.6391 1.0995 1.0444 Total 373 272 6810 7611 547 619 404 547 211 486 425 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0253 -0.0355 0.06410 0.0274 0.0396 0.06911 0.0558 0.0557 -0.0589 0.0091 0.0212 RMSE( ) 0.0323 -0.0454 0.0595 0.0777 0.0696 0.09411 0.0778 0.0789 -0.07910 0.0151 0.0292 Bias( ) 0.2053 0.1962 0.37111 0.28610 0.2246 0.2619 0.2094 0.2428 0.1821 0.2257 0.2155 50 RMSE( ) 0.2663 0.2502 0.38610 0.51411 0.3417 0.3448 0.2724 0.3459 0.2341 0.3036 0.2785 CP( ) 0.9428 0.9143 0.9459 0.94610 0.9386 0.9204 0.9132 0.9305 0.8781 0.95411 0.9427 CP( ) 0.9437 0.9164 0.9458 0.95110 0.9346 0.9163 0.9102 0.9315 0.8711 0.95411 0.9509 AW( ) 0.8053 0.7182 1.46611 1.13110 0.8997 1.0199 0.8064 0.9628 0.6271 0.8946 0.8365 AW( ) 0.5243 0.4602 0.75210 1.01511 0.6687 0.6798 0.5274 0.6899 0.3981 0.5926 0.5495 Total 333 241 7411 7310 517 639 364 608 252 496 405 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0124 -0.04011 0.0135 -0.0062 0.0216 0.0349 0.0288 0.0247 -0.03710 0.0041 0.0093 RMSE( ) 0.0145 -0.05110 0.0093 0.0041 0.0356 0.0449 0.0418 0.0387 -0.05211 0.0062 0.0144 Bias( ) 0.1383 0.1415 0.24211 0.19510 0.1526 0.1669 0.1352 0.1598 0.1271 0.1537 0.1394 100 RMSE( ) 0.1803 0.1814 0.25510 0.34011 0.2208 0.2197 0.1782 0.2329 0.1631 0.2056 0.1825 CP( ) 0.9478 0.8881 0.9499 0.9355 0.9447 0.9293 0.9344 0.9426 0.8962 0.94910 0.95111 CP( ) 0.9468 0.8861 0.94810 0.9396 0.9447 0.9334 0.9283 0.9385 0.8862 0.9468 0.95111 AW( ) 0.5444 0.5132 0.94511 0.74810 0.5936 0.6489 0.5263 0.6248 0.4541 0.6067 0.5555 AW( ) 0.3524 0.3282 0.49110 0.65111 0.4328 0.4277 0.3413 0.4529 0.2891 0.3996 0.3635 Total 394 363 6911 568 547 579 332 5910 291 475 486 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0083 -0.03411 -0.0094 -0.0179 0.0105 0.0158 0.0147 0.0116 -0.02410 0.0021 0.0052 RMSE( ) 0.0092 -0.04411 -0.0134 -0.0249 0.0155 0.0208 0.0207 0.0176 -0.03410 0.0021 0.0093 Bias( ) 0.0973 0.1056 0.17311 0.13910 0.1045 0.1109 0.0922 0.1108 0.0901 0.1087 0.0994 200 RMSE( ) 0.1253 0.1345 0.18110 0.24011 0.1498 0.1457 0.1202 0.1609 0.1151 0.1426 0.1284 Sanku Dey, Andre F. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0574 -0.0011 0.25511 0.0937 0.0906 0.15010 0.1288 0.1339 -0.0715 0.0262 0.0443 RMSE( ) 0.1245 0.0241 0.30810 0.39811 0.2648 0.3079 0.2477 0.2346 -0.1053 0.0842 0.1114 Bias( ) 0.3263 0.2992 0.77211 0.4479 0.3877 0.46510 0.3565 0.4328 0.2821 0.3766 0.3564 20 RMSE( ) 0.5843 0.5132 0.8678 1.25011 0.8889 0.90210 0.6344 0.7027 0.4411 0.6776 0.6595 CP( ) 0.9479 0.8741 0.9334 0.9233 0.94710 0.9407 0.9375 0.9396 0.9052 0.94911 0.9418 CP( ) 0.9469 0.8751 0.9334 0.9233 0.94710 0.9447 0.9355 0.9416 0.8982 0.9448 0.94711 AW( ) 0.3773 0.3774 0.66811 0.53110 0.4056 0.4349 0.3582 0.4258 0.3281 0.4217 0.3815 AW( ) 0.2444 0.2413 0.34810 0.46011 0.2918 0.2857 0.2302 0.3099 0.2101 0.2766 0.2485 Total 363 424 588 6611 577 6210 322 588 281 476 424 Overall Total 134 102 4010 4010 287 379 123 338 51 236 205 Comparison of estimation methods for unit-Gamma distribution 784 CP( ) 0.9387 0.9469 0.8912 0.94610 0.9286 0.9004 0.8901 0.9185 0.8963 0.94911 0.9468 CP( ) 0.9447 0.95410 0.8701 0.9458 0.9146 0.8873 0.8752 0.9045 0.9024 0.95611 0.9509 aw( ) 0.6643 0.5802 1.47811 0.8538 0.7587 0.93210 0.7366 0.8949 0.4731 0.7215 0.6994 AW( ) 2.3273 1.9542 3.43910 4.69811 3.4209 3.4168 2.6256 2.9497 1.5201 2.5995 2.5104 Total 35 3 292 649 7511 588 649 394 567 191 486 415 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0183 -0.0245 0.06511 0.0142 0.0326 0.05310 0.0478 0.0489 -0.0467 0.0101 0.0234 RMSE( ) 0.0413 -0.0342 0.0685 0.12811 0.0999 0.10210 0.0928 0.0767 -0.0766 0.0311 0.0474 Bias( ) 0.1853 0.1812 0.33811 0.25110 0.2036 0.2248 0.1914 0.2369 0.1691 0.2047 0.1935 50 RMSE( ) 0.3083 0.2852 0.4079 0.71811 0.43810 0.3918 0.3174 0.3375 0.2701 0.3567 0.3386 CP( ) 0.9468 0.9263 0.9436 0.9479 0.9447 0.9324 0.9232 0.9375 0.8971 0.94910 0.95211 CP( ) 0.9458 0.9334 0.9509 0.95110 0.9366 0.9223 0.9122 0.9355 0.8831 0.95311 0.9457 aw( ) 0.3703 0.3412 0.67011 0.49310 0.4076 0.4558 0.3714 0.4709 0.2991 0.4087 0.3865 AW( ) 1.2343 1.0702 1.5948 2.67811 1.69310 1.5979 1.2394 1.4097 0.9271 1.4056 1.3035 Total 343 222 7010 7411 608 608 364 567 191 506 475 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0093 -0.02911 0.0114 -0.0072 0.0176 0.0249 0.0207 0.0228 -0.02910 0.0011 0.0125 RMSE( ) 0.0235 -0.0428 0.0041 0.0203 0.04910 0.0439 0.0427 0.0356 -0.05111 0.0132 0.0214 Bias( ) 0.1293 0.1325 0.21811 0.17110 0.1356 0.1458 0.1252 0.1559 0.1201 0.1387 0.1294 100 RMSE( ) 0.2114 0.2063 0.2629 0.42011 0.27410 0.2468 0.2032 0.2186 0.1891 0.2377 0.2155 CP( ) 0.9457 0.9052 0.9499 0.9414 0.9445 0.9446 0.9313 0.9498 0.9051 0.95411 0.95210 CP( ) 0.9446 0.9022 0.95210 0.9509 0.9405 0.9384 0.9313 0.9468 0.8931 0.95311 0.9447 aw( ) 0.2524 0.2442 0.42811 0.33910 0.2706 0.2938 0.2443 0.3119 0.2161 0.2777 0.2585 AW( ) 0.8254 0.7522 1.0279 1.68911 1.06610 0.9928 0.7893 0.9056 0.6681 0.9347 0.8475 Total 364 353 6411 608 587 608 302 608 271 536 455 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0052 -0.02911 -0.0074 -0.0159 0.0105 0.0138 0.0106 0.0107 -0.02110 0.0001 0.0073 RMSE( ) 0.0113 -0.04211 -0.0144 -0.0155 0.0249 0.0207 0.0218 0.0176 -0.03510 0.0081 0.0092 Bias( ) 0.0893 0.0987 0.15311 0.12610 0.0945 0.1018 0.0862 0.1069 0.0851 0.0986 0.0894 200 RMSE( ) 0.1443 0.1526 0.18510 0.30211 0.1819 0.1658 0.1392 0.1505 0.1331 0.1657 0.1464 CP( ) 0.9456 0.8841 0.9455 0.9253 0.95010 0.9456 0.9394 0.9468 0.9142 0.95010 0.9509 CP( ) 0.95111 0.8801 0.9448 0.9293 0.9479 0.9447 0.9344 0.9425 0.9052 0.94810 0.9446 aw( ) 0.1753 0.1775 0.30311 0.24210 0.1866 0.1988 0.1662 0.2139 0.1551 0.1937 0.1774 AW( ) 0.5664 0.5463 0.72810 1.17111 0.7119 0.6608 0.5312 0.6126 0.4821 0.6437 0.5735 Total 35 3 455 6311 629 629 608 302 557 281 496 374 Overall Total 134 122 4111 3910 328 339 122 297 41 246 195 Table 6: Simulation results for = 1.0 and  = 1.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Sanku Dey, Andre F. B. 3 Monte Carlo Simulations Menezes and Josmar Mazucheli 785 Bias( ) 0.0684 -0.0031 0.26711 0.1358 0.1176 0.17810 0.1469 0.1197 -0.0905 0.0292 0.0513 RMSE( ) 0.0974 -0.0031 0.26710 0.31211 0.2218 0.2579 0.2087 0.1756 -0.1195 0.0442 0.0743 Bias( ) 0.3663 0.3272 0.82611 0.55410 0.4418 0.5399 0.4014 0.4035 0.2931 0.4217 0.4136 20 RMSE( ) 0.4793 0.4242 0.81710 1.04311 0.7118 0.7379 0.5344 0.5525 0.3781 0.5587 0.5546 CP( ) 0.9449 0.9428 0.8903 0.9387 0.9246 0.8934 0.8841 0.9145 0.8872 0.95111 0.94410 CP( ) 0.9428 0.9459 0.8772 0.9307 0.9156 0.8894 0.8681 0.9105 0.8833 0.95611 0.94810 aw( ) 1.4593 1.2432 3.08411 2.0909 1.7588 2.14910 1.6346 1.7037 0.9861 1.6005 1.5524 AW( ) 1.9403 1.6182 3.11210 3.91911 2.7738 2.9309 2.1886 2.2847 1.2641 2.1475 2.0764 Total 373 272 6810 7411 588 649 384 476 191 507 465 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0192 -0.0335 0.06911 0.0223 0.0396 0.0609 0.0538 0.0507 -0.06210 0.0101 0.0234 RMSE( ) 0.0282 -0.0444 0.0625 0.0737 0.0738 0.08511 0.0769 0.0696 -0.08310 0.0151 0.0333 Bias( ) 0.2003 0.1942 0.37411 0.28810 0.2248 0.2509 0.2054 0.2237 0.1781 0.2216 0.2125 50 RMSE( ) 0.2613 0.2492 0.38210 0.51111 0.3378 0.3379 0.2694 0.2906 0.2321 0.2987 0.2805 CP( ) 0.94910 0.9172 0.9406 0.9437 0.9448 0.9274 0.9203 0.9365 0.8841 0.95211 0.9448 CP( ) 0.9478 0.9183 0.9467 0.95311 0.9406 0.9224 0.9122 0.9385 0.8771 0.95010 0.9489 aw( ) 0.8003 0.7192 1.47311 1.12110 0.9008 1.0109 0.8044 0.8806 0.6241 0.8967 0.8385 AW( ) 1.0453 0.9202 1.50810 2.01711 1.3438 1.3509 1.0524 1.1596 0.7941 1.1847 1.1025 Total 343 221 7111 7010 608 649 384 486 262 507 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0062 -0.03610 0.0114 -0.0073 0.0216 0.0289 0.0268 0.0227 -0.03911 0.0061 0.0145 RMSE( ) 0.0124 -0.04810 0.0051 0.0072 0.0387 0.0399 0.0388 0.0326 -0.05411 0.0073 0.0215 Bias( ) 0.1373 0.1404 0.24011 0.19010 0.1517 0.1619 0.1342 0.1476 0.1281 0.1548 0.1445 100 RMSE( ) 0.1804 0.1803 0.24810 0.33311 0.2209 0.2128 0.1762 0.1916 0.1651 0.2017 0.1895 CP( ) 0.94810 0.8972 0.9469 0.9394 0.9427 0.9395 0.9353 0.9438 0.8931 0.95111 0.9426 CP( ) 0.95011 0.8992 0.9499 0.9447 0.9426 0.9394 0.9293 0.9425 0.8821 0.95010 0.9448 aw( ) 0.5404 0.5152 0.94311 0.74710 0.5937 0.6449 0.5253 0.5806 0.4531 0.6078 0.5595 AW( ) 0.7044 0.6562 0.98010 1.30711 0.8679 0.8508 0.6813 0.7616 0.5781 0.7997 0.7305 Total 424 353 6511 588 588 6110 322 506 281 557 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0011 -0.03111 -0.0116 -0.0159 0.0083 0.0148 0.0137 0.0115 -0.02710 0.0022 0.0084 RMSE( ) 0.0032 -0.04211 -0.0165 -0.0177 0.0166 0.0188 0.0199 0.0164 -0.03610 0.0021 0.0123 Bias( ) 0.0953 0.1026 0.17411 0.13910 0.1037 0.1109 0.0932 0.0995 0.0901 0.1098 0.0994 200 RMSE( ) 0.1243 0.1326 0.17910 0.24311 0.1479 0.1448 0.1202 0.1285 0.1161 0.1427 0.1284 CP( ) 0.94911 0.8901 0.9304 0.9273 0.9479 0.9447 0.9305 0.94810 0.9012 0.9468 0.9446 CP( ) 0.95011 0.8841 0.9335 0.9243 0.9477 0.9446 0.9324 0.9479 0.8972 0.94810 0.9478 aw( ) 0.3743 0.3794 0.66711 0.53210 0.4047 0.4339 0.3572 0.3986 0.3271 0.4218 0.3835 AW( ) 0.4854 0.4833 0.69410 0.92411 0.5849 0.5708 0.4602 0.5216 0.4181 0.5527 0.4985 Total 383 435 629 6411 578 6310 332 506 281 517 394 Comparison of estimation methods for unit-Gamma distribution 786 Overall Total 134 112 4111 4010 328 389 123 246 51 287 195 Table 7: Simulation results for = 2.0 and  = 1.0. 3 Monte Carlo Simulations B. Menezes and Josmar Mazucheli 787 CP( ) 0.95111 0.8711 0.9354 0.9273 0.9458 0.94710 0.9446 0.9447 0.9002 0.9469 0.9385 CP( ) 0.94810 0.8681 0.9314 0.9273 0.9438 0.9459 0.9385 0.9437 0.9002 0.94911 0.9426 aw( ) 0.7993 0.7994 1.41411 1.16510 0.8726 0.9289 0.7552 0.8907 0.6911 0.9008 0.8135 AW( ) 0.4564 0.4553 0.68810 0.81611 0.5337 0.5348 0.4292 0.5499 0.3921 0.5166 0.4645 Total 384 425 599 6610 547 6711 332 558 291 506 353 Overall Total 154 112 4111 389 287 3910 112 338 41 246 175 Table 8: Simulation results for = 0.5 and  = 2.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0704 -0.0071 0.22011 0.1357 0.1216 0.1819 0.1538 0.18910 -0.0865 0.0262 0.0503 RMSE( ) 0.0804 -0.0031 0.2108 0.22110 0.1686 0.2209 0.1827 0.23411 -0.1005 0.0332 0.0603 Bias( ) 0.3913 0.3592 0.66611 0.56210 0.4627 0.5399 0.4074 0.5188 0.3271 0.4336 0.4235 20 RMSE( ) 0.4433 0.4182 0.65910 0.81911 0.5937 0.6419 0.4764 0.6218 0.3701 0.5106 0.4905 CP( ) 0.9408 0.9367 0.9095 0.9439 0.9276 0.9004 0.8903 0.8892 0.8731 0.95511 0.94410 CP( ) 0.9438 0.9417 0.8965 0.9449 0.9246 0.8944 0.8842 0.8933 0.8721 0.95311 0.94910 aw( ) 2.9923 2.5672 4.74011 4.20110 3.5447 4.0149 3.3056 3.8388 2.0791 3.2095 3.1274 AW( ) 1.7223 1.4772 2.3248 3.08911 2.2787 2.38110 1.9186 2.3299 1.1811 1.8715 1.8174 Total 363 242 6910 7711 527 639 404 598 161 486 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0324 -0.0416 0.06510 0.0252 0.0385 0.06811 0.0608 0.0659 -0.0597 0.0121 0.0263 RMSE( ) 0.0363 -0.0464 0.0557 0.0515 0.0536 0.08111 0.0698 0.08110 -0.0719 0.0131 0.0302 Bias( ) 0.2224 0.2072 0.38411 0.31010 0.2406 0.2759 0.2143 0.2578 0.1961 0.2437 0.2325 50 RMSE( ) 0.2524 0.2362 0.37210 0.44111 0.2997 0.3189 0.2433 0.3148 0.2241 0.2816 0.2625 CP( ) 0.9406 0.9152 0.9459 0.95111 0.9427 0.9285 0.9193 0.9244 0.8731 0.95110 0.9438 CP( ) 0.9417 0.9072 0.9489 0.95611 0.9416 0.9234 0.9173 0.9234 0.8611 0.95110 0.9478 aw( ) 1.7194 1.5052 2.96711 2.45610 1.9397 2.1789 1.7133 2.0548 1.3191 1.9216 1.7895 AW( ) 0.9824 0.8572 1.42910 1.73411 1.2037 1.2609 0.9773 1.2528 0.7461 1.1056 1.0275 Total 364 221 7711 7110 517 679 343 598 221 476 415 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0144 -0.04111 0.0123 -0.0061 0.0206 0.0308 0.0307 0.0329 -0.04110 0.0062 0.0155 RMSE( ) 0.0164 -0.04710 0.0073 0.0021 0.0286 0.0368 0.0357 0.0409 -0.04811 0.0052 0.0175 Bias( ) 0.1483 0.1504 0.25811 0.20910 0.1596 0.1749 0.1442 0.1667 0.1351 0.1688 0.1535 100 RMSE( ) 0.1683 0.1734 0.25110 0.29411 0.1977 0.2008 0.1622 0.2069 0.1571 0.1936 0.1745 CP( ) 0.94911 0.8892 0.9489 0.9386 0.94810 0.9335 0.9313 0.9324 0.8841 0.9478 0.9457 CP( ) 0.94811 0.8852 0.94710 0.9416 0.9448 0.9375 0.9283 0.9304 0.8751 0.9459 0.9417 aw( ) 1.1564 1.0832 1.99011 1.64210 1.2776 1.3819 1.1133 1.3228 0.9541 1.3037 1.1895 AW( ) 0.6604 0.6162 0.96610 1.14911 0.7857 0.7968 0.6343 0.8139 0.5411 0.7486 0.6805 Total 444 373 6711 567 567 6010 302 599 271 486 444 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0062 -0.03711 -0.0115 -0.0169 0.0114 0.0147 0.0148 0.0126 -0.02710 0.0011 0.0093 RMSE( ) 0.0072 -0.04111 -0.0134 -0.0189 0.0145 0.0178 0.0167 0.0146 -0.03010 -0.0001 0.0103 Bias( ) 0.1013 0.1116 0.18211 0.15310 0.1127 0.1179 0.0962 0.1105 0.0951 0.1168 0.1074 200 RMSE( ) 0.1153 0.1275 0.17910 0.21411 0.1379 0.1347 0.1091 0.1368 0.1092 0.1316 0.1214 Sanku Dey, Andre F. 3 Monte Carlo Simulations Comparison of estimation methods for unit-Gamma distribution 788 Bias( ) 0.0063 -0.02711 -0.0085 -0.0199 0.0096 0.0128 0.0117 0.0032 -0.02010 0.0031 0.0064 RMSE( ) 0.0124 -0.04311 -0.0123 -0.0216 0.0258 0.0269 0.0237 0.0062 -0.03310 0.0061 0.0145 Bias( ) 0.0893 0.0966 0.15611 0.12710 0.0965 0.1018 0.0842 0.1019 0.0821 0.0997 0.0914 200 RMSE( ) 0.1444 0.1516 0.18810 0.30311 0.1829 0.1718 0.1373 0.1001 0.1322 0.1627 0.1485 CP( ) 0.9479 0.8901 0.9364 0.9233 0.9479 0.9458 0.9457 0.9375 0.9192 0.95111 0.9436 CP( ) 0.95110 0.8861 0.9334 0.9223 0.9468 0.9376 0.9365 0.9377 0.9082 0.95511 0.9489 aw( ) 0.1753 0.1785 0.30311 0.24110 0.1856 0.1988 0.1672 0.2059 0.1551 0.1937 0.1774 aw ( ) 1.1325 1.0914 1.45910 2.33311 1.4249 1.3298 1.0653 1.0262 0.9651 1.2827 1.1516 Total 414 456 588 6310 609 6310 362 373 291 527 435 Overall Total 153 122 389 4011 348 3910 153 205 41 257 216 Table 9: Simulation results for = 1.0 and  = 2.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0595 -0.0031 0.19911 0.0384 0.0757 0.1309 0.1198 0.15210 -0.0736 0.0212 0.0353 RMSE( ) 0.1365 0.0231 0.2438 0.1986 0.2087 0.25811 0.2499 0.25110 -0.0954 0.0502 0.0793 Bias( ) 0.3313 0.2962 0.61911 0.3708 0.3546 0.4139 0.3515 0.49610 0.2731 0.3557 0.3494 20 RMSE( ) 0.5643 0.5062 0.7057 0.84211 0.7169 0.71710 0.6166 0.7088 0.4501 0.5834 0.5855 CP( ) 0.9447 0.9468 0.9034 0.96511 0.9396 0.9225 0.8973 0.8871 0.8942 0.95210 0.9489 CP( ) 0.9417 0.9549 0.8863 0.99611 0.9396 0.9095 0.8712 0.8651 0.9064 0.96710 0.9538 aw( ) 0.6333 0.5632 1.13211 0.6948 0.6716 0.7979 0.6807 0.85210 0.4661 0.6655 0.6464 aw ( ) 4.1464 3.6222 4.9238 5.86711 5.05810 5.0419 4.4187 4.1053 2.9571 4.3206 4.2025 Total 373 272 639 7011 578 6710 476 537 201 465 414 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0204 -0.0315 0.05711 0.0021 0.0336 0.0509 0.0468 0.0437 -0.05110 0.0112 0.0143 RMSE( ) 0.0454 -0.0383 0.0655 0.0888 0.10010 0.10511 0.0939 0.0746 -0.0747 0.0291 0.0322 Bias( ) 0.1884 0.1802 0.32511 0.2399 0.2056 0.2278 0.1853 0.26010 0.1661 0.2127 0.1895 50 RMSE( ) 0.3093 0.2862 0.3928 0.60811 0.42710 0.4019 0.3124 0.3426 0.2661 0.3527 0.3245 CP( ) 0.9488 0.9193 0.9447 0.94910 0.9416 0.9305 0.9284 0.9192 0.8961 0.9489 0.95411 CP( ) 0.9487 0.9295 0.9508 0.96311 0.9356 0.9224 0.9152 0.9183 0.8911 0.95410 0.9509 aw( ) 0.3703 0.3392 0.64811 0.4589 0.4026 0.4488 0.3704 0.49310 0.2971 0.4077 0.3815 aw ( ) 2.4683 2.1342 3.0908 4.51111 3.26910 3.1269 2.4754 2.5405 1.8591 2.7707 2.5586 Total 363 242 6910 7011 608 639 384 496 231 507 465 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0112 -0.03110 0.0136 -0.0124 0.0167 0.0249 0.0238 0.0123 -0.03311 0.0051 0.0135 RMSE( ) 0.0204 -0.0467 0.0111 0.0183 0.0478 0.05311 0.0489 0.0235 -0.05310 0.0132 0.0246 Bias( ) 0.1273 0.1314 0.22111 0.17410 0.1406 0.1508 0.1242 0.1619 0.1181 0.1457 0.1325 100 RMSE( ) 0.2075 0.2033 0.2669 0.42911 0.27210 0.2568 0.2034 0.1912 0.1861 0.2387 0.2186 CP( ) 0.94911 0.8991 0.9448 0.9334 0.9397 0.9356 0.9355 0.9283 0.9052 0.9469 0.94710 CP( ) 0.95311 0.9112 0.9499 0.9376 0.9407 0.9294 0.9263 0.9295 0.8951 0.94910 0.9458 aw( ) 0.2524 0.2432 0.42911 0.33310 0.2706 0.2928 0.2443 0.3229 0.2151 0.2787 0.2585 aw ( ) 1.6444 1.4982 2.0689 3.29911 2.12610 2.0018 1.5873 1.6525 1.3351 1.8657 1.6986 Total 445 312 6411 598 619 6210 373 414 281 506 517 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Table 8: Simulation results for = 0.5 and  = 2.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0684 -0.0111 0.23511 0.0836 0.0927 0.16510 0.1398 0.1409 -0.0835 0.0302 0.0333 RMSE( ) 0.1004 -0.0071 0.23711 0.2009 0.1546 0.23310 0.1988 0.1937 -0.1085 0.0473 0.0452 Bias( ) 0.3603 0.3252 0.68511 0.4439 0.4097 0.49510 0.3956 0.4278 0.2861 0.3925 0.3784 20 RMSE( ) 0.4773 0.4262 0.68310 0.76311 0.5998 0.6499 0.5205 0.5497 0.3771 0.5206 0.4854 CP( ) 0.9478 0.9427 0.9013 0.96611 0.9396 0.9064 0.8851 0.9085 0.8932 0.95710 0.9539 CP( ) 0.9437 0.9478 0.8832 0.96811 0.9326 0.9025 0.8731 0.8964 0.8913 0.96010 0.9579 aw( ) 1.3883 1.2012 2.44111 1.6269 1.5197 1.79710 1.5066 1.6148 0.9851 1.4755 1.4194 aw ( ) 3.6193 3.1102 4.75110 5.40411 4.4398 4.6299 3.9136 4.0797 2.5181 3.8545 3.7024 Total 35 3 252 6910 7711 557 679 415 557 191 466 394 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0254 -0.0356 0.06611 0.0152 0.0355 0.0559 0.0457 0.0528 -0.05710 0.0111 0.0193 RMSE( ) 0.0393 -0.0434 0.0616 0.0575 0.0627 0.08211 0.0698 0.0709 -0.07410 0.0161 0.0262 Bias( ) 0.2034 0.1942 0.37011 0.27010 0.2226 0.2469 0.2013 0.2298 0.1801 0.2267 0.2105 50 RMSE( ) 0.2694 0.2502 0.38010 0.47111 0.3238 0.3329 0.2643 0.2866 0.2361 0.3017 0.2745 CP( ) 0.9458 0.9122 0.9406 0.95211 0.9427 0.9325 0.9253 0.9253 0.8851 0.9519 0.95210 CP( ) 0.9468 0.9163 0.9446 0.95911 0.9447 0.9275 0.9122 0.9244 0.8801 0.95710 0.9509 aw( ) 0.8054 0.7172 1.42811 1.06410 0.8916 0.9989 0.7963 0.8988 0.6271 0.8937 0.8335 aw ( ) 2.1094 1.8412 2.92610 3.74011 2.6428 2.6689 2.0923 2.2726 1.6011 2.3587 2.1845 Total 394 231 7110 7110 548 669 323 527 262 496 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0125 -0.03610 0.0092 -0.0093 0.0176 0.0258 0.0227 0.0259 -0.03911 0.0061 0.0124 RMSE( ) 0.0205 -0.04510 0.0042 0.0041 0.0336 0.0399 0.0358 0.0337 -0.05211 0.0103 0.0164 Bias( ) 0.1373 0.1404 0.24611 0.18910 0.1456 0.1599 0.1332 0.1507 0.1281 0.1548 0.1415 100 RMSE( ) 0.1825 0.1803 0.25710 0.32911 0.2159 0.2138 0.1752 0.1826 0.1651 0.2067 0.1814 CP( ) 0.9489 0.8962 0.9416 0.9384 0.94810 0.9437 0.9385 0.9343 0.8921 0.9448 0.94911 CP( ) 0.9468 0.8932 0.9416 0.9457 0.9479 0.9385 0.9313 0.9384 0.8831 0.94811 0.94710 Sanku Dey, Andre F. B. 3 Monte Carlo Simulations Menezes and Josmar Mazucheli 789 aw( ) 0.5444 0.5152 0.94011 0.74410 0.5916 0.6419 0.5233 0.5927 0.4541 0.6088 0.5575 aw ( ) 1.4174 1.3162 1.95810 2.59911 1.7289 1.7018 1.3583 1.4786 1.1581 1.6017 1.4515 Total 434 353 589 578 6110 6311 332 496 281 537 485 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0062 -0.03411 -0.0105 -0.0169 0.0094 0.0106 0.0117 0.0138 -0.02410 0.0021 0.0073 RMSE( ) 0.0093 -0.04511 -0.0154 -0.0199 0.0165 0.0178 0.0167 0.0166 -0.03310 0.0041 0.0092 Bias( ) 0.0963 0.1057 0.17711 0.13710 0.1016 0.1099 0.0912 0.0995 0.0901 0.1088 0.0984 200 RMSE( ) 0.1264 0.1346 0.18210 0.23911 0.1479 0.1458 0.1173 0.1161 0.1162 0.1427 0.1285 CP( ) 0.9509 0.8701 0.9293 0.9314 0.95111 0.9468 0.9376 0.9365 0.9062 0.95110 0.9467 CP( ) 0.94810 0.8761 0.9294 0.9263 0.9479 0.9435 0.9436 0.9447 0.9002 0.95211 0.9448 aw( ) 0.3773 0.3774 0.66811 0.53210 0.4057 0.4329 0.3562 0.4036 0.3291 0.4218 0.3815 aw ( ) 0.9774 0.9633 1.39010 1.84611 1.1689 1.1388 0.9192 1.0036 0.8391 1.1067 0.9905 Total 383 445 588 6711 609 6110 352 445 291 537 394 Overall Total 144 112 379 4011 348 3910 123 256 51 267 185 Table 10: Simulation results for = 2.0 and  = 2.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0704 -0.0101 0.21911 0.1176 0.1227 0.17810 0.1599 0.1478 -0.0875 0.0362 0.0463 RMSE( ) 0.0844 -0.0071 0.21010 0.1898 0.1626 0.21211 0.1909 0.1757 -0.1005 0.0432 0.0583 Bias( ) 0.3813 0.3402 0.64911 0.5109 0.4588 0.51710 0.4205 0.4336 0.3181 0.4417 0.4144 20 RMSE( ) 0.4383 0.3972 0.63210 0.70511 0.5758 0.5949 0.4825 0.4996 0.3621 0.5087 0.4804 CP( ) 0.9458 0.9457 0.9095 0.96011 0.9326 0.9044 0.8802 0.8933 0.8721 0.95310 0.9509 CP( ) 0.9488 0.9457 0.8974 0.95811 0.9296 0.9005 0.8762 0.8913 0.8711 0.9489 0.95010 aw( ) 2.9373 2.5372 4.59711 3.6649 3.3298 3.79410 3.2136 3.2647 2.0691 3.1205 3.0284 aw ( ) 3.3693 2.9052 4.42410 5.05511 4.1538 4.3689 3.6866 3.7487 2.3511 3.5905 3.4884 Total 363 242 7210 7611 578 689 445 476 161 476 414 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0263 -0.0415 0.06811 0.0374 0.0466 0.06710 0.0588 0.0517 -0.0589 0.0131 0.0232 RMSE( ) 0.0333 -0.0464 0.0606 0.0668 0.0605 0.08211 0.0679 0.0617 -0.07010 0.0161 0.0292 Bias( ) 0.2164 0.2072 0.38511 0.31410 0.2437 0.2729 0.2153 0.2215 0.1961 0.2508 0.2266 50 RMSE( ) 0.2514 0.2342 0.37510 0.43811 0.3018 0.3159 0.2433 0.2555 0.2231 0.2887 0.2606 CP( ) 0.9447 0.9112 0.9479 0.94810 0.9386 0.9274 0.9263 0.9305 0.8731 0.95011 0.9458 CP( ) 0.9457 0.9092 0.94810 0.95211 0.9356 0.9223 0.9254 0.9275 0.8631 0.9469 0.9458 aw( ) 1.7084 1.5072 2.96911 2.41210 1.9468 2.1669 1.7073 1.7735 1.3201 1.9187 1.7826 aw ( ) 1.9584 1.7152 2.86210 3.38711 2.4118 2.5079 1.9493 2.0445 1.4921 2.2127 2.0496 Total 363 211 7811 7510 548 649 363 445 252 517 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0145 -0.04010 0.0093 -0.0011 0.0227 0.0349 0.0288 0.0216 -0.04011 0.0052 0.0134 RMSE( ) 0.0164 -0.04410 0.0052 0.0041 0.0307 0.0419 0.0318 0.0266 -0.04811 0.0053 0.0175 Comparison of estimation methods for unit-Gamma distribution 790 790 Comparison of estimation methods for unit-Gamma distribution Bias( ) 0.1474 0.1505 0.25511 0.20910 0.1607 0.1799 0.1392 0.1433 0.1351 0.1698 0.1526 100 RMSE( ) 0.1694 0.1695 0.24810 0.28611 0.1998 0.2059 0.1582 0.1663 0.1541 0.1947 0.1746 CP( ) 0.9489 0.8872 0.9436 0.9447 0.94810 0.9343 0.9344 0.9365 0.8821 0.94811 0.9458 CP( ) 0.94610 0.8922 0.9457 0.9416 0.9459 0.9313 0.9345 0.9334 0.8781 0.94811 0.9458 aw( ) 1.1564 1.0862 1.98311 1.64710 1.2797 1.3879 1.1123 1.1605 0.9551 1.3028 1.1876 aw ( ) 1.3214 1.2372 1.93110 2.29911 1.5748 1.5999 1.2643 1.3365 1.0821 1.4967 1.3596 Total 445 384 609 577 6311 609 352 373 281 577 496 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0073 -0.03611 -0.0126 -0.0147 0.0115 0.0179 0.0158 0.0094 -0.02610 0.0001 0.0062 RMSE( ) 0.0092 -0.03911 -0.0125 -0.0167 0.0156 0.0209 0.0168 0.0104 -0.03010 -0.0011 0.0093 Bias( ) 0.1045 0.1117 0.18311 0.15110 0.1106 0.1219 0.0963 0.0911 0.0952 0.1158 0.1044 200 RMSE( ) 0.1205 0.1266 0.17810 0.21111 0.1358 0.1379 0.1103 0.1051 0.1082 0.1327 0.1184 CP( ) 0.9427 0.8691 0.9304 0.9263 0.95010 0.9335 0.9406 0.9448 0.9022 0.95411 0.9459 CP( ) 0.9395 0.8711 0.9324 0.9283 0.94810 0.9416 0.9417 0.9438 0.8972 0.95511 0.9479 aw( ) 0.7995 0.7994 1.41211 1.16710 0.8727 0.9329 0.7562 0.7913 0.6911 0.9008 0.8116 aw ( ) 0.9125 0.9124 1.37610 1.63411 1.0688 1.0719 0.8582 0.9113 0.7841 1.0327 0.9286 Total 373 456 619 6210 608 6511 394 322 301 547 435 Overall Total 143 132 3911 389 358 389 143 165 51 277 206 Table 11: Simulation results for = 0.5 and  = 3.0. 3 Monte Carlo Simulations Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 791 Total 41 5 261 7311 649 638 6510 374 333 292 547 436 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0104 -0.03510 0.0083 -0.0157 0.0136 0.0229 0.0198 0.0011 -0.03511 0.0032 0.0115 RMSE( ) 0.0245 -0.05110 0.0011 0.0054 0.0377 0.0459 0.0388 -0.0022 -0.05811 0.0053 0.0276 Bias( ) 0.1284 0.1336 0.21811 0.17310 0.1357 0.1489 0.1233 0.1222 0.1181 0.1428 0.1295 100 RMSE( ) 0.2125 0.2044 0.2639 0.41511 0.26510 0.2528 0.1993 0.0341 0.1872 0.2317 0.2186 CP( ) 0.9487 0.8992 0.9509 0.9363 0.9456 0.9384 0.9435 0.98211 0.8981 0.9498 0.95310 CP( ) 0.9405 0.9022 0.9509 0.9467 0.9446 0.9333 0.9374 0.95211 0.8921 0.95010 0.9478 aw( ) 0.2525 0.2423 0.42111 0.31910 0.2677 0.2909 0.2434 0.2322 0.2141 0.2778 0.2576 aw ( ) 2.4725 2.2383 3.0249 4.45611 3.10310 2.9408 2.3564 0.0431 1.9932 2.7597 2.5466 Total 404 404 6210 6311 598 598 393 312 301 537 526 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0043 -0.02911 -0.0086 -0.0209 0.0064 0.0128 0.0107 0.0011 -0.02310 0.0022 0.0075 RMSE( ) 0.0153 -0.04411 -0.0164 -0.0259 0.0196 0.0228 0.0217 -0.0001 -0.03810 0.0012 0.0165 Bias( ) 0.0884 0.0987 0.15811 0.12610 0.0936 0.1019 0.0853 0.0842 0.0831 0.0998 0.0905 200 RMSE( ) 0.1474 0.1496 0.19210 0.30311 0.1809 0.1708 0.1353 0.0021 0.1302 0.1607 0.1485 CP( ) 0.9498 0.8861 0.9374 0.9213 0.9509 0.9435 0.9446 0.97411 0.9112 0.95210 0.9487 CP( ) 0.9468 0.8871 0.9334 0.9243 0.9479 0.9375 0.9426 0.96411 0.9132 0.94910 0.9447 aw( ) 0.1754 0.1776 0.30311 0.23910 0.1857 0.1989 0.1673 0.1632 0.1551 0.1938 0.1775 aw ( ) 1.7035 1.6364 2.18010 3.43011 2.1259 1.9858 1.5943 0.0221 1.4422 1.9137 1.7306 Total 394 476 609 6611 598 609 383 301 301 547 455 Overall Total 174 133 4010 4010 328 379 174 92 51 267 236 Table 12: Simulation results for = 1.0 and  = 3.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0366 -0.0143 0.13911 0.0021 0.0457 0.10110 0.1019 0.0164 -0.0798 -0.0042 0.0215 RMSE( ) 0.0796 -0.0081 0.1419 0.0475 0.0967 0.16310 0.17911 0.0193 -0.1198 -0.0092 0.0404 Bias( ) 0.3003 0.2872 0.50011 0.3368 0.3215 0.3869 0.3257 0.39210 0.2691 0.3246 0.3184 20 RMSE( ) 0.4754 0.4473 0.5369 0.61811 0.5258 0.55510 0.4887 0.3051 0.4182 0.4875 0.4876 CP( ) 0.9577 0.9486 0.9405 0.96811 0.9588 0.9394 0.9163 0.9032 0.8891 0.9589 0.96110 CP( ) 0.9746 0.9725 0.9404 0.99811 0.99610 0.9757 0.9242 0.9283 0.9091 0.9868 0.9869 aw( ) 0.5763 0.5292 0.90911 0.6057 0.5965 0.6869 0.6118 0.73110 0.4511 0.5976 0.5854 aw ( ) 4.9854 4.6533 5.3468 6.28111 5.63810 5.5349 5.0625 3.5091 4.0052 5.1577 5.0666 Total 394 252 6810 65 9 608 6810 527 343 241 455 486 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0194 -0.0337 0.04910 -0.0113 0.0266 0.0479 0.0408 -0.0041 -0.05211 0.0042 0.0205 RMSE( ) 0.0466 -0.0455 0.0477 0.0343 0.0738 0.09211 0.0799 -0.0021 -0.08310 0.0112 0.0454 Bias( ) 0.1854 0.1802 0.31311 0.23110 0.1957 0.2229 0.1833 0.1936 0.1671 0.2038 0.1935 50 RMSE( ) 0.3135 0.2813 0.3738 0.52311 0.38010 0.3779 0.2984 0.1211 0.2672 0.3357 0.3206 CP( ) 0.9487 0.9202 0.9519 0.9486 0.9498 0.9374 0.9363 0.98211 0.8901 0.95610 0.9475 CP( ) 0.9436 0.9272 0.9579 0.97411 0.9558 0.9314 0.9273 0.9415 0.8891 0.95710 0.9497 aw( ) 0.3634 0.3362 0.59411 0.4179 0.3826 0.42510 0.3623 0.3857 0.2961 0.3928 0.3745 aw ( ) 3.5375 3.1293 4.0988 5.37811 4.30310 4.1619 3.5094 1.0831 2.7432 3.8407 3.6636 Bias( ) 0.1474 0.1505 0.25511 0.20910 0.1607 0.1799 0.1392 0.1433 0.1351 0.1698 0.1526 100 RMSE( ) 0.1694 0.1695 0.24810 0.28611 0.1998 0.2059 0.1582 0.1663 0.1541 0.1947 0.1746 CP( ) 0.9489 0.8872 0.9436 0.9447 0.94810 0.9343 0.9344 0.9365 0.8821 0.94811 0.9458 CP( ) 0.94610 0.8922 0.9457 0.9416 0.9459 0.9313 0.9345 0.9334 0.8781 0.94811 0.9458 aw( ) 1.1564 1.0862 1.98311 1.64710 1.2797 1.3879 1.1123 1.1605 0.9551 1.3028 1.1876 aw ( ) 1.3214 1.2372 1.93110 2.29911 1.5748 1.5999 1.2643 1.3365 1.0821 1.4967 1.3596 Total 445 384 609 577 6311 609 352 373 281 577 496 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0073 -0.03611 -0.0126 -0.0147 0.0115 0.0179 0.0158 0.0094 -0.02610 0.0001 0.0062 RMSE( ) 0.0092 -0.03911 -0.0125 -0.0167 0.0156 0.0209 0.0168 0.0104 -0.03010 -0.0011 0.0093 Bias( ) 0.1045 0.1117 0.18311 0.15110 0.1106 0.1219 0.0963 0.0911 0.0952 0.1158 0.1044 200 RMSE( ) 0.1205 0.1266 0.17810 0.21111 0.1358 0.1379 0.1103 0.1051 0.1082 0.1327 0.1184 CP( ) 0.9427 0.8691 0.9304 0.9263 0.95010 0.9335 0.9406 0.9448 0.9022 0.95411 0.9459 CP( ) 0.9395 0.8711 0.9324 0.9283 0.94810 0.9416 0.9417 0.9438 0.8972 0.95511 0.9479 aw( ) 0.7995 0.7994 1.41211 1.16710 0.8727 0.9329 0.7562 0.7913 0.6911 0.9008 0.8116 aw ( ) 0.9125 0.9124 1.37610 1.63411 1.0688 1.0719 0.8582 0.9113 0.7841 1.0327 0.9286 Total 373 456 619 6210 608 6511 394 322 301 547 435 Overall Total 143 132 3911 389 358 389 143 165 51 277 206 Table 11: Simulation results for = 0.5 and  = 3.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0445 -0.0233 0.13711 0.0344 0.0626 0.1249 0.13210 0.1118 -0.0817 -0.0001 0.0232 RMSE( ) 0.0644 -0.0252 0.1308 0.0755 0.1036 0.16910 0.17111 0.1379 -0.1077 0.0051 0.0303 Bias( ) 0.3273 0.3022 0.51211 0.3758 0.3465 0.4039 0.3657 0.40410 0.2971 0.3454 0.3516 20 RMSE( ) 0.4193 0.3812 0.5059 0.56011 0.4848 0.51510 0.4546 0.4707 0.3761 0.4425 0.4354 CP( ) 0.9567 0.9455 0.9526 0.97811 0.9598 0.9424 0.9033 0.8972 0.8811 0.96610 0.9599 CP( ) 0.9657 0.9566 0.9384 0.99611 0.9719 0.9495 0.9042 0.9093 0.8871 0.98110 0.9708 aw( ) 1.2353 1.1192 1.87211 1.3468 1.2946 1.47210 1.3197 1.3889 0.9531 1.2825 1.2574 aw ( ) 4.5704 4.2062 5.22510 5.90611 5.1908 5.2199 4.7106 4.5083 3.5861 4.7837 4.6665 Total 363 242 7011 6910 568 669 527 516 201 435 414 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0224 -0.0406 0.0558 0.0132 0.0345 0.06211 0.05910 0.0417 -0.0569 0.0081 0.0223 RMSE( ) 0.0343 -0.0485 0.0526 0.0454 0.0588 0.08711 0.07610 0.0557 -0.0719 0.0131 0.0282 Bias( ) 0.2023 0.1972 0.34511 0.26410 0.2236 0.2469 0.2084 0.2278 0.1821 0.2237 0.2185 50 RMSE( ) 0.2643 0.2512 0.35810 0.44711 0.3278 0.3319 0.2664 0.2685 0.2331 0.3007 0.2796 Comparison of estimation methods for unit-Gamma distribution 792 p CP( ) 0.9478 0.9073 0.9519 0.95611 0.9416 0.9345 0.9204 0.8992 0.8801 0.95510 0.9437 CP( ) 0.9498 0.9112 0.95410 0.97011 0.9436 0.9275 0.9164 0.9143 0.8821 0.9529 0.9477 aw( ) 0.7913 0.7102 1.29611 0.96210 0.8526 0.9539 0.7974 0.8668 0.6271 0.8667 0.8195 aw ( ) 3.0855 2.7342 3.95610 4.78211 3.7018 3.7359 3.0844 3.0313 2.4061 3.3947 3.1906 Total 373 241 7511 7010 538 689 446 435 241 497 414 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0114 -0.04111 0.0063 -0.0041 0.0167 0.0329 0.0308 0.0135 -0.03710 0.0062 0.0136 RMSE( ) 0.0175 -0.05211 0.0011 0.0072 0.0257 0.0449 0.0398 0.0176 -0.04710 0.0093 0.0154 Bias( ) 0.1373 0.1415 0.23611 0.19410 0.1537 0.1639 0.1362 0.1374 0.1291 0.1548 0.1436 100 RMSE( ) 0.1784 0.1795 0.24710 0.33311 0.2209 0.2148 0.1753 0.1481 0.1662 0.2057 0.1846 CP( ) 0.95110 0.8932 0.95111 0.9396 0.9428 0.9395 0.9304 0.9053 0.8861 0.9499 0.9417 CP( ) 0.95211 0.8872 0.9508 0.9447 0.9396 0.9355 0.9274 0.9193 0.8861 0.9509 0.95110 aw( ) 0.5434 0.5122 0.92711 0.72710 0.5887 0.6449 0.5273 0.5736 0.4551 0.6078 0.5575 aw ( ) 2.1205 1.9642 2.89010 3.74211 2.5599 2.5528 2.0444 2.0093 1.7441 2.3947 2.1726 Total 465 404 6511 588 609 6210 363 312 271 537 506 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0063 -0.03411 -0.0137 -0.0126 0.0064 0.0179 0.0158 0.0021 -0.02410 0.0022 0.0075 RMSE( ) 0.0094 -0.04211 -0.0177 -0.0166 0.0125 0.0249 0.0198 0.0052 -0.03210 0.0041 0.0073 Bias( ) 0.0954 0.1057 0.17411 0.14310 0.1056 0.1119 0.0933 0.0791 0.0912 0.1088 0.0965 200 RMSE( ) 0.1234 0.1346 0.18010 0.24511 0.1509 0.1468 0.1183 0.0721 0.1182 0.1437 0.1245 CP( ) 0.95310 0.8701 0.9324 0.9243 0.9478 0.9406 0.9335 0.96011 0.9032 0.9457 0.9539 CP( ) 0.95210 0.8751 0.9304 0.9243 0.9427 0.9416 0.9428 0.9335 0.8952 0.9459 0.95211 aw( ) 0.3764 0.3775 0.66511 0.53310 0.4037 0.4359 0.3583 0.3302 0.3281 0.4218 0.3816 aw ( ) 1.4665 1.4494 2.07810 2.76711 1.7459 1.7178 1.3813 0.8661 1.2592 1.6587 1.4846 Total 444 465 6410 609 558 6410 413 241 312 496 507 Overall Total 154 122 4311 379 338 3810 195 143 51 257 216 Table 13: Simulation results for = 2.0 and  = 3.0. 3 Monte Carlo Simulations n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0605 -0.0232 0.14410 0.0594 0.0896 0.1379 0.15411 0.1208 -0.0987 0.0111 0.0313 RMSE( ) 0.0754 -0.0302 0.1298 0.1015 0.1187 0.15810 0.17111 0.1359 -0.1136 0.0111 0.0353 Bias( ) 0.3563 0.3252 0.52511 0.4119 0.3958 0.44610 0.3947 0.3856 0.3091 0.3845 0.3754 20 RMSE( ) 0.4063 0.3692 0.49810 0.54811 0.4788 0.4929 0.4357 0.4235 0.3531 0.4306 0.4214 CP( ) 0.9557 0.9475 0.9526 0.98511 0.9578 0.9374 0.8972 0.9183 0.8731 0.96710 0.9639 CP( ) 0.9557 0.9516 0.9404 0.99611 0.9568 0.9425 0.8942 0.9203 0.8701 0.97510 0.9639 aw( ) 2.6143 2.3532 3.75711 2.9169 2.7737 3.08410 2.7818 2.7596 1.9761 2.7255 2.6634 aw ( ) 4.3853 3.9492 5.15410 5.63911 4.9468 5.0589 4.5436 4.5245 3.3471 4.5827 4.4734 Total 35 3 232 7010 7111 608 669 547 45 5 191 455 404 Table 13: Simulation results for = 2.0 and  = 3.0. Sanku Dey, Andre F. B. 3 Monte Carlo Simulations Menezes and Josmar Mazucheli 793 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0274 -0.0435 0.0548 0.0233 0.0436 0.06310 0.0619 0.0517 -0.06611 0.0101 0.0222 RMSE( ) 0.0323 -0.0506 0.0475 0.0474 0.0598 0.07510 0.0699 0.0597 -0.08011 0.0141 0.0282 Bias( ) 0.2123 0.2042 0.35911 0.29210 0.2417 0.2649 0.2164 0.2165 0.1891 0.2438 0.2286 50 RMSE( ) 0.2445 0.2362 0.34710 0.40611 0.3028 0.3029 0.2434 0.2423 0.2171 0.2807 0.2636 CP( ) 0.9477 0.9062 0.9519 0.96211 0.9396 0.9295 0.9163 0.9274 0.8681 0.95510 0.9488 CP( ) 0.9478 0.9072 0.95610 0.96111 0.9356 0.9335 0.9213 0.9254 0.8621 0.9569 0.9447 aw( ) 1.6893 1.4982 2.70611 2.15210 1.8648 2.0549 1.6924 1.7325 1.3071 1.8637 1.7466 aw ( ) 2.8984 2.5512 3.90310 4.43811 3.4458 3.5409 2.8883 2.9455 2.2161 3.2127 3.0036 Total 373 231 7411 7110 578 669 394 405 282 507 436 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0145 -0.04210 0.0113 -0.0021 0.0226 0.0308 0.0319 0.0227 -0.04311 0.0072 0.0124 RMSE( ) 0.0175 -0.04910 0.0072 0.0061 0.0307 0.0369 0.0358 0.0256 -0.05111 0.0093 0.0154 Bias( ) 0.1464 0.1485 0.25611 0.20810 0.1617 0.1719 0.1423 0.1372 0.1341 0.1648 0.1546 100 RMSE( ) 0.1684 0.1695 0.25110 0.29211 0.1989 0.1978 0.1613 0.1531 0.1542 0.1907 0.1746 CP( ) 0.94810 0.8912 0.9459 0.9437 0.9438 0.9416 0.9293 0.9334 0.8851 0.95511 0.9395 CP( ) 0.94710 0.8862 0.9405 0.9459 0.9436 0.9448 0.9303 0.9364 0.8771 0.95211 0.9437 aw( ) 1.1575 1.0822 1.96411 1.61110 1.2777 1.3799 1.1143 1.1504 0.9521 1.3028 1.1866 aw ( ) 1.9835 1.8442 2.86410 3.37311 2.3558 2.3819 1.9023 1.9554 1.6171 2.2477 2.0346 Total 486 384 6110 609 588 6611 353 322 291 577 445 n Qtd AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS Bias( ) 0.0083 -0.03611 -0.0136 -0.0157 0.0105 0.0158 0.0169 0.0094 -0.02810 0.0021 0.0062 RMSE( ) 0.0094 -0.04211 -0.0146 -0.0177 0.0135 0.0188 0.0199 0.0093 -0.03410 0.0031 0.0082 Bias( ) 0.1014 0.1116 0.18211 0.15310 0.1127 0.1169 0.0983 0.0831 0.0942 0.1148 0.1045 200 RMSE( ) 0.1164 0.1276 0.17810 0.21311 0.1389 0.1338 0.1103 0.0901 0.1082 0.1317 0.1185 CP( ) 0.94810 0.8731 0.9334 0.9283 0.9468 0.9469 0.9376 0.9365 0.9032 0.95111 0.9447 CP( ) 0.9458 0.8661 0.9294 0.9283 0.9427 0.9479 0.9385 0.9406 0.8982 0.95311 0.95010 aw( ) 0.8005 0.7994 1.40911 1.16610 0.8717 0.9299 0.7562 0.7773 0.6891 0.9008 0.8106 aw ( ) 1.3705 1.3644 2.05910 2.44811 1.6008 1.6039 1.2902 1.3253 1.1711 1.5527 1.3886 Total 434 446 629 629 568 6911 393 261 302 547 434 Overall Total 164 132 4010 399 328 4010 175 132 61 267 196 Table 14: Overall performance of the estimation methods Scenario AD AD2 AD2L AD2R ADR CvM MLE MOM MPS OLS WLS (α=0.5,β=0.5) 154 102 4311 3810 307 379 102 307 51 246 195 (α=1.0, β=0.5) 134 102 4010 4010 287 379 123 338 51 236 205 (α=2.0, β=0.5) 133 102 3810 4111 297 379 133 338 51 246 195 Comparison of estimation methods for unit-Gamma distribution 794 (α=0.5, β=1.0) 134 122 4111 3910 328 339 122 297 41 246 195 (α=1.0, β=1.0) 134 112 4111 4010 328 389 123 246 51 287 195 (α=2.0,β=1.0) 154 112 4111 389 287 3910 112 338 41 246 175 (α=0.5, β=2.0) 153 122 389 4011 348 3910 153 205 41 257 216 (α=1.0, β=2.0) 144 112 379 4011 348 3910 123 256 51 267 185 (α=2.0, β=2.0) 143 132 3911 389 358 389 143 165 51 277 206 (α=0.5, β=3.0) 174 133 4010 4010 328 379 174 92 51 267 236 (α=1.0,β=3.0) 154 122 4311 379 338 3810 195 143 51 257 216 (α=2.0, β=3.0) 164 132 4010 399 328 4010 175 132 61 267 196 Total 1734 1382 48111 47010 3798 4529 1643 2796 581 3027 2355 Data set I 0.265, 0.269, 0.297, 0.315, 0.3235, 0.338, 0.379, 0.379, 0.392, 0.402, 0.412, 0.416, 0.418, Data set II 0.090, 0.149,0.183,0.117,0.122,0.167,0.190,0.164,0.204,0.162,0.151,0.148,0.229, 0.232, 0.173,0.153,0.204,0.263,0.200,0.145,0.114,0.291,0.240,0.162,0.281,0.179, 0.192, 0.133,0.225,0.341,0.312,0.276,0.198,0.327,0.154,0.276,0.177,0.439,0.164, 0.254, 0.329,0.230,0.464,0.420,0.201,0.263,0.182,0.200 Table 16: Descritive measures for data sets I and II. Data set I Data set II n 20 48 mean 0.4321 0.2181 std-dev 0.1253 0.0835 min 0.2650 0.0903 median 0.4070 0.1989 max 0.7400 0.4641 Table 16: Descritive measures for data sets I and II. Data set I Data set II The parameter estimates and their corresponding bootstrap confidence intervals under various methods considered in this paper for the two data sets are summarized in Tables 17 and 18. We also present the results of formal goodness-of-fit tests, the Kolmogorov-Smirnov (KS) statistic along with p-value, in order to show that the unit-Gamma can be used to model these The parameter estimates and their corresponding bootstrap confidence intervals under various methods considered in this paper for the two data sets are summarized in Tables 17 and 18. We also present the results of formal goodness-of-fit tests, the Kolmogorov-Smirnov (KS) statistic along with p-value, in order to show that the unit-Gamma can be used to model these data sets. statistic along with p-value, in order to show that the unit-Gamma can be used to model these data sets. From Table 17 we can see that all estimates provides a good fit to the data sets. We also observe that the MPS and AD2 estimators give the shortest confidence intervals for both the parameters α and β, respectively. From Table 17 we can see that all estimates provides a good fit to the data sets. We also observe that the MPS and AD2 estimators give the shortest confidence intervals for both the parameters α and β, respectively. The results in Table 18 indicate that the CvM estimates do not provide a good fit to this data set as per K-S statistic is concerned. We also observe that OLS has the lowest value of K-S. It is also noteworthy, that AD2 and ADR have the shortest confidence intervals for both α and β. The results in Table 18 indicate that the CvM estimates do not provide a good fit to this data set as per K-S statistic is concerned. We also observe that OLS has the lowest value of K-S. It is also noteworthy, that AD2 and ADR have the shortest confidence intervals for both α and β. The overall performance of the estimators of a and /3 with respect to width of the parametric bootstrap confidence intervals are presented in Table 17. 4 Applications In this section, we use two real data sets to demonstrate the performance of different methods of estimation considered in this paper. The first data set is from Dumonceaux and Antle (1973) and corresponds to 20 observations of the maximum flood level (in millions of cubic feet per second) for Susquehanna River at Harrisburg, Pennsylvania. The second data set corresponds to twelve core samples from petroleum reservoirs that were sampled by four cross-sections, and there are 48 observations. Each core sample was measured for permeability and each cross-section has the following variables: the total area of pores, the total perimeter of pores and shape. It should be noted that this data can be found in R Core Team (2017), especially on data. frame named rock. Both data sets are in the Table 15 and some descriptive measures are reported in Table 16. Further, we note that these data sets were studied by Mazucheli et al.(2018) to illustrate the applicability of the unit-Gamma distribution in order to compare the second-order bias corrections of the maximum likelihood estimators. Table 15: Maximum flood level data and petroleum reservoirs data. Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 795 0.423, 0.449, 0.484, 0.494, 0.613, 0.654, 0.740 0.423, 0.449, 0.484, 0.494, 0.613, 0.654, 0.740 Data set II 0.090, 0.149,0.183,0.117,0.122,0.167,0.190,0.164,0.204,0.162,0.151,0.148,0.229, 0.232, 0.173,0.153,0.204,0.263,0.200,0.145,0.114,0.291,0.240,0.162,0.281,0.179, 0.192, 0.133,0.225,0.341,0.312,0.276,0.198,0.327,0.154,0.276,0.177,0.439,0.164, 0.254, 0.329,0.230,0.464,0.420,0.201,0.263,0.182,0.200 Data set II 0.090, 0.149,0.183,0.117,0.122,0.167,0.190,0.164,0.204,0.162,0.151,0.148,0.229, 0.232, 0.173,0.153,0.204,0.263,0.200,0.145,0.114,0.291,0.240,0.162,0.281,0.179, 0.192, 0.133,0.225,0.341,0.312,0.276,0.198,0.327,0.154,0.276,0.177,0.439,0.164, 0.254, 0.329,0.230,0.464,0.420,0.201,0.263,0.182,0.200 L(U) CL lower (upper) confidence limit. Data set II 0.090, 0.149,0.183,0.117,0.122,0.167,0.190,0.164,0.204,0.162,0.151,0.148,0.229, 0.232, 0.173,0.153,0.204,0.263,0.200,0.145,0.114,0.291,0.240,0.162,0.281,0.179, 0.192, 0.133,0.225,0.341,0.312,0.276,0.198,0.327,0.154,0.276,0.177,0.439,0.164, 0.254, 0.329,0.230,0.464,0.420,0.201,0.263,0.182,0.200 We considered the p-bootstrap method based on B = 1000 resamples, Efron (1982b), to construct the confidence intervals for α and β . Table 17: Parameter estimates, 95% confidence intervals based on parametric Bootstrap and K- S test: data set I Method α LCL UCL β LCL UCL KS (p-value) Comparison of estimation methods for unit-Gamma distribution 796 MLE 8.7332 5.4251 18.8766 9.7275 5.9152 21.0504 0.1955 (0.4294) MPS 6.2605 3.0916 10.3881 6.9502 3.2673 11.3440 0.2081 (0.3519) MOM 8.3938 5.1258 17.1975 9.2678 5.3816 20.3412 0.1874 (0.4837) OLS 12.8657 6.0086 30.0672 13.7425 6.2067 31.3893 0.1274 (0.9017) WLS 12.6654 6.3662 26.9534 13.5638 6.4821 30.5281 0.1260 (0.9085) CvM 15.2902 8.1805 40.6867 16.3930 8.6397 43.6603 0.1357 (0.8552) AD 9.1007 4.9838 19.0116 9.8457 5.3477 20.6942 0.1592 (0.6909) ADR 6.6690 3.7733 16.3230 7.0020 3.8323 17.4604 0.1504 (0.7559) AD2R 4.7501 2.1651 15.3588 4.7887 1.9332 17.0276 0.2066 (0.3605) AD2L 17.7944 7.7244 61.0730 18.9778 8.3134 62.2243 0.1416 (0.8172) AD2 6.2011 3.0918 11.9963 7.0293 3.2679 13.8680 0.2289 (0.2454) L(U) CL lower (upper) confidence limit. Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 797 Table 18: Parameter estimates, 95% confidence intervals based on parametric Bootstrap and K-S test: data set II Method α LCL UCL β LCL UCL KS (p-value) MLE 17.9541 12.8359 29.0063 11.3115 8.0495 18.2736 0.1365 (0.3331) MPS 15.3776 7.1628 27.0135 9.6316 4.3030 16.9532 0.1298 (0.3937) MOM 15.8115 9.3381 34.5760 9.8926 5.7186 21.5892 0.1275 (0.4162) OLS 19.6011 8.8391 42.6476 12.0676 5.3705 26.4850 0.0947 (0.7829) WLS 19.8300 9.9533 43.1044 12.3017 6.0218 26.9789 0.1077 (0.6341) CvM 15.2857 8.2430 40.5391 16.3882 8.3749 43.2302 0.7456 (0.0000) AD 18.4562 12.0864 29.3272 11.4635 7.4358 18.5932 0.1118 (0.5862) ADR 15.0523 9.9705 25.1797 9.2342 5.9850 16.0918 0.1183 (0.5131) AD2R 11.0919 5.9193 22.5897 6.6442 3.2657 14.4050 0.1706 (0.1221) AD2L 30.3925 15.2310 57.8613 18.6428 9.3745 35.2371 0.1124 (0.5794) AD2 14.5794 8.3875 21.3646 9.3427 5.4558 14.0784 0.1655 (0.1441) L(U)CL lower (upper) confidence limit. Table 19: Width of the parametric Bootstrap confidence intervals. Method Data set I Data set II Width of α Width of β Width of α Width of β MLE 13.4515 15.1353 16.1704 10.2241 MPS 7.2965 8.0768 19.8507 12.6501 MOM 12.0717 14.9595 25.2379 15.8707 OLS 24.0586 25.1825 33.8084 21.1146 WLS 20.5872 24.0460 33.1511 20.9571 CvM 32.5062 35.0207 32.2960 34.8553 AD 14.0277 15.3465 17.2408 11.1574 ADR 12.5497 13.6281 15.2093 10.1068 AD2R 13.1937 15.0944 16.6704 11.1393 AD2L 53.3486 53.9109 42.6302 25.8625 AD2 8.9046 10.6001 12.9771 8.6226 5 Concluding Remarks Table 19: Width of the parametric Bootstrap confidence intervals. 5 Concluding Remarks In this paper, we have performed an extensive simulation study to compare eleven aforementioned methods of estimation. We have compared estimators with respect to bias and rootmean-squared error. Besides, we have obtained the coverage probability and the average Comparison of estimation methods for unit-Gamma distribution 798 widthof the Bootstrap confidence intervals. To illustrate the use of these methods of estimation,we provided two real data examples where the parameters of a two-parameter unit- Gammadistribution have been obtained. Furthermore, we have obtained estimates for the parameters α and β along with 95% confidence intervals and width of CIs based on parametric Bootstrap confidence intervals. The simulation results show that MPS estimators is the best performing estimator in terms of biases and RMSE. The next best performing estimators is the AD2, followed by MLE. The real data applications show that the MPS and AD2 estimators give the shortest confidence intervals for α and β, respectively for the data set-I and AD2 and ADR have the shortest confidence intervals for the data set-II. 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________________________________ ÁGORA FILOSÓFICA______________________________________________ Para una crítica inmanente del liberalismo. Theodor Adorno y la cuestión del individuo en Mínima Moralia. Gisela Catanzaro1 DOI 10.20399/P1982-999X.2016v1n3pp62-80 Resumen: A diferencia de lo que sucede con otros pensadores asociados al marxismo occidental -de G. Lukacs a L. Althusser, pasando por distintos miembros de la Escuela de Frankfurt-, las reflexiones ético-políticas de Th. Adorno suelen remitirnos a la tradición liberal del pensamiento, en particular en lo que respecta a su categoría central: la de individuo. En efecto, tan persistente como la problematización de la autonomía, la crítica ideológica de la personalidad autoritaria o el trazado de la genealogía violenta del sí mismo, parecería ser en su obra la negativa a realizarlas yendo “más allá” del individuo o apelando a alguna otra lógica que lo complemente. Sin embargo, lejos de la realidad aproblemática e inmediatamente reconocible por todo el mundo, el "individuo" adorniano es -como en Althusser, aunque por otros motivosuna suerte de enigma que exige desciframiento y que, si nos reenvía al liberalismo, lo hace en todo caso a uno "fuera de quicio", acosado por sus propios fantasmas. Aportar algunas claves interpretativas para leer esa figura cifrada es la tarea que nos proponemos abordar aquí partiendo de una relectura de Minima Moralia. Palabras clave: Th. Adorno – Individuo – Liberalismo – Minima Moralia Abstract:In contrast to other Occidental Marxists –from G. Lukacs to L. Althusser going through different members of the Frankfurt School-, Adorno’s ethic-political reflections usually send us back to the liberal tradition of political thought, starting with its main cathegory: the individual. As persistent as his problematization of authonomy, his ideological critique of authoritarian personality or his drawing of the violent geneology of the self, is his negative to perform them going beyond the individual or appealling to any other logic in order to “complement” it. However, far from that aproblematic reality that anyone can immediately recognize, Adorno’s “individual” is a kind of enigma that requires to be deciphered and which send us back to a “liberalism” but “out of joint”, “haunted” by its own ghosts. To contribute with some interpretative clues in order to read that weird figure is the task we asume here departing from Minima Moralia Keywords: Th. Adorno – Individual – Liberalism – Minima Moralia Introducción Si, urgidos por los desafíos emergentes de nuestra actual coyuntura, volvemos a interrogar la obra de Theodor Adorno respecto de aquello que, no sólo en el totalitarismo sino también en la democracia, amenaza a esta última, nos veremos inmediatamente confrontados con una serie de preocupaciones prioritariamente 1 Doctora en Ciencias Sociales por la Universidad de Buenos Aires, Argentina, Investigadora adjunta del Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) en el Instituto de Investigaciones Gino Germani y profesora en las carreras de Ciencia Política y Sociología de la Facultad de Ciencias Sociales UBA. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 62 ________________________________ ÁGORA FILOSÓFICA______________________________________________ asociadas a -y conjugadas en los lenguajes de- la tradición liberal del pensamiento político. Desde sus reflexiones sobre la “personalidad autoritaria” a Mínima Moralia, pero también desde Actualidad de la filosofía a Consignas, la “primacía del universal”, el colectivo homogeneizante, y una totalidad tan opresiva como ideológica, retornan una y otra vez como los peligros concretos frente a los cuales un pensamiento atento a lo falso de la presunción de reconciliación, se ve obligado a intentar rescatar lo que se halla en inminencia de desaparición: la autonomía; la consideración de cada uno frente al reconocimiento igualitario de todos; el individuo. Es indudable que esas reflexiones “ético-políticas” 2 elaboradas por Adorno exceden el marco liberal de su formulación ya sea allí donde enlazan la denunciada intolerancia frente a la singularidad con la lógica equivalencial desplegada por el capital, como donde esa crítica ético-política del todo se conjuga inextricablemente con una crítica dialéctica del conocimiento dominante en la que son expuestos los límites idealistas-empiristas de aquél pensamiento liberal. Sin embargo, no menos notoria resulta la asimetría existente entre las dimensiones ético-política -por una parte- y teórico-metodológica -por otra parte- de la crítica adorniana de la totalidad. Porque mientras la disputa con el “positivismo” se da como despliegue de la dialéctica interna de aquél, pero, precisamente, desde la no inmanencia -esto es: desde una aproximación “dialéctica”-, la reflexión ético-política de Adorno parecería, en cambio, no abandonar en ningún momento el “campo de la problemática” dispuesto por el liberalismo. Esto último resulta particularmente notorio en el caso de Mínima Moralia, donde más que de una reflexión ético-política, parecería necesario hablar de una reflexión moral dada a expensas de lo político en un gesto máximamente próximo a esa tradición liberal que según Carl Schmitt “elude o ignora al Estado y a la política de un modo genuinamente sistemático, y en su lugar se mueve en el seno de una polaridad típica y recurrente entre dos esferas heterogéneas, las de ética y economía”: Una tradición prosigue Schmitt- cuya “desconfianza crítica hacia el Estado y la política se explica fácilmente por los principios de un sistema para el cual el individuo es y debe seguir siendo tanto terminus a quo como terminus ad quem”.3 Mínima Moralia, texto que al igual que otros escritos por Adorno se resiste a tomar cualquier disociación de “principios” (políticos, morales, económicos…) como 2 3 Más abajo discutimos la posibilidad de sostener esta categoría respecto a Mínima Moralia. SCHMITT, C.. El concepto de lo político. Madrid: Alianza, 1998, p. 98-99. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 63 ________________________________ ÁGORA FILOSÓFICA______________________________________________ principio, acusa sin embargo una disociación real de ética y política en una época donde según él se “extrema la antítesis de vida pública y existencia individual”4 y donde un drástico desacople de escalas hace que la dimensión en que transcurre lo político sólo pueda ser registrada automáticamente pero no ya experimentada en términos morales por el individuo5. Aunque no “por principio” el ámbito de reflexión de este texto se encontraría, en efecto, y en consonancia con el liberalismo denunciado por Schmitt, acotado a esa dimensión privada de la vida en la cual el individuo emerge como figura central, y donde la política queda perfilada, en el mejor de los casos, exclusivamente de un modo negativo en las alusiones a una oscura e impermeable “Razón de Estado”. Así, podría sostenerse que un pensamiento de lo político tendría que prescindir de Adorno. O bien que, aún cuando no se tratara sencillamente de sancionar su caducidad o la necesidad de su abandono, una reapropiación de los planteos de Adorno a la luz de la cuestión de la democracia en nuestra actualidad precisaría ser conciente de la limitación liberal de su pensamiento para eventualmente buscar complementar sus reflexiones con otros aportes que permitieran pensar aquello que él no puede pensar, así como formular la necesaria crítica ideológica del liberalismo y de todas las categorías que conforman su andamiaje conceptual: empezando por la evidencia del individuo y el presupuesto de la armonía social invisiblemente garantizada. Sin embargo, la intuición orientadora de las páginas que siguen sugiere que es precisamente en la elaboración compleja y sutil de tales críticas donde el planteo de Adorno revela su mayor actualidad. Y esto no sólo debido a los contenidos que provee, sino a la misma modalidad de su reflexión que, en tanto crítica situada e inmanente, podría ayudarnos a determinar algunas limitaciones de ciertas tendencias actuales de la filosofía política. 1.- Pensamientos finitos e inacabados “Quien quiera conocer la verdad sobre la vida inmediata tendrá que estudiar su forma alienada” 4 ADORNO, Th.. Minima Moralia. Madrid: Editora Nacional, 2002, p. 171. “Quien comete acciones que, según las normas reconocidas, son contrarias a la rectitud, como la venganza contra los enemigos o la falta de compasión, apenas es conciente de la culpa, y sólo mediante un penoso esfuerzo puede imaginársela […] Sin embargo, cuando se piensa en faltas personales de tacto, microorganismos de injusticia que probablemente nadie notó […] pueden llenar al delincuente de continuo arrepentimiento e insoportable mala conciencia, y en ocasiones de tan sofocante vergüenza que sería incapaz de confesárselo a nadie, ni aún a sí mismo”. ADORNO, Th.. Minima Moralia. Op. cit., p. 171. 5 UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 64 ________________________________ ÁGORA FILOSÓFICA______________________________________________ Adorno, Th. Minima Moralia 6 “A la vista de la conformidad totalitaria que proclama directamente la eliminación de la diferencia como razón es posible que hasta una parte de la fuerza social liberadora se haya contraído temporalmente a la esfera de lo individual” Adorno, Th. Minima Moralia7 Entre las características habituales de las formas lingüísticas implicadas en los clichés socialmente aceptados se destaca su atemporalidad. Sin constituir una condición necesaria ni suficiente de su contextura ideológica, las autoevidencias en que se traman nuestros sentidos comunes parecerían, las más de las veces, querer valer para todos los tiempos y conseguirlo gracias al borramiento de lo que Walter Benjamin llamaba “el índice histórico de la imágenes”8, o bien -como se diría en una prosa más próxima al análisis estructural- gracias a la supresión en el enunciado de las marcas de la situación de enunciación. 65 Leídos sobre este trasfondo -que, como veremos en seguida, dista de ser caprichoso- en los fragmentos de Minima Moralia que escogimos como epígrafes y puntos de partida de nuestra reflexión, resalta ante todo el anclaje histórico de lo dicho y pensado. Un anclaje que si para Adorno no “justifica” al pensamiento al mostrarlo como parte de una totalidad significativa pretérita que garantizaría de antemano su sentido y su valor, sí “ancla” negativamente lo dicho al dejar expuestas las marcas de su finitud, y tornar legible -por contrapartida- la violencia implicada en la pretensión de eternidad de los enunciados que se quieren “sin tiempo ni lugar”. Muchos de esos enunciados provienen de la Filosofía. A ellos responde Adorno poniendo de relieve el carácter “referido” de la reflexión, que se produce “a la vista” de algo que le ha salido al paso, como respuesta a una estructuración actual del mito, y no como un pensamiento desplegado en sus propios términos válidos para todo tiempo y lugar. Como crítica de una tal violencia eternizante podrían concebirse, de hecho, los 6 ADORNO, Th.. Minima Moralia. Op. cit., p. 7. Ibidem, p. 10. 8 BENJAMIN, W.. “La obra de los Pasajes - Convoluto N” en La dialéctica en suspenso. Santiago de Chile: ARCIS-LOM, 1996, p. 122. 7 UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 ________________________________ ÁGORA FILOSÓFICA______________________________________________ persistentes cuestionamientos realizados por Adorno al idealismo ya sea en sus formulaciones científico-logicistas o en las (neo)ontológicas, y que alcanzan también a las corrientes formalistas de la Sociología. En todos los casos se trata para él de modos de pensamiento que de diversas formas “afectan eternidad” en tanto rechazan su propio ser suscitados, y a los cuales opone la dialéctica en tanto problematización del origen que relee como mediado aquello que en cada caso se querría dato último. Atendiendo a estas consideraciones “epistemológicas”, lo primero que cabría señalar a propósito de la aproximación adorniana al individuo, es su carácter no “original” sino urgido. Y esto en un doble sentido: si por un lado la consideración del “individuo” se le impone al intérprete materialista -antes que escogerla él entre un acervo de tópicos siempre disponibles para el ejercicio de una Filosofía eterna- y en ese sentido lo urge, además lo hace cuando él mismo está urgido, esto es, en el momento de su decadencia, cuando tanto la centralidad de la categoría de individuo como su misma existencia han perdido evidencia. Enseguida volveremos sobre ello a propósito de Mínima Moralia. Y no obstante cabría anticipar que, en toda la obra de Adorno, es una cierta urgencia en que se halla la experiencia individual la que a su vez urge un pensamiento que sólo se da como pensamiento emplazado. “Emplazado”, sin embargo, y esto es lo fundamental, menos en una “corriente del pensamiento”, que en una situación en la cual quedan expuestas también- las insuficiencias de la “corriente de pensamiento” que hace de esa figura del individuo su motivo central. Dicho de otro modo, en la “emplazada” reflexión adorniana sobre el individuo quedan expuestas las insuficiencias en el trato dado al individuo por parte del liberalismo, lo cual produce la situación paradójica de no poder ser liberal precisamente por fidelidad a aquello que el liberalismo tiene de verdadero pero que sistemáticamente traiciona al plantearlo como principio y como algo con lo que es posible contar. 2.- Desbordes del liberalismo En la persecución de intereses absolutamente particulares por parte de cada individuo puede estudiarse con la mayor precisión UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 66 ________________________________ ÁGORA FILOSÓFICA______________________________________________ la esencia de lo colectivo en la sociedad falsa. Adorno, Th. Minima Moralia9 La figura actual de lo colectivo en sí misma carece de lenguaje. Hoy ningún colectivo al que confiar la expresión del sujeto es ya sujeto. Adorno, Th. Minima Moralia10 Desde el mismo comienzo de Mínima Moralia el individuo aparece a la vez como un lugar de llegada y como algo no con-temporáneo o anacrónico, algo que no es del todo “presente” en el sentido de que no coincide con los tiempos. Es un lugar de llegada, en primer término, para la reflexión crítica que encuentra en la experiencia individual la única instancia en que aún es posible leer la persistencia del antagonismo al que más arriba aludimos como antítesis de vida pública y existencia individual- en el presente. Paradójicamente, no obstante, esa instancia individual “resistente”11 se muestra como particularmente reveladora en tanto anacrónica, es decir, donde y cuando el individuo ya ha sido sobrepasado y perdido algo de su con-temporaneidad. “Cuando el individuo, como todos los procedimientos individualistas de producción, aparece históricamente anticuado y a la zaga de la técnica, le llega de nuevo, en cuanto sentenciado, el momento de decir la verdad frente al vencedor”12, escribe Adorno. Y esta relación condicional que la categoría de individuo mantiene con la verdad es un primer índice de su “distancia” con la tradición liberal. Mientras para esta última el individuo constituye una categoría primera, válida para todos los tiempos y que remite a una evidencia actual, para Adorno es la experiencia que el individuo tiene de sí mismo en la edad de su decadencia - la experiencia de su nulidad- la que contribuye “a un conocimiento que él simplemente encubría durante el tiempo en que, como categoría dominante, se afirmaba sin fisuras.”13 Así, el individuo resulta un punto de llegada en este primer sentido porque, a la inversa, en su postulación como originario, como terminus a quo -digamos para retomar 9 ADORNO, Th. Minima Moralia. Op. cit., p. 38. Ibidem, p. 212. 11 Más adelante retomaremos la cuestión del carácter individual de la resistencia. 12 ADORNO, Th. Minima Moralia. Op. cit., p. 122. 13 Ibidem, p. 10. 10 UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 67 ________________________________ ÁGORA FILOSÓFICA______________________________________________ el planteo de Schmitt-, no sólo vuelve a afirmarse para Adorno el culto de la inmediatez que consagra lo primero sólo gracias al rechazo de su propio ser mediado, sino también porque al dar por descontada la existencia y centralidad de la instancia individual en la configuración actual del mundo, el pensamiento que parte de él resulta necesariamente ciego a su padecimiento e impotencia, así como a ese estado actual del mundo que los produce. Dicho de otro modo, la afirmación del individuo como primero y dado es ideológica. Lo es porque desconoce (en general) la mediación social de lo individual; pero lo es sobre todo porque, tras la afirmación de la centralidad eterna del individuo y la celebración de su esplendor, queda velada -en particular- la producción social de su nulidad en la sociedad de masas, y, junto con ella, queda velado el carácter falsamente reconciliado de la totalidad, lo falso de la “armonía” presupuesta en el pensamiento liberal. Aquí quedan planteados los términos de una de las críticas centrales de Adorno al liberalismo político y sobre la que volveremos más adelante: por sus mismos puntos de partida, éste resulta incapaz de realizar aquello que afirma, esto es, su voluntad de hacer justicia a la instancia individual. Pero precisamente esa alusión a una reconciliación inactual nos remite -antes- al segundo sentido en que el individuo sería, para Adorno, un punto de llegada; esto es: en tanto afirmación de la singularidad irreductible frente a la lógica actualmente dominante de la equivalencia y la sustituibilidad generalizadas. El término clave en la configuración de este segundo problema es “homogeneización”. El individuo es inactual, en esta nueva dimensión, menos por encontrarse ya en decadencia que por no haber devenido aún. Es “inactual”, en otros términos, en tanto la “libre comunicación de lo diferente” -como la llama a veces Adorno- supondría una ruptura de la igualación homogeneizante a la que lo singular está actualmente sometido bajo el imperio de la sociedad equivalencial. Es de esa lógica homogeneizante que emerge, como uno de sus productos, el individuo aislado que concurre al mercado guiado por su propio interés egoísta y que, a los fines del intercambio, resulta esencialmente sustituible por cualquier otro ejemplar de la especie. Pero “el pensamiento dialéctico -escribe Adorno- se opone a toda cosificación también en el sentido de negarse a confirmar a cada individuo en su UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 68 ________________________________ ÁGORA FILOSÓFICA______________________________________________ aislamiento y separación”14. Lo que hace, en cambio, es definir su aislamiento como producto de lo general: Precisamente en su individuación refleja el individuo la ley social inexplícita de la sin embargo bien conducida explotación […] Si hoy parece persistir un vestigio de lo humano únicamente en el individuo en tanto que perece, ese vestigio exhorta a poner fin a esa fatalidad que individúa a los hombres únicamente para poder separarlos tanto más perfectamente en su aislamiento.15 Lejos de constituir la última palabra sobre el individuo, el aislamiento es para Adorno la cicatriz dejada por aquello que, en la sociedad, posterga indefinidamente la posibilidad real de una individuación no cosificada, y que constriñe en cambio al individuo a la identidad sin mixtura y “auténtica” de lo siempre igual. Veamos esto más de cerca. En la crítica adorniana “aislamiento” y “homogeneización” no forman parte de sistemas heterogéneos y exteriormente enfrentados entre sí, sino que aparecen como elementos inherentes a la dinámica de la equivalencia. Allí donde triunfa, es decir, donde llega a todas partes y consigue configurar el mundo a su medida, ésta produce el aislamiento del individuo típico de las sociedades industriales avanzadas; las mismas sociedades que, a su vez, proveen la contrafigura de la masa como la instancia en la que esos átomos aislados se funden. Esa fusión, sin embargo, no puede conceptualizarse sin más como algo que le sobrevenga a las mónadas desde afuera, sino que ya estaba implicada en la misma sustituibilidad de los ejemplares operante en la constitución de los átomos aislados. Por ello, no menos que la masa, el individuo aislado constituye en Mínima Moralia un auténtico exponente del triunfo de la equivalencia y su lógica homogeneizante. Así como aislamiento y homogeneización son interpretados por Adorno menos como alternativas que como elementos constitutivos de una misma dialéctica, el individuo aislado y la masa son concebidos como síntomas de la misma deformación social de lo singular y de lo colectivo en ese presente histórico. De esto último no se deriva, sin embargo, la necesidad de postular la simple identidad de las figuras del individuo aislado y la masa. Ello implicaría pasar por alto su desigual dominancia en coyunturas históricas determinadas así como el nítido acento 14 15 ADORNO, Th. Minima Moralia. Op. cit., p. 63. Ibidem, p. 141-142. Volveremos sobre este fragmento en el tercer apartado. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 69 ________________________________ ÁGORA FILOSÓFICA______________________________________________ colocado por Adorno sobre la crítica de la totalidad en una coyuntura marcada por los totalitarismos, el monopolio, la razón de Estado, los medios masivos de comunicación, etc.. Pero la crítica adorniana de la interpretación liberal que entiende el aislamiento y la homogeneización, el individuo atomístico y la masa, como elementos simplemente externos y alternativos, crítica que se despliega mostrando -en cambio- la profunda solidaridad existente entre ellos en una sociedad determinada y de la cual son producto, esa crítica no podría satisfacerse con la mera postulación de una inversión por la cual el término dominado pasara a la posición dominante. No se trata de promover el fortalecimiento16 del individuo aislado contra la masa. Antes bien, de lo que se trataría para una perspectiva de la reconciliación pendiente, sería de un individuo y un colectivo transmutados, diversos de lo que han llegado a ser en cada caso. Podemos ahora ahondar algo más en el segundo sentido en que Mínima Moralia nos presenta al individuo como punto de llegada. Si más arriba sostuvimos que la lectura crítica descubre en la experiencia individual un potencial expresivo de la violencia acaecida en tanto conserva parcialmente las marcas del antagonismo, el individuo es asimismo un lugar de llegada en tanto no-contemporáneo también en el sentido de lo que no ha llegado a ser. El individuo como lugar de llegada es, aquí, diverso al individuo que es (el individuo aislado y confinado en su aislamiento). La marca de su inactualidad lo distingue, como sucedía en el primer caso, de esa individualidad que, en tanto categoría dominante “se afirmaba sin fisuras”, como figura rutilante del presente. Pero mientras la individualidad decadente revelaba como ideológica ante todo la confianza liberal en la vivacidad, pujanza y espontaneidad de lo ya mutilado, vuelto impotente y dirigido, en este segundo caso se llama ideología a la pretensión de que ha sido consumado lo que aún permanece pendiente. Una individuación pendiente que no puede decirse sencillamente en los términos de una liberación (negativa) de lo singular respecto del colectivo, esto es, a expensas de lo social trans-individual, sino que alude antes bien a otro tipo de relación social basada en aquello a lo que Adorno se refiere en el siguiente pasaje con el término “mímesis”: La autenticidad no es otra cosa que el obstinado y altanero encastillarse en la forma monadológica que la opresión 16 Al proyecto de un tal fortalecimiento Adorno respondería del mismo modo que frente a la apología neo-romántica de la corporalidad y la naturaleza que desconoce su devenir corpus, cadáver, en el decurso del proceso histórico civilizatorio tal como este se ha consumado. Al respecto, se pueden consultar los fragmentos filosóficos del final de Dialéctica de la Ilustración, en particular, “Interés por el cuerpo” y “Hombre y animal”. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 70 ________________________________ ÁGORA FILOSÓFICA______________________________________________ social imprime al hombre. Lo que no quiere marchitarse prefiere llevar el estigma de lo inauténtico. Entonces vive de la herencia mimética. Lo humano se aferra a la imitación: un hombre se hace verdaderamente hombre sólo cuando imita a otros hombres. En este comportamiento, forma primaria del amor, olfatean los sacerdotes de la autenticidad la pista de aquella utopía que podría sacudir la estructura del dominio.17 El individuo como lugar de llegada es indiferente a la (falsa) alternativa entre encasillamiento (en el sí mismo) y fusión (en la masa), una dualidad modelada en el patrón de la mismidad pura y sin mixtura cuyo cuestionamiento Adorno emprende aquí y en otros textos bajo el título de una crítica de la “autenticidad”. La lógica impulsada por esta última garantiza la reproducción de la estructura de dominio en tanto genera las identidades en las que aquél se sostiene, es decir, en tanto reprime las múltiples afinidades entre lo existente puestas de relieve en el impulso mimético, en favor de la producción de interioridades homogéneas y sin ventanas cuya única forma de afirmarse en la existencia depende de la posibilidad de sostener la independencia del sí mismo respecto de lo otro, y de la eliminabilidad de la presencia de lo otro en el sí mismo. La jerga de la autenticidad, así como la dicotomía amigo-enemigo a partir de la cual Carl Schmitt piensa la especificidad de lo político, no sólo no dicen la última palabra sobre “la” política18, sino que constituyen para Adorno expresiones de un “empobrecimiento de las relaciones entre las personas [...que tiende] ya antes de toda formación política de la voluntad y toda fijación de rótulos, al facismo.” Su modelo es el de la administración y la mirada evaluadora del manager al que las personas con las que entra en contacto se le convierten de antemano en obstaculizadores o facilitadores, y finalmente en admisibles o eliminables19. Pero el sentido de esta relación social empobrecida, señala Adorno, 17 ADORNO, Th. Minima Moralia. Op. cit., p. 146. Entre otras cosas porque no hay “la” política sino configuraciones históricas de la politicidad, planteadas en determinadas y singulares relaciones con lo que no es política: la moral, la economía, el conocimiento, el arte, lo ideológico. 19 “Quien hace del juicio sobre las aptitudes un asunto personal, ve a los enjuiciados, por una especie de necesidad tecnológica, como de los suyos o de los otros, como individuos de su especie o de otra, como 18 UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 71 ________________________________ ÁGORA FILOSÓFICA______________________________________________ ya se anunciaba en el “término catolicismo, una palabra griega para la latina totalidad -que los nacionalsocialistas han hecho realidad- [...y que] significa hacer equivaler lo diferente a lo adverso.” Si “lo auténtico” es -en la obra de Adorno- la forma vigente en que se constituyen de modo dominante las identidades individuales y colectivas, “mímesis”20 nombra tanto un individuo como un colectivo trasmutados. 3.- Armonía y totalitarismo liberal La doble perspectiva sobre la individualidad que hemos visto desplegarse en Mínima Moralia, esa aproximación bifocal que la trata como punto de llegada (y no de partida) deteniéndose en los aspectos contradictorios de su in-actualidad, le permite a Adorno extrañar la categoría liberal del individuo sin confrontarla en ningún momento desde el exterior. En algún sentido -que no obstante enseguida intentaremos desabsolutizar- podríamos decir que en este texto el individuo se vuelve espectral: entre lo “ya inactual” de una autonomía con la que dista de ser evidente que podamos contar, y lo “todavía inactual” de una individualidad no estandarizada, la confianza liberal en el individuo que es, se muestra en su complicidad -a pesar de las apariencias y declaraciones- con las fuerzas sociales que perpetúan indefinidamente el sometimiento de lo particular. Paralelamente, el individuo sólo parecería verdadero en tanto espectro, espectralizado, no siendo enteramente algo que actualmente es ¿Sería lo mismo dictaminar sencillamente la falsedad tout court de la categoría de individuo? ¿Sostener que, en tanto presencia, el individuo es falso, además de pieza clave de un discurso ideológico, y que más convendría pasar a otra cosa? Esta pregunta nos sugiere dos respuestas negativas de muy diversa índole y que resumiríamos (mal) del siguiente modo: a) no, porque ello haría imposible la crítica, y b) no, porque la crítica no es todo. Antes de terminar, consideremos brevemente estas dos negativas a “ir más allá” del individuo. sus cómplices o sus víctimas”. Esta y todas las citas del presente párrafo pertenecen al fragmento 85 de Minima Moralia. Op. cit., p. 123-124. 20 O “amor” que por momentos parecen sinónimos: “El amor es la capacidad de percibir lo semejante en lo desemejante.” Minima Moralia. Op. cit., p. 182. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 72 ________________________________ ÁGORA FILOSÓFICA______________________________________________ La simple eliminación de la categoría de individuo -o una renominación menos mítica de lo mentado por él- impediría, en primer lugar, formular la crítica de lo que funciona como ideología en un presente determinado, y aún cuando un nuevo nombre fuera capaz de llamar mejor a aquello a lo que el individuo alude, las fuerzas sociales que con el bautismo quedarían por fuera de la nominación, no por ello dejarían de ser efectivas21. Lo que oprime al individuo -también allí donde confía en su existencia, es decir, en el liberalismo- precisa ser nombrado, conceptualizado; y es precisamente esa conceptualización la que no se da a expensas de la categoría de individuo (más allá de ella, sustituyéndola) sino con la elaboración de su crítica. Pero además en Mínima Moralia esa crítica tiene la potencia de descubrir oscuras afinidades entre el liberalismo y lo que se presenta en primera instancia como su “absolutamente otro”: la pretensión “totalitaria” de una totalidad autosuficiente y armónica, dotada de una lógica transindividual propia y superior a la de los individuos. Adorno descubre esta afinidad secreta en su crítica a una figura que no cesa de invocar: la dialéctica hegeliana. Si, como mencionábamos más arriba, rechaza la hipostatización de la “categoría básica de la sociedad burguesa, el individuo”22 y reclama un conocimiento de la sociedad de la que es producto, lo mediador -la sociedad- tampoco puede ser afirmado según él como un primero al modo en que finalmente sucede en la totalización propuesta por Hegel. Del mismo modo en que sostuvimos que la “elitista” mónada aislada dista de ser la última palabra de Adorno sobre la individuación, es preciso enfatizar los límites que la totalización encuentra en su argumento y que -amén de la alusión crítica al cristianismo referida en el punto anterior- se resumen en el siguiente fragmento: También donde la no ingenuidad se concibe en el sentido teóricamente responsable de lo que mira más allá, de lo que no se detiene en el fenómeno aislado, de lo que piensa la totalidad, hay una zona oscura. Es simplemente aquel seguir sin poder detenerse, aquel tácito reconocimiento del primado de lo general frente a lo particular en que consiste no solamente el engaño del idealismo que hipostatiza los conceptos, sino también su inhumanidad, que, apenas captado, rebaja lo particular a 21 A propósito de esto siguen siendo esclarecedoras algunas de las más vibrantes páginas de Radiografía de la pampa. La “barbarie” de Sarmiento nombraba mal el peligro, dice allí Ezequiel Martínez Estrada, pero cuando todo fue “civilización” el mal ya no pudo ser nombrado y zonas enteras de la realidad cayeron en la irrealidad. 22 ADORNO, Th. Minima Moralia. Op. cit., p. 9. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 73 ________________________________ ÁGORA FILOSÓFICA______________________________________________ estación de tránsito para finalmente resignarse con demasiada rapidez, no sin dolor y sin muerte, en aras de una conciliación que meramente existe en la reflexión -es, en última instancia, la frialdad burguesa, que con excesiva complacencia suscribe lo inevitable. El conocimiento sólo puede extenderse hasta donde de tal modo se aferra al individuo que, por efecto de la insistencia su aislamiento se quiebra.23 En la primacía de lo general donde concluye el gesto totalizador del idealismo, se revela para Adorno su perfil apologético de la sociedad opresiva: la postulación de una “conciliación que meramente existe en la reflexión” y de cuya no-verdad sólo la experiencia individual podría dar testimonio. Por eso “casi podría decirse que […] la verdad depende del tempo, de la perseverancia y duración del permanecer en el individuo”24, es decir, de la permanencia de la reflexión no en la categoría rutilante de individuo, sino en esa experiencia individual que guarda las huellas de la sociedad antagonista y a la cual el pensamiento debe aferrarse puesto que es allí donde, a pesar de todo, según Minima Moralia ésta puede ser mejor leída como lo que es. Pero si en el Hegel apologeta de la totalidad se conjugan, por una parte, un límite epistemológico: el desconocimiento de aquello que la totalidad es (antagonismo) debido a la hipostatización de la reconciliación universal, y un límite ético: la “inhumanidad” de un sistema condenado a “seguir sin poder detenerse” a pesar del dolor y la muerte de lo particular, esto no sucede para Adorno a expensas del pensamiento liberal sino precisamente debido al liberalismo de Hegel: 23 Ibidem, p. 66 Ibidem, p. 69. El tema de la permanencia dialéctica en el individuo resulta sumamente sugerente a la luz de un comentario que realiza Fredric Jameson a propósito de la relación y diferencia entre dialéctica y deconstrucción. Según sostiene el autor en Valencias de la dialéctica, libro donde persigue su “parecido de familia” pero intentando comprender también su conflicto, “ambas trabajan para llevar a la luz las incoherencias estructurales de la ‘idea’ o de las ‘posiciones’ o interpretaciones conceptuales que constituyen el objeto de su crítica. Pero allí donde la dialéctica hace una pausa, esperando que la nueva solución ‘dialéctica’ se congele a su vez y se convierta en una idea o ideología a la que la dialéctica puede volver a ‘aplicarse’ [...], la deconstrucción se apresura, deshaciendo la incoherencia que había estado denunciando y demostrando que ese resultado analítico aparente es él mismo una nueva incoherecia y una nueva ‘contradicción’ que a su vez debe ser desarmado.” JAMESON, F..Valencias de la diléctica. Buenos Aires: Eterna cadencia, 2013, p. 39. Se trata de una diferencia de tempos, sin duda, pero una que, a su vez, parecería alertar sobre la diferencia entre un modo de la crítica que privilegia la deconstrucción de las interpretaciones ya producidas sobre un objeto, y otro modo de la crítica que vuelve a recomenzar una otra vez su movimiento a partir de las nuevas aristas que el objeto le va revelando a través de las diversas interpretaciones producidas a propósito del mismo pero sin reducirlo a ellas. Tanto en la figura del tratado invocada por Benjamin en el prólogo epistemocrítico de El origen del drama Barroco alemán, como en la consigna adorniana de la “primacía del objeto” parecería retornar una suerte de reclamo excesivo del material en relación a las interpretaciones ya provistas que orienta a la reflexión sobre algo otro y distinto del propio discurso crítico sobre el cual se vuelve, por el contrario, el gesto deconstructivo. 24 UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 74 ________________________________ ÁGORA FILOSÓFICA______________________________________________ El gesto displicente con que Hegel, en contradicción con su propia teoría, trata continuamente a lo individual proviene, de un modo harto paradójico, de su necesaria adscripción al pensamiento liberal. La representación de una totalidad armónica a través de sus antagonismos le obliga a atribuir a la individuación, por más que la determine siempre como momento impulsor del proceso, sólo un rango inferior en la construcción del todo.25 En la sociedad no reconciliada la representación de la armonía es totalitaria antes de hablar en favor de la centralización del poder o aún oponiéndose a ella. Aquí no se trata sólo de que, al presuponerlo como evidencia, el liberalismo resulte intrínsecamente incapaz de atender a la decadencia del individuo y de formular la crítica del mundo (homogeneizante, identitario, equivalencial) que la produce. En la negación del conflicto y la afirmación de la armoniosa composición de los intereses egoístas de cada uno -fundante del liberalismo pero que subyace también a la razón objetiva hegelianael individuo en cierto sentido ha sido siempre-ya abandonado en tanto postulado como esencialmente amoldado a y digerible por la lógica del todo. Adorno muestra que “liberalismo” y “primacía de lo general” no constituyen alternativas exteriores; muestra que Hegel espectraliza al individuo en el mismo momento en que, junto a la tradición liberal, confía en la racionalidad del Espíritu absoluto, su propia versión de la mano invisible. Y muestra también que el liberal presupuesto de la armonía es totalitario aún antes de que el liberalismo muestre su impotencia -o bien su funcionalidad- frente al totalitarismo. Pero precisamente porque en esa espectralización prematura Adorno lee los rastros de la “frialdad burguesa, que con excesiva complacencia suscribe lo inevitable”, él mismo se resiste a la espectralización, insistiendo en la necesidad de no reducir al individuo a un puro no ser. Si en la reflección crítica de Adorno el individuo es inactual decadente o por venir, es también siempre más que un puro espectro. O bien: su no ser, no menos que su ser, conlleva algo de ilusión: Quien quiera conocer la verdad sobre la vida inmediata tendrá que estudiar su forma alienada, los poderes objetivos que determinan la existencia individual hasta en sus zonas más ocultas. Quien habla con inmediatez de lo inmediato apenas se 25 ADORNO, Th. Minima Moralia. Op. cit., p. 9. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 75 ________________________________ ÁGORA FILOSÓFICA______________________________________________ comporta de manera diferente a la de aquellos escritores de novelas que adornan a sus marionetas con imitaciones de las pasiones de otros tiempos cual alhajas baratas y hacen actuar a personajes que no son nada más que piezas de la maquinaria como si aún pudieran obrar como sujetos y como si algo dependiera de sus acciones. La visión de la vida ha devenido en la ideología que crea la ilusión de que ya no hay vida.26 La autonomía individual como presupuesto y evidencia de la vivacidad de la vida es ideológica. Pero también la comprobación de que ya no hay vida es ilusión. Y es aquí donde se perfila la segunda razón adorniana para -como si dijéramos- no “abandonar al individuo”, para evitar dictaminar sencillamente la falsedad de la categoría de individuo o limitarse a sostener que, en tanto presencia, el individuo es falso. El individuo decíamos en el punto anterior- no puede caer como nombre sin arrastrar también a la ilegibilidad la violencia actual que lo somete, y que lo somete no necesariamente bajo los nombres de la violencia y las invocaciones épicas, sino en la pacífica referencia a las armonías preestablecidas. Pero hay más, porque la crítica no es todo, o mejor: porque el individuo no es sólo el jeroglífico que la crítica descifra sino que es también una débil fuerza de resistencia, poco más que un “vestigio” -como lo llamaba Adorno en uno de los fragmentos citados más arriba- pero un vestigio que “exhorta”: “Si hoy parece persistir un vestigio de lo humano únicamente en el individuo en tanto que perece, ese vestigio exhorta a poner fin a esa fatalidad que individúa a los hombres únicamente para poder separarlos tanto más perfectamente en su aislamiento.”27 Sin provenir de una presencia plena, rutilante, dominante, la exhortación a poner fin a un tipo determinado de relación social empobrecedora, es tan real y actual como las fuerzas dominantes en función de las cuales se da la estructuración del todo. Adorno se resiste a la tentación de poner las cosas en términos de presencia o ausencia y busca en cambio otra modulación de la presencia/ausencia: la presencia frágil o “débil” -como la llamaba Benjamin-, el vestigio de presencia -como leemos aquí-. Porque no sólo lo que oprime debe ser nombrado, sino que también lo que padece, resiste y “exhorta” exige una nominación, en Mínima Moralia “individuo” no es sólo sido-advenidero sino también nombre de una débil resistencia actual. A lo que -no obstante- inmediatamente hay que agregar: lo que resiste en el individuo no es sólo de naturaleza individual: 26 27 Ibidem, p. 7. Enfatizado por mí. Ya citado en nota 14: ADORNO, Th. Minima Moralia. Op. cit., p. 141-142. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 76 ________________________________ ÁGORA FILOSÓFICA______________________________________________ El rechazo de la confusión reinante en la cultura presupone que se participa de ella lo suficiente como para sentirla palpitar, por así decirlo, entre los propios dedos, mas al propio tiempo presupone que de dicha participación se han extraído fuerzas para denunciarla. Tales fuerzas, que se presentan como fuerzas de resistencia individual, no son por ello de índole meramente individual. La conciencia intelectual en la que se concentran tiene un momento social en la misma medida que lo tiene el superyo moral. Dicho momento se decanta en una representación de la sociedad justa y sus ciudadanos.28 Si el individuo no es mero espectro, sino también resistencia y exhortación aquí y ahora, hay sin embargo más que individuo en la resistencia “individual”. Hay una “fuerza social liberadora” temporalmente “contraída” en una esfera -la de lo individualdonde, estrictamente hablando, para Adorno, no puede ser política pero tampoco moral sino, a lo sumo, contener “microorganismos de justicia”. Sólo en la figura de lo colectivo -esa figura que, decía Adorno “actualmente carece de lenguaje” y no puede expresar al sujeto- habría política y moral en un sentido del que aquella actualidad sólo conoce vestigios. Y bueno es reconocerlos como tales, diría Adorno a un liberalismo para el que lo privado curiosamente ha dejado de estar privado -marcado por la limitación- para constituirse en ideal, pero también a un “realismo” que toma por la esencia de lo político lo que no constituye sino un producto particular de ciertas relaciones sociales empobrecidas: la capacidad de configurar de una vez el mundo en la cuadrícula del o bien, o bien. 4.- Para terminar Adorno nos arrojaría entonces un liberalismo, pero uno “fuera de quicio”, acosado por sus propios fantasmas. A la inversa, no sería desde un más allá del individuo o apelando a alguna otra lógica que lo complemente sino -como si dijéramos“extremando su concepto”, como se elaborarían en su obra la genealogía violenta del “sí mismo”, la crítica de la “personalidad”, o la problematización de la autonomía. Por contrapartida -no obstante- el individuo en cuestión ya no podría identificarse con 28 Ibidem, p. 21. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 77 ________________________________ ÁGORA FILOSÓFICA______________________________________________ aquella categoría para nosotros familiar, que promete incesantemente remitirnos a una realidad aproblemática e inmediatamente reconocible por todo el mundo; categoría “simple” de la cual se podría disponer en el proceso de comprensión de realidades “más complejas”. El “individuo” en juego en la obra de Adorno sería -antes bien, como en la obra de Althusser aunque por otros motivos- una suerte de enigma que exige desciframiento, y descifrar ese individuo que es más y otra cosa que el individuo “normal”/normalizado pero también más que un fantasma con el fin de complejizar nuestras teorizaciones actuales sobre la democracia, fue la tarea que nos propusimos abordar aquí partiendo de una relectura de Mínima Moralia. En tal sentido, y retomando ahora algunos de los elementos desplegados, digamos que Adorno extraña el modo en que solemos pensar los problemas de la democracia por lo menos de tres modos. En primer lugar, y a propósito del carácter situado del pensamiento y la no “eternidad” de las categorías filosóficas, “individuo” pero también “colectivo”, “política”, “totalitarismo” o “democracia” dejan de constituirse como los tópicos perennes de la filosofía, para tener que ser leídos en su constelación y remitidos al tipo de relaciones sociales involucradas en la definición de su concepto en cada caso. (Por ejemplo: relaciones sociales reducidas al modelo contractual, empobrecidas en términos dicotómicos, etc.). En segundo lugar, el tema de la no originariedad de ninguna de estas categorías se asocia a lo relativo de su valor de verdad. Pero “relativo” no “en general”, al modo de un relativismo que aceptaría gustoso que cada uno privilegie la que quiera y monte su edificio a partir de allí, sino en el sentido que mentábamos el carácter condicional de la relación que la categoría de individuo sostiene con la verdad de acuerdo a su posición decadente o triunfal; subordinada, central o periférica; rutilante o vetusta; celebrada o vapuleada, en coyunturas precisas. Apelando a la noción de ideología, podríamos reformular lo anterior diciendo que una interrogación de aquellos términos que mentábamos más arriba no puede darse independientemente de lo que funciona como ideología en un momento determinado. Esto es, no puede darse sin interrogar la posición que esos términos tienen, o no, en la configuración de los ideologemas centrales de una época y que pueden convertir al individuo en una ideología, pero también a la (anti)política, la tecnocracia, la nación e incluso la democracia (sobre todo si es liberal y del consenso) en ideologías. UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 78 ________________________________ ÁGORA FILOSÓFICA______________________________________________ En tercer lugar, y como decíamos a propósito de la doble valencia de la inactualidad del individuo en Mínima Moralia, pero también del mismo presupuesto de la armonía esencial de lo social que lo rebaja de antemano, surge la necesidad de no osificar el concepto de lo ideológico en una unidad sin tensiones ni reducirlo a un sólo y mismo nivel: si en la primer entrada que planteamos ideológica es la confianza en que aún goza de buena salud lo que ya ha fenecido y se fortalece un cadáver (confianza en que es presente lo ya inactual), en la segunda se llama ideología a la pretensión de que la promesa se ha cumplido (pretensión de que ya es presente lo aún pendiente), y en el tercero se muestra la solidaridad entre esas absolutizaciones del individuo y una representación dominante de la sociedad: en el postulado (liberal) de la armonía social se elabora la aniquilación del individuo antes de que el liberalismo se pronuncie respecto a él. Finalmente, en el entrelazamiento de estos elementos resultan legibles las distancias de Adorno, en tanto pensador singular, con las tradiciones más incisivas de la teoría política. En primer lugar, distancia con la tradición liberal en la que Adorno parecería instalarse y que es objetada tanto por Schmitt como por el marxismo: tampoco para Adorno el individuo es un terminus a quo, y es más bien la experiencia que el individuo tiene de sí mismo en la edad de su decadencia la que contribuye a formular la crítica del individuo como ideología en su época de esplendor. Sin embargo, a diferencia de Schmitt, para Adorno el individuo en el sentido de una individuación no domesticada bajo la lógica de la comparabilidad y la competencia sino modulada por las potenciales afinidades y el impulso mimético, sí es un extraño terminus a quo, punto de llegada deseable. No obstante, ese advenimiento dista de representar un simple triunfo del individuo sobre la totalidad, e implica en cambio una trasmutación de ambos que interviene críticamente sobre el par “individuo como principio”/ “sociedad armónica” sostenido por el liberalismo (aunque vigente mucho más allá de él en figuras antiliberales o no liberales que sostienen la primacía de alguna objetividad transindividual dotadora de sentido a un real carente de él). En último término, y a diferencia de ciertas tendencias postfundacionalistas de la teoría política, la figura del individuo que en planteo de Adorno es “extrañada” y no sencillamente negada, es a su vez más que un puro vacío o no-ser actual, más que una pura espectralidad amenazante. Como intentamos mostrar, en la crítica de los supuestos, principios y evidencias del individuo Adorno no se limita a la pura espectralización sino que aquél emerge, en UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2 79 ________________________________ ÁGORA FILOSÓFICA______________________________________________ cambio, como una fuerza de resistencia real y limitada que “exhorta” desde su acotamiento a “poner fin a esa fatalidad que individúa a los hombres únicamente para poder separarlos tanto más perfectamente en su aislamiento”.29 Correo electrónico: giselacatanzaro@yahoo.com Dirección postal: Santiago del Estero 674, piso 6, CABA, Argentina. 80 29 Citado más arriba (nota 14). UNIVERSIDADE CATÓLICA DE PERNAMBUCO Ano 16 • n. 3(especial) • jul./dez. 2016-2
https://openalex.org/W4307553301
https://www.frontiersin.org/articles/10.3389/fonc.2022.1010122/pdf
English
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Meta analysis of indocyanine green fluorescence in patients undergoing laparoscopic colorectal cancer surgery
Frontiers in oncology
2,022
cc-by
7,126
OPEN ACCESS OPEN ACCESS EDITED BY Gaetano Gallo, Sapienza University of Rome, Italy REVIEWED BY George Pappas-Gogos, Democritus University of Thrace, Greece Pietro Fransvea, Scientific Institute for Research, Hospitalization and Healthcare (IRCCS), Italy Nunzio Velotti, University of Naples Federico II, Italy *CORRESPONDENCE Xiangquan Lai 1587415141@qq.com Tianbao Xiao prof_xiaotianbao@163.com SPECIALTY SECTION This article was submitted to Surgical Oncology, a section of the journal Frontiers in Oncology RECEIVED 08 August 2022 ACCEPTED 03 October 2022 PUBLISHED 26 October 2022 CITATION Deng J, Hu W, Li Y, Xiong K, Yue T, Lai X and Xiao T (2022) Meta analysis of indocyanine green fluorescence in patients undergoing laparoscopic colorectal cancer surgery. Jia Deng 1, Wenting Hu 1,2, Yang Li 2,3, Kai Xiong 1, Tinghui Yue 1, Xiangquan Lai 3* and Tianbao Xiao 3* Jia Deng 1, Wenting Hu 1,2, Yang Li 2,3, Kai Xiong 1, Tinghui Yue 1, Xiangquan Lai 3* and Tianbao Xiao 3* 1College of Clinical Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang, China, 2College of Pharmacy, Dali University, Dali, China, 3Colorectal and Anal Surgery, The First Affiliated Hospital of Guizhou, University of Traditional Chinese Medicine, Guiyang, China This meta-analysis intended to systematically evaluate the clinical implications of indocyanine green fluorescence (ICG) in patients undergoing laparoscopic colorectal surgery. PubMed, MEDLINE, Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), Wanfang Database, VIP Medical Information System and China Biomedical Database were synthetically searched for studies published from inception to April 14, 2022. The randomized controlled trials comparing ICG-use with controls were selected. The incidence of anastomotic leakage (AL), lymph node detection, operation duration, intraoperative bleeding, postoperative morbidity, and hospitalization time were evaluated in summary analysis, and calculated the corresponding 95% confidence intervals (CI). Subsequently, in addition to subgroup analyses, studies for heterogeneity, sensitivity, and publication bias were carried out. Consequently, 3453 patients in the enrolled 15 studies were included; 1616 patients were allocated to the experimental group, and 1837 patients were assigned to the control group. The ICG group had a significantly decreased risk of AL (RR: 0.50, 95% CI: 0.37–0.67) and shorter hospitalization time (SMD: -0.31, 95% CI: -0.54–0.08) compared to the control group. Meanwhile, the ICG showed clearly better lymph node detection (SMD: 0.19, 95% CI: 0.02–0.36). However, when the content of operation duration (SMD: - 0.07, 95% CI: -0.30–0.15) and intraoperative bleeding (SMD: -0.16, 95% CI: - 0.35–0.04) were compared, no statistical significance was found. TYPE Systematic Review PUBLISHED 26 October 2022 DOI 10.3389/fonc.2022.1010122 TYPE Systematic Review PUBLISHED 26 October 2022 DOI 10.3389/fonc.2022.1010122 TYPE Systematic Review PUBLISHED 26 October 2022 DOI 10.3389/fonc.2022.1010122 OPEN ACCESS Furthermore, the pooled analysis of postoperative morbidity was not statistically significant (RR:0.79, 95% CI: 0.58–1.08). The results of the subgroup analysis of AL indicated that there may be regional variations in AL (RR: 0.50, 95% CI: 0.37– 0.67) but not in postoperative morbidity (RR: 0.79, 95% CI: 0.58–1.08). In conclusion, the application of ICG in laparoscopic colorectal surgery can CITATION Deng J, Hu W, Li Y, Xiong K, Yue T, Lai X and Xiao T (2022) Meta analysis of indocyanine green fluorescence in patients undergoing laparoscopic colorectal cancer surgery. Front. Oncol. 12:1010122. doi: 10.3389/fonc.2022.1010122 CITATION Deng J, Hu W, Li Y, Xiong K, Yue T, Lai X and Xiao T (2022) Meta analysis of indocyanine green fluorescence in patients undergoing laparoscopic colorectal cancer surgery. Front. Oncol. 12:1010122. doi: 10.3389/fonc.2022.1010122 COPYRIGHT © 2022 Deng, Hu, Li, Xiong, Yue, Lai and Xiao. 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. 01 Frontiers in Oncology frontiersin.org Deng et al. Deng et al. 10.3389/fonc.2022.1010122 10.3389/fonc.2022.1010122 effectively reduce the AL, lymph node detection, and hospitalization time. However, more multicenter large-sample randomized controlled trials are required to further confirm its advantages. The meta-analysis was registered in PROSPERO (no. CRD42022288054). indocyanine green, laparoscopic surgery, meta-analysis, colorectal cancer, systematic review Protocol registration This agreement was previously registered in PROSPERO in December 2021. (Number: CRD42022288054, https://www.crd. york.ac.uk/PROSPERO/display_record.php?RecordID=288054). Introduction including the accurate intraoperative localization of the tumor and the rigorous cleaning of the lymphatic drainage area. In addition, since ICG can be used to observe the intestinal blood supply at the anastomosis, it has significant practical advantages in lowering the incidence of postoperative anastomotic leakage (AL) (13). However, this technique is still in the exploratory stage in the diagnosis and treatment of CRC, so there are no standardized application criteria and operation specifications, and few guidelines and consensus for reference. Systematic observation and analysis are lacking in the application value of ICG. In this study, we will investigate the efficacy of ICG in laparoscopic colorectal surgery and provide reliable evidence- based medical evidence for its wide application in clinical practice. Colorectal cancer (CRC) is one of the most prevalent gastrointestinal malignancies with high incidence and mortality, seriously threatening human life and health. The latest global statistics show 1,148,515 new cases of colon cancer and 732,210 new cases of rectal cancer in 2020, with a 9.4% mortality rate for CRC patient (1). In addition, mortality from CRC is projected to increase significantly by 71.5% (rectal cancer) and 60% (colon cancer) in 2035 (2). In the Chinese population, CRC is also one of the most common cancers and has shown a pattern of increasing incidence and mortality rates over time (3). Radical surgery (R0/R1 resection) remains to be a primary procedure for the management of CRC and recommended by the National Comprehensive Cancer Network (NCCN) and European Society for Medical Oncology (ESMO) guidelines. Laparoscopic colorectal surgery has become a standard and mainstay operation for the treatment of localized CRC with the continuous development of minimally invasive technology (4, 5). In comparation with open abdominal surgery, laparoscopic surgery has significant advantages, such as minimally invasiveness, little bleeding, quickly postoperative recovery and improvement in patient care quality. However, the blindness of lymphatic clearance and the inability to touch become the biggest challenges to be solved in the laparoscopy. Furthermore, the technical challenges of laparoscopic surgery for CRC also have been raised by robotic technology with advantages of articulating wrists, lack of hand tremors, and decreasing the learning curve (6). Therefore, an auxiliary technology is urgent for laparoscopic colorectal surgery to improve its precision and individuality. Frontiers in Oncology Quality evaluation The quality of the included studies was evaluated by two researchers independently, and conflicts among them were resolved through discussion or third-party consensus. Risk bias assessments were produced using RevMan 5.3 software based on the Cochrane Risk of Bias Assessment Tool. The evaluation included the following seven major components: random sequence generation; allocation concealment; blinding of patients and testers; blinding of outcomes assessors; incomplete results; selective reporting; and other bias (such as potential bias related to special research design, declaration of fraud, etc.). Finally, judgments should be made in this study, such as “low bias risk,” “high bias risk,” and “uncertain bias risk.” Qualification criteria The Cochrane Handbook for Systematic Reviews of Interventions and the PRISMA Statement were consulted for this study. Meanwhile, the “PICOS” principles were used as a guide for this study’s inclusion and exclusion standards. Studies must meet the following inclusion criteria: (i) Patients with laparoscopic colorectal surgery for CRC (including colon and rectal cancer) were of different sex, age, race, and nationality. (ii) The experimental group was allocated in laparoscopic colorectal surgery by ICG, while the control group was assigned in laparoscopic colorectal cancer without ICG. (iii) The effectiveness between conventional laparoscopic colorectal surgery and laparoscopic colorectal surgery with ICG was compared. (iv) At least one of these outcome indicators must Indocyanine green (ICG) is a near-infrared light contrast agent with good biocompatibility. It is excited by external light at a wavelength of 750–800 nm, and in-turn emits near-infrared light of a longer wavelength for visualization of tissues and organs (7). Since Nagata first applied ICG in colorectal surgery in 2006, considerable research value and good application prospects in the adjuvant diagnosis and therapy of CRC have been demonstrated by this technology (8–12). More and more attention has been paid to the role of ICG in laparoscopy, Frontiers in Oncology 02 frontiersin.org frontiersin.org 10.3389/fonc.2022.1010122 Deng et al. 10.3389/fonc.2022.1010122 time, and intraoperative bleeding. The original author was contacted if the necessary information was not available, and the data was considered missing if there was no response. Furthermore, the disagreements between two researchers were settled through discussion or consensus, and a third researcher was requested to solve the conflict if required. be reported, including the main outcome indicators such as AL and lymph node detection. The secondary outcome indicators include postoperative morbidity, operation duration, hospitalization time, and intraoperative bleeding. (v) The design of these studies were prospective randomized controlled trials (RCTs). Studies were asked to meet the following exclusion criteria: (i) Full text or specific values of the required indicators were not available. (ii) Comparison of the efficacy of two procedures was not included; (iii) duplicate publications; (iv) case reports, conference reports, reviews, animal experimental papers, and meta-analysis. Search methodology Two researchers conducted a search in the following databases: pubMed, MEDLINE, Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), Wanfang Database, VIP Medical Information System, and China Biomedical Database (CBM). Meanwhile, the period was limited from the database’s creation until April 14, 2022. The search terms were composed of the following medical themes (MeSH) and additional conditions: (colorectal cancer/colorectal neoplasms/colorectal tumor) AND (indocyanine green/ fluorescence/spectroscopy near-infrared) AND (randomized controlled experiments/clinical trials). Furthermore, manual studies would be conducted to find potential references. Language was not an obstacle to publication. Data analysis The data was statistically analyzed using Stata 14.0 (Stata Corporation). Relative risk (RR) and standardized mean difference (SMD) were used as effect statistics for dichotomous and continuous variables, and 95% confidence intervals (CI) were calculated. The I2 test was initially used to investigate heterogeneity. If P ≥0.1 and I2 ≤50%, heterogeneity between studies was small and a fixed-effect model was used; if P < 0.1 and I2 > 50%, heterogeneity existed between studies and a random-effect model was used for meta-analysis. Subgroup analysis was carried out based on the various included study areas, and sensitivity analysis was carried out using the leave- one-out method for outcome indicators with high heterogeneity, as well as the contour-enhanced funnel plot. Research selection The program Endnote™, Version X8 (Thompson Reuters) was used to combine all search results. Repeated studies were manually removed. Two researchers independently screened the original studies and then read the full text to select the literature that met the inclusion criteria. Disagreements should be settled through discussion or by reaching an agreement with a third party. Frontiers in Oncology Quality evaluation of included studies The methodological quality assessment of the 15 included studies was displayed in Figure 2. The formation of random sequences was fully recognized in all of the selected studies, as shown in Figure 2A. Meanwhile, allocation concealment was ambiguous. None of studies mentioned the application of blinding, and the evaluation of performance bias was regarded Literature selection results The data extraction was completed independently by two researchers who cross-checked the results using a uniform data extraction form. The following information was included in the data extraction: (i) The research ID, including the first author’s name and publication date. (ii) The research participant, including the number and age of the participants. (iii) The treatment program for the experimental and control groups. (iv) the main outcome indicators, such as AL and lymph node detection, and the secondary outcome indicators included postoperative morbidity, operation duration, hospitalization Initially, 523 potentially relevant papers were screened, and 50 duplicate items were completely removed. Furthermore, 423 papers were excluded after reading the titles and abstracts because they were not suitable for the inclusion criteria. 35 articles were retained after carefully reviewing the abstracts and full texts of the remaining 50 articles. Finally, 15 studies were enrolled after rigorous screening using inclusion and exclusion criteria (Figure 1). Frontiers in Oncology 03 frontiersin.org frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 FIGURE 1 Literature screening process. Evidence quality The GRADEpro, which was developed by the Cochrane Collaboration Network, was used to assess the quality of evidence. A classification of the evidence was made for these studies and based on their limitations, inconsistencies, indirectness, imprecision, and publication bias. The quality of the evidence was classified as high, moderate, low, or very low. The quality of evidence was moderate for these outcome indicators due to a lack of blinding in RCTs. The profile of GRADE evidence was displayed in Figure 3A, and a detailed summary of the findings was shown in Figure 3B. Study characteristics as high risk (Figure 2B). There were no studies with incomplete or biased results. The methodological quality of all selected studies remained low due to the complete lack of blinding. A total of 3453 individuals (14–28) were enrolled in 15 included studies; 1616 of these patients were in the ICG group and 1837 were in the control group. The fundamental features of all included studies were displayed in Table 1. ICG was administered through an intravenous injection (IV) in 12 trials (14, 15, 17–21, 24–28) and as a submucosal injection in 3 trials (16, 22, 23). The AL result was reported in 10 trials (14, 15, 17– 21, 25, 27, 28). There were 5 trials that reported lymph node detection (22–24, 26, 28). Postoperative morbidity was observed in 8 trials (14–16, 22–24, 26, 28). Operation time was reported by 9 trials (16, 21–28), hospitalization time was reported by 8 trials (16, 19, 21–24, 26, 28), and intraoperative bleeding was reported by 7 trials (21–24, 26–28). Frontiers in Oncology Meta-analysis of AL Heterogeneity was examined first before the pooled analysis. Ten studies displayed low significant heterogeneity (P=0.405, I2 = 3.9%) so a fixed-effect model was used to combination. The result showed a significantly lower incidence of AL [RR=0.50, 04 Frontiers in Oncology frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 TABLE 1 Basic characteristics of the included studies in the meta-analysis. TABLE 1 Basic characteristics of the included studies in the meta analysis. Research ID Sample number (n) Ages (year) Tumor types Dose of ICG Route of medication Outcomes study control study control Alekseev M (14) 187 190 Range:21-86 Range:66-85 CRC 0.2 mg/kg iv ①③ De Nardi, P (15) 118 122 Range: 29-88 CRC 0.3 mg/kg iv ①③ Park JH (16) 114 228 67.91 ± 8.94 66.81 ± 10.18 CRC 0.5-1 ml submucosal injection ③④⑤ Ishii M (17) 223 265 Range:30–90 Range:27–93 CRC 5 mg iv ① Bonadio L (18) 33 33 71.85 ± 11.1 69.03 ± 11.3 CRC 0.2 mg/kg iv ① Kin C (19) 173 173 58.2 ± 13.2 58.1 ± 13.2 CRC 3 ml iv ①⑤ Ren P (20) 63 82 NR CRC 2.5 mg/ml iv ① Wu G.C (21) 130 130 66.63 ± 4.72 67.53 ± 4.59 CRC 2.5 mg/ml iv ①④⑤⑥ Zhang J.F (22) 68 77 60.2 ± 12.5 58.4 ± 14.1 CRC 2.5 mg/ml submucosal injection ②③④⑤⑥ Zhou SC (23) 12 30 60.3 ± 9.6 58.5 ± 9.5 RC 0.1 mg/ml submucosal injection ②③④⑤⑥ Su H (24) 84 105 59.1 ± 11.1 60.2 ± 9.8 CC 3 ml iv ②③④⑤⑥ Tsang YP (25) 62 69 69.82 ± 9.89 67.71 ± 11.65 CRC 10 mg iv ①④ Ge L (26) 36 22 56.4 ± 9.6 58.1 ± 11.0 CC 3 ml iv ②③④⑤⑥ Foo CC (27) 253 253 66.6 ± 10.6 67.2 ± 11.0 CRC 5 mg iv ①④⑥ Jing D.S (28) 60 58 56.7 ± 7.42 54.4 ± 7. 64 CC 2.5 mg/ml iv ①②③④⑤⑥ NR, not reported; IV, intravenous injection; ①, AL; ②, Lymph node detection; ③,Postoperative morbidity; ④,Operation duration;⑤, Hospitalization time; ⑥, Intraoperative bleeding. 95% CI (0.37–0.67), Z=4.49, P=0.000] in the ICG group. Meanwhile, a subgroup analysis of AL was conducted by different regions. The studies were divided into two subgroups based on their geographical location: Asia and Europe/America. Four studies were reported from Europe/America [RR=0.64, 95% CI (0.44–0.95), Z=2.25, P=0.025] and six from Asia [RR=0.34, 95% CI (0.21–0.57), Z=4.11, P=0.000]. Meta-analysis of hospitalization time The test of heterogeneity was first conducted before performing a pooled analysis, and the result showed that there was obviously high heterogeneity (P = 0.000, I2 = 76,3%) among these studies. A random-effect model was chosen to combine, and the pooled analysis showed that hospitalization time in the Meta-analysis of AL The subgroup analysis revealed statistically significant differences [RR=0.50, 95% CI (0.37–0.67), Z=4.49, P=0.000] across regions (Figure 4). low heterogeneity across the five studies that reported lymph node detection outcomes (P = 0.192, I2 = 34.4%). A fixed-effects model was used to combine the pooled analysis, and the result revealed that this indicator was noticeably higher in the ICG group than in the control group [SMD=0.19, 95% CI (0.02–0.36), Z=2.16, P=0.031]. However, a subgroup analysis was not performed due to the small number of enrolled studies (Figure 6). Meta-analysis of postoperative morbidity The test of heterogeneity was first conducted before performing a pooled analysis, and there was obviously high heterogeneity (P = 0.000, I2 = 79.9%) across the nine studies that reported operation duration. A random-effect was used to do the pooled analysis. The result revealed that there was no statistically significant difference among these studies (SMD = -0.07, 95% CI (-0.30–0.15), Z = 1.53, P = 0.126). However, a subgroup analysis was not conducted because of the same distribution (Figure 7). The test of heterogeneity in the pooled analysis was examined first, and there was no significant heterogeneity (P=0.970, I2 = 0.0%) in the eight studies. The pooled analysis of postoperative morbidity was performed by a fixed-effects model, and there was no statistically significant [RR= 0.79, 95% CI (0.58–1.08), Z = 1.46, P = 0.143] in the ICG group compared with the control group. Meanwhile, a subgroup analysis was performed, the result revealed no significant variance [RR = 0.79, 95%CI (0.58–1.08), Z=1.46, P=0.143] between the different locations (Figure 5). Meta-analysis of lymph node detection The test of heterogeneity was first conducted before performing a pooled analysis, and there was significantly 05 Frontiers in Oncology frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 B A FIGURE 2 Methodological quality graphs and summaries: (A) Summary of risk of bias; (B) Graph of risk of bias. A B quality graphs and summaries: (A) Summary of risk of bias; (B) Graph of risk of bias. B FIGURE 2 Methodological quality graphs and summaries: (A) Summary of risk of bias; (B) Graph of risk of bias. B FIGURE 2 Methodological quality graphs and summaries: (A) Summary of risk of bias; (B) Graph of risk of bias. ICG group and the control group [SMD = -0.16, 95% CI (-0.35–0.04), Z = 1.54, P = 0.122]. A subgroup analysis was not carried out in this meta-analysis since intraoperative bleeding was distributed similarly across regions (Figure 9). ICG group was considerably decreased (SMD = -0.31, 95% CI (0.54–0.08), Z = 5.96, P = 0.000). A subgroup analysis was not carried out due to the small number of included studies coming from America/Europe (Figure 8). Sensitivity analysis for robustness of pooled analysis The test of heterogeneity was first examined before performing a pooled analysis, and the result showed that there was moderate heterogeneity (P = 0.021, I2 = 59.7%) among these studies. Consequently, a pooled analysis was carried out using a random-effect model. The result showed that there was no statistically significant difference between the The sensitivity analysis was conducted by the leave-one-out method to evaluate the robustness of the combined results (hospitalization time, operation duration, and intraoperative bleeding) in the study. The robustness of the hospitalization time may have been impacted if Wu.G.C. (21) was excluded from the Frontiers in Oncology 06 frontiersin.org frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 B A FIGURE 3 Level of quality of evidence: (A) GRADE evidence profile; (B) Summary table of findings. A B 3 of quality of evidence: (A) GRADE evidence profile; (B) Summary table of findings. FIGURE 3 Level of quality of evidence: (A) GRADE evidence profile; (B) Summary table of findings. In addition, the sensitivity analysis of intraoperative bleeding (Figure 10C) demonstrated that the robustness and reliability of the pooled analysis would not be significantly impacted by any study. Finally, the pooled analysis was generally reliable and robust to some degree according to all the sensitivity analysis findings. study, but there were no opposing results (estimated SMD = -0.19, 95% CI: -0.31–0.08; Figure 10A). According to the sensitivity analysis of operation duration, Tsang Y.P. (25)’s exclusion from the study may have an impact in this study but not the opposite way around (estimated SMD = -0.16, 95% CI: -0.33–0.01; Figure 10B). Frontiers in Oncology 07 frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 FIGURE 4 Risk ratio (RR) forest plot of AL and subgroup analysis of AL. (A): Forest plot of AL. (B): Subgroup analysis of AL. FIGURE 4 Risk ratio (RR) forest plot of AL and subgroup analysis of AL. (A): Forest plot of AL. (B): Subgroup analysis of AL. FIGURE 4 Risk ratio (RR) forest plot of AL and subgroup analysis of AL. (A): Forest plot of AL. (B): Subgroup analysis of AL. Contour-enhanced funnel plot to detect the potential source of publication bias 0.05, P < 0.1, or P > 0.1). The contour-enhanced funnel plots of hospitalization time (Figure 11A), operative duration (Figure 11B), and intraoperative bleeding (Figure 11C), all missing RCTs were within the region of low statistical significance (P >0.1), which suggested that the asymmetry of the funnel plots was due to publication bias. The counter-enhanced funnel plots were used to investigate the cause of publication bias, which contours represent statistically significant conventional milestones (P < 0.01, P < Frontiers in Oncology 08 frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 B A forest plot of postoperative morbidity and subgroup analysis of postoperative morbidity. (A): Forest plot of postoperative Subgroup analysis of postoperative morbidity. A B est plot of postoperative morbidity and subgroup analysis of postoperative morbidity. (A): Forest plot of postoperat FIGURE 5 Risk ratio (RR) forest plot of postoperative morbidity and subgroup analysis of postoperative morbidity. (A): Forest plot of postoperative morbidity. (B): Subgroup analysis of postoperative morbidity. Frontiers in Oncology Discussion nodes effectively under the laparoscopy. Meanwhile, due to the potential of laparoscopy to reduce postoperative pain and promote the recovery of digestive function, the impact on the abdominal cavity becomes small. Furthermore, laparoscopy makes the images larger, the surgery more precise, and decreases intraoperative bleeding and surgical injuries (7, 32, 33). However, there are still With the rapid progress of the modern minimally invasive techniques, the efficacy of laparoscopic colorectal surgery in the treatment of CRC has been confirmed (29–31). The surgeon would observe the abdomen clearly, peel off the lesion, and detect lymph Frontiers in Oncology 09 frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 FIGURE 6 Standard mean difference (SMD) forest plot of lymph node detection. FIGURE 6 Standard mean difference (SMD) forest plot of lymph node detection. many difficulties in laparoscopic colorectal cancer, among which the main difficulty is to accurately determine the anastomosis blood flow during the operation. The primary cause of AL is the insufficient anastomotic blood flow during surgery. AL can be reduced if the operation program is revised based on anastomotic blood supplies in time (34). Currently, the perfusion of the digestive segment during laparoscopy mainly depends on the surgeon’s subjective opinion, such as monitoring the color of the anastomosis and observing the incision margin, etc. There is a possibility of AL due to errors in judgment objectively (35). Furthermore, it is still a difficulty to detect lymph nodes completely in laparoscopic colorectal surgery. It is essential to evaluate the quality of surgery by detecting lymph nodes thoroughly in operation, especially for lymph nodes, which may FIGURE 7 Standard mean difference (SMD) forest plot of operation duration. URE 7 ndard mean difference (SMD) forest plot of operation duration. FIGURE 7 Standard mean difference (SMD) forest plot of operation duration. Frontiers in Oncology frontiersin.org 10 10 Frontiers in Oncology frontiersin.org frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 FIGURE 8 Standard mean difference (SMD) forest plot of hospitalization time. FIGURE 8 Standard mean difference (SMD) forest plot of hospitalization time. ICG is a mildly toxic fluorescent substance that has been used in colorectal surgery more and more in recent years to monitor intestinal perfusion and visualize the surgical area, thus the incidence of AL would effectively reduce (36, 37). From 1998 to 2003, Kudszus et al. Discussion enrolled 402 patients in a control group without ICG who had colorectal surgery and a test group of patients who underwent ICG-assisted surgery from 2003 to 2008. The test group suggested that patients with poor perfusion underwent reanastomosis with the proximal bowel free, and the final be ignored by using standard dissection protocols based on experience. Therefore, it is urgent to find out a technique that can accurately perform laparoscopic surgery and improve the effectiveness of surgical therapy while guiding and displaying anastomosis blood flow and lymph node detection in real time throughout the entire operation process. Therefore, it will be an important technological breakthrough to find a method that can guide and determine anastomosis blood flow and lymph node detection for laparoscopic colorectal surgery. FIGURE 9 Standard mean difference (SMD) forest plot of intraoperative bleeding. FIGURE 9 Standard mean difference (SMD) forest plot of intraoperative bleeding. FIGURE 9 Standard mean difference (SMD) forest plot of intraoperative bleeding. 11 11 Frontiers in Oncology frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 B C A FIGURE 10 Sensitivity analysis via leave-one-out procedure each time. (A): Sensitivity analysis of hospitalization time. (B): Sensitivity analysis of operation duration. (C): Sensitivity analysis of intraoperative bleeding. FIGURE 10 Sensitivity analysis via leave-one-out procedure each time. (A): Sensitivity analysis of hospitalization time. (B): Sensitivity analysis of operation duration. (C): Sensitivity analysis of intraoperative bleeding. percentage of AL was observed in both the test and control groups of 7.5% (15/201) and 3.5% (7/201), which indicated that ICG reduced the incidence of AL (38). The use of ICG in colorectal anastomosis procedures is increasing, and all of them have shown good results (37, 39, 40). The application of ICG provides a new option for lymphatic tracing technology (41, 42). Lymph nodes can be detected by ICG tracing up to 65.5%–100% for CRC, which helps guide lymph node dissection and raise the probability of having more positive lymph nodes (31, 43–46). The study by Nishigori et al. found that ICG altered lymph node dissection in 23.5% (4/21) of cases, and all nodes >5 mm in diameter were identified (47). hospitalization time. ICG assesses anastomotic blood flow more objectively before and after intestinal anastomosis in laparoscopic colorectal surgery and reduces the incidence of AL, which is consistent with the findings of Jafari MD et al. (38). Frontiers in Oncology Discussion Meanwhile, ICG has some advantages in lymph node tracing, such as the ability to precisely locate the distribution of metastatic lymph nodes and anterior lymph nodes, particularly extra-regional lymph nodes, and guide intraoperative lymph node dissection, improve lymph node detection rate after surgery, and reduce the number of missed positive lymph nodes (48, 49). Furthermore, ICG can precisely locate the lesion with less intraoperative trauma during laparoscopic colorectal surgery to reduce the hospitalization time. In this study, there was no statistical difference in the postoperative morbidity, It was found that the use of ICG in laparoscopic colorectal surgery reduced AL, enhanced lymph node detection, and reduced Frontiers in Oncology 12 frontiersin.org Deng et al. 10.3389/fonc.2022.1010122 A B C FIGURE 11 Contour-enhanced funnel plots of hospitalization time, operation duration and intraoperative bleeding. (A): Contour-enhanced funnel plot of hospitalization time (B): Contour-enhanced funnel plot of operation duration. (C): Contour-enhanced funnel plot of intraoperative bleeding. A B C C FIGURE 11 Contour-enhanced funnel plots of hospitalization time, operation duration and intraoperative bleeding. (A): Contour-enhanced funnel plot of hospitalization time (B): Contour-enhanced funnel plot of operation duration. (C): Contour-enhanced funnel plot of intraoperative bleeding. intraoperatively, as well as to determine the blood supply to the intestine at the anastomosis, which has important practical value for reducing AL and improving lymph node detection. intraoperative bleeding, and operation duration between the two groups, which suggested that the application of ICG did not affect the surgical operation and postoperative recovery of the patients. In conclusion, the use of ICG in laparoscopic colorectal surgery is reliable and efficient, with the primary benefits of improving lymph node detection, reducing AL, and shortening hospitalization time. Data availability statement Although strict inclusion and exclusion criteria for the meta- analysis literature were established, the following issues remain: (i) the quantity of articles was insufficient and the included literature’s quality was average; (ii) although the inclusion studies were RCTs, not all of them mentioned the use of blinding; (iii) some indicators have a significant degree of heterogeneity when combined, which could affect the article’s results; and (iv) according to the findings of the subgroup analysis, regional differences in AL outcomes may exist. These factors may have an impact on the final conclusions, which must be validated further by a large sample, multicenter, prospective randomized controlled trials. The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors. Frontiers in Oncology Acknowledgment All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. We are grateful to the authors for providing detailed data for this meta-analysis, as well as to all colleagues in this study for their hard work. References Int J Colorectal Dis (2020) 35(2):269– 75. doi: 10.1007/s00384-019-03482-0 5. Fujii S, Akagi T, Inomata M, Katayama H, Mizusawa J, Ota M, et al. Transitional impact of short- and long-term outcomes of a randomized controlled trial to evaluate laparoscopic versus open surgery for colorectal cancer from Japan clinical oncology group study JCOG0404. Ann Gastroenterol Surg (2019) 3(3):301–9. doi: 10.1002/ags3.12245 5. 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Quantifying a Sustainable Management Space for Human Use of Coastal Groundwater under Multiple Change Pressures
Water resources management
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Water Resour Manage (2016) 30:4063–4080 DOI 10.1007/s11269-016-1363-1 Quantifying a Sustainable Management Space for Human Use of Coastal Groundwater under Multiple Change Pressures K. Mazi1,2,3 & A. D. Koussis2,3 & G. Destouni1,3 Received: 1 July 2015 /Accepted: 18 May 2016 / Published online: 9 June 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Received: 1 July 2015 /Accepted: 18 May 2016 / Published online: 9 June 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Received: 1 July 2015 /Accepted: 18 May 2016 / Published online: 9 June 2016 # The Author(s) 2016. This article is published wi # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract In the densely populated coastal regions of the world, loss of groundwater due to seawater intrusion, driven by changes of climate, sea level, land use and water use, may critically impact many people. We analytically investigate and quantify the limits constraining a coastal aquifer’s sustainable management space, in order to avoid critical loss of the coastal groundwater resource by seawater intrusion. Limiting conditions occur when the intrusion toe reaches the pumping wells, well intrusion, or the marine-side groundwater divide, complete intrusion; in both cases the limits are functions of the seaward groundwater flow remaining after the human groundwater extractions. The study presents a screening-level approach to the quantification of the key natural and human-determined controls and sustainability limits for the human use of coastal groundwater. The physical and geometrical characteristics of the coastal aquifer along with the natural conditions for recharge and replenishment of the coastal groundwater are the key natural controls of the sustainable management space for the latter. The groundwater pumping rates and locations are the key human-determined controls of this space. The present approach to combining and accounting for both of these types of controls is Highlights • A general screening-level framework for evaluating sustainable coastal aquifer management options. • Identified critical conditions in an exploited coastal aquifer: well, or complete aquifer intrusion. • Simple & general Sustainable Management Space quantified based on key natural & pumping controls. • Sustainability bounds are directly related to remaining groundwater flow after pumping. • Rule of thumb is derived for 1st-order regional threat and management assessment of coastal aquifers. Electronic supplementary material The online version of this article (doi:10.1007/s11269-016-1363-1) contains supplementary material, which is available to authorized users. Electronic supplementary material The online version of this article (doi:10.1007/s11269-016-1363-1 contains supplementary material, which is available to authorized users. * K. 1 Department of Physical Geography, Bolin Centre for Climate Research, Stockholm University, SE-106 91 Stockholm, Sweden 2 Institute for Environmental Research and Sustainable Development, National Observatory of Athens, I. Metaxa & Vas. Pavlou Str, GR-152 36 Athens, Palea Penteli, Greece Highlights • A general screening-level framework for evaluating sustainable coastal aquifer management options. • Identified critical conditions in an exploited coastal aquifer: well, or complete aquifer intrusion. • Simple & general Sustainable Management Space quantified based on key natural & pumping controls. • Sustainability bounds are directly related to remaining groundwater flow after pumping. • Rule of thumb is derived for 1st-order regional threat and management assessment of coastal aquifers. Electronic supplementary material The online version of this article (doi:10.1007/s11269-016-1363-1) contains supplementary material, which is available to authorized users. 3 Navarino Environmental Observatory (NEO), Messinia, GR-240 01 Kalamata, Greece 2 Institute for Environmental Research and Sustainable Development, National Observatory of Athens, I. Metaxa & Vas. Pavlou Str, GR-152 36 Athens, Palea Penteli, Greece 3 Navarino Environmental Observatory (NEO), Messinia, GR-240 01 Kalamata, Greece SE-106 91 Stockholm, Sweden 2 Institute for Environmental Research and Sustainable Development, National Observatory of Athens I. Metaxa & Vas. Pavlou Str, GR-152 36 Athens, Palea Penteli, Greece 3 N i E i l Ob (NEO) M i i GR 240 01 K l G 1 Introduction In 2001, over half the world’s population lived within 200 km of a coastline, and eight of the ten largest cities in the world are located by the coast; http://www.oceansatlas.org/. In the typically densely populated coastal regions, people rely often, and largely, on groundwater for drinking, food production and their economies. This is true especially for semi-arid regions and more so for arid coastal regions, which may be water-stressed or even water-deprived when densely inhabited; the Middle East and North Africa are pertinent examples (Leas et al. 2014). If coastal aquifers are exploited intensively, i.e., the groundwater abstractions substan- tially modify the aquifer conditions (Llamas and Custodio 2003), the naturally occurring seawater intrusion (SWI) increases and can threaten large-scale contamination of the coastal groundwater resource. Through progressive salinization, the groundwater will then become non-potable and the seawater may eventually invade pumping wells entirely; potable water standard is 500 ppm TDS, corresponding to water mixture with 2 % seawater of average-ocean salinity (35,000 ppm TDS). Coastal regions are also exposed to other hydro-climatic changes and associated alterations of aquifer forcing on both the land and the marine side. Land-side changes may be related to climate change and to various changes in land- and water-use (Destouni et al. 2013; Jaramillo and Destouni 2014), not least due to agricultural irrigation that accounts for two thirds of world-water use (Postel 1997; Jaramillo and Destouni 2015). Such land-side changes are in addition to the direct intensive and rising use of coastal groundwater by increasing coastal populations and tourism (Llamas and Custodio 2003; Parry et al. 2007; Ferguson and Gleeson 2012). Marine-side changes are related to climate-driven sea-level change. In particular, SWI in coastal aquifers is sensitive to altered aquifer forcing, in terms of groundwater level and seaward flow. Such alterations are implied by increased groundwater use (Llamas and Custodio 2003; Post 2005; Ferguson and Gleeson 2012; Mazi 2014), but also by overall hydro-climatic variability (Prieto and Destouni 2005; Prieto et al. 2006) and climate-driven change, such as reduction in groundwater recharge (Döll 2009; Small 2005) and sea-level rise (Nicholls and Klein 2005; Masterson and Garabedian 2007; Werner and Simmons 2009; Webb and Howard 2011; Loáiciga et al. 2012; Mazi et al. 2013). If the aquifer- forcing regime is thusly altered, certain thresholds or tipping points (Lenton 2011) can be non- linearly approached and ultimately crossed, causing drastic shifts in SWI (Mazi et al. 2013). Quantifying a Sustainable Management Space for Human Use of Coastal Groundwater under Multiple Change Pressures Mazi kmazi@noa.gr * K. Mazi kmazi@noa.gr 1 Department of Physical Geography, Bolin Centre for Climate Research, Stockholm University, SE-106 91 Stockholm, Sweden 4064 K. Mazi et al. simple, yet general. The approach is applicable across different scales and regions, and for historic, current and projected future conditions of changing hydro-climate, sea level, and human freshwater use. The use of this approach is also concretely demonstrated for the natural and human-determined controls and limits of the sustainable management space for two specific Mediterranean aquifers. Keywords Seawater intrusion . Coastal aquifer. Sustainable water management. Sustainability limits . Groundwater use . Mediterranean aquifers 1 Introduction The high non-linearity of the SWI response to forcing changes (Werner and Simmons 2009) implies that, after a tipping point has been crossed, even a minor further forcing change can greatly enhance SWI into the coastal aquifer. To relatively simply address and quantify the SWI response to forcing changes, Koussis et al. (2012) developed an analytical framework that extends previous analytical solutions A Sustainable Management Space for the Use of Coastal Groundwater 4065 (Strack 1976). This extended framework has been used, for instance, to assess the proximity of prominent Mediterranean aquifers to the site-specific critical thresholds that determine the above-mentioned tipping points under prevailing aquifer conditions (Mazi et al. 2014, Mazi (Strack 1976). This extended framework has been used, for instance, to assess the proximity of prominent Mediterranean aquifers to the site-specific critical thresholds that determine the above-mentioned tipping points under prevailing aquifer conditions (Mazi et al. 2014, Mazi l = L Sea Submarine discharge Hsea Zone 1: Fresh- water Zone 2: Interface l = lw qw qr Interface toe r l = 0 φ Inland boundary Well (a) Groundwater divide qr l = 0 Interface toe r Zone 1: Fresh- water Zone 2: Interface l = L φ Hsea l = lw Sea Submarine discharge qw Well Inland boundary (b) Fig. 1 Critical seawater intrusion in a coastal aquifer: a intrusion into water supply wells, with the toe of seawater-freshwater interface reaching the well location; b complete intrusion into the coastal aquifer, with the interface toe reaching the coastal groundwater divide (point of maximum resistance to intrusion), which may be located between the pumping well and the coast (Mazi 2014) l = L Sea Submarine discharge Hsea Zone 1: Fresh- water Zone 2: Interface l = lw qw qr Interface toe r l = 0 φ Inland boundary Well (a) Inland boundary (a) Zone 2: Interface φ l = L l = lw l = 0 φ Groundwater divide qr l = 0 Interface toe r Zone 1: Fresh- water Zone 2: Interface l = L φ Hsea l = lw Sea Submarine discharge qw Well Inland boundary (b) Fig. 1 Introduction 1 Critical seawater intrusion in a coastal aquifer: a intrusion into water supply wells, with the toe of seawater-freshwater interface reaching the well location; b complete intrusion into the coastal aquifer, with the interface toe reaching the coastal groundwater divide (point of maximum resistance to intrusion), which may be located between the pumping well and the coast (Mazi 2014) Groundwater divide qr l = 0 Interface toe r Zone 1: Fresh- water Zone 2: Interface l = L φ Hsea l = lw Sea Submarine discharge qw Well Inland boundary (b) Fig. 1 Critical seawater intrusion in a coastal aquifer: a intrusion into water supply wells, with the toe of seawater-freshwater interface reaching the well location; b complete intrusion into the coastal aquifer, with the interface toe reaching the coastal groundwater divide (point of maximum resistance to intrusion), which may be located between the pumping well and the coast (Mazi 2014) (b) Groundwater divide Fig. 1 Critical seawater intrusion in a coastal aquifer: a intrusion into water supply wells, with the toe of seawater-freshwater interface reaching the well location; b complete intrusion into the coastal aquifer, with the interface toe reaching the coastal groundwater divide (point of maximum resistance to intrusion), which may be located between the pumping well and the coast (Mazi 2014) 4066 K. Mazi et al. 2014). Such thresholds may concern intrusion to key water supply wells (Fig. 1a) or complete intrusion up to the groundwater divide (Fig. 1b) in a coastal aquifer. 2014). Such thresholds may concern intrusion to key water supply wells (Fig. 1a) or complete intrusion up to the groundwater divide (Fig. 1b) in a coastal aquifer. In general, analytical interface-flow solutions, although far less complete than variable-density solutions, offer clarity of interpretation, require few data and are far more readily evaluated, hence also better amenable to stochastic analyses. Such analytical solutions are thus suitable for first-order regional-scale assessments of coastal aquifer vulnerability to SWI (Mazi et al. 2014), as well as for screening of aquifer management scenarios (Koussis et al. 2012). Numerical variable-density solu- tions can complement screening-level solutions by studying in detail cases of partic- ular interest, accounting for irregular multi-dimensional geometries and hydrogeological heterogeneities, provided adequate field data support the detailed simulations (Sanford and Pope 2010). 1 Introduction Controlling the threats to coastal groundwater discussed above requires monitoring and managing of the coastal aquifers, not only for meeting present-day water demand and supply, but also regarding long-term sustainability. Sustainability is defined as the ethical obligation to ensure that the current use of a resource does not compromise its use by future generations (World Commission on Environment and Development 1987). Complex hydrogeology, variable natural and human-controlled forcing and demand, all exacer- bated by uncertainty, make meeting this sustainability obligation for the groundwater resource a formidable task. Hydrogeological concepts relating to such aquifer man- agement include an ambiguous safe yield (Llamas and Custodio 2003) as a possible simple rule against overdraft, with the latter defined as groundwater withdrawals exceeding the aquifer recharge, and various other vulnerability indicators (Werner et al. 2012). Executing this management task employs then typically advanced flow models, combined with optimisation considering technological options and socio-economics (Koussis et al. 2010a, 2010b; Stigter et al. 2015; Zuurbier et al. 2016). To complement such detailed and site-specific management approaches, the present study develops a general screening-level framework for evaluating sustainable management options for coastal groundwater under various possible, current and future, aquifer and hydro-climatic conditions. This is done in terms of a set of key, inter-linked yet readily calculated, natural and human-controlled limits to the use of coastal groundwater for avoiding its critical loss by SWI. Such framework development and its quantification are needed and useful for first-order regional assessment of main threats and management options for the regional resource of coastal fresh groundwater (Ferguson and Maxwell 2012), for example for water-stressed or water-deprived coastal regions such as the Middle East and North Africa. In this study, natural and human-controlled limits are derived and define a sustainable management space (SMS) for the human use of coastal groundwater resources. This general concept is here also concretely applied to and quantified for the Israel Coastal Aquifer (ICA) and the Cyprus Akrotiri Aquifer (CAA). The concrete exemplification of the use of the general screening framework for these two regional aquifers of the Southeastern Mediterranean is motivated by their importance as freshwater sources for the local populations and for this region’s economic prosperity (details in Mazi et al. 2014). The Southeastern Mediterranean region is particularly threatened by reduced groundwater recharge (Döll 2009; Bring et al. 1 Introduction 2015) along with many additional change pressures from human activity developments over at least the last 50 years (Mazi et al. 2014), which have all affected and will continue to affect SWI into the coastal groundwater resources of this region. 2 Method In this study we use and further develop the analytical framework of Koussis et al. (2012), enhanced and refined in Koussis et al. (2015). The conceptualization of the coastal groundwater system (Fig. 1) represents an inclined (sinφ) unconfined aquifer, with base depth at the coast Hsea below sea-level, average hydraulic conductivity K, and length L to a land-boundary, with either known flow, e.g., zero-flow at an impervious boundary or groundwater divide, or known hydraulic head; the ℓ-axis follows the aquifer base, starting at Hsea below the intersection of the sea surface. The aquifer is recharged by precipitation (and possibly artificially) minus evapotrans- piration at resulting net rate r, in addition to land-boundary inflow qb. Human control conditions are quantified in terms of a representative regional groundwater pumping rate qw at a representative (e.g., flow-weighted average) location from the coast ℓw (Destouni and Prieto 2003; Prieto and Destouni 2005; Koussis et al. 2010a, 2010b). SWI is quantified by the toe of a nominal seawater-freshwater interface located at distance ℓT from the coast, interpreted as the 50 %-salinity isoline in the transition zone between fresh groundwater and seawater. Online Material 1 summarises the analytical model. A Sustainable Management Space for the Use of Coastal Groundwater 4067 2.1 Determining a Sustainable Management Space (SMS) Identification and quantification of a regional SMS departs from the groundwater flow qr directly after the location ℓw of groundwater pumping at rate qw: qr ¼ r L−ℓw ð Þ−qw−qb: ð1Þ ð1Þ We shall henceforth call qr the groundwater outflow remaining after pumping, with the understanding that it can be truly remaining, i.e. flowing seawards: qr > 0, or flowing towards the well: qr < 0; thus, qr > 0 is hydraulically negative (flux in the –ℓdirection). This sign reversal is management-suitable. That groundwater flow qr links to the fresh groundwater outflow to the sea qSD through the relation qSD = −rL + qw + qb = −qr – rℓw (Mazi et al. 2013); qSD corresponds to the freshwater component of total submarine groundwater discharge (Destouni and Prieto 2003). The limits on the groundwater extraction (qw) that are allowed by the natural regional groundwater recharge (r) and are required for sustain- able groundwater management are thus quantifiable in terms of the groundwater flux qr that must remain after pumping so that the fresh groundwater outflow to the sea, qSD, can resist critical SWI. The limits on qr are derived and quantified in this study, considering for simplicity a no-flow inland boundary condition. This simplification represents a conservative SMS assessment: it expects the total recharge (to the land boundary, rL) of coastal groundwater alone (without groundwater inflow qb through the land boundary) to yield a fresh groundwater flux to the sea (qSD) that suffices to resist critical SWI. To fully control SWI, however, monitoring of the groundwater table (hydraulic head) and management triggered by too low groundwater levels are also needed to assess boundary flows and complement the flux-based management approach exempli- fied in this study (Werner et al. 2011). 4068 K. Mazi et al. Based on the analytical framework of Koussis et al. (2012), we can identify the following general conditions for the critical case of SWI into the representative well location ℓw/L (illustrated in Fig. 1a; red dashed line in Fig. 2a) ℓT L ¼ ℓw L for 0 ≤qr > qmax r ð2aÞ ð2aÞ and the critical case of complete SWI intrusion into the coastal aquifer up to its defining coastal groundwater divide (illustrated in Fig. 1b; blue solid line in Fig. 2a; eq. 2.1 Determining a Sustainable Management Space (SMS) In a the relative intrusion toe location ℓT/L is shown for given groundwater recharge rate r and aquifer extent L under variable groundwater flow remaining directly after pumping qr, and in b qr is shown for given L and variable r qr lT/L qr max = r(L- lw) qr min = - r lw 0 Limiting condition: lT = lw Limiting condition: lT = ldiv Complete intrusion Well intrusion Limiting conditions lT/L = lw/L -qr (a) (a) Limiting condition: lT = l - qr + qr Limiting conditions Complete intrusion Well intrusion r (b) 0 (b) Limiting conditions A Sustainable Management Space for the Use of Coastal Groundwater 4069 flow qr is then qr max = r(L – ℓw), which applies to zero pumping (qw = 0) in the coastal aquifer. Well intrusion (Fig. 1a) is also the limiting critical case for any qr ≥0, because positive qr in Eq. 2b for the alternative critical case of complete aquifer intrusion implies ℓT/L > ℓw/L. That is, Eq. 2b for qr ≥0 yields deeper SWI than ℓw/L, implying that the critical well intrusion limit ℓT/ L = ℓw/L (Fig. 1a) is reached before the alternative critical limit ℓT/L = ℓdiv/L for complete intrusion (Fig. 1b). For the case of complete aquifer intrusion (Fig. 1b; Eq. 2b; blue solid line in Fig. 2a), Eq. 2b implies that the critical location of the interface toe (ℓT/L) is a function of qr min < qr ≤0, i.e., of negative qr until a lowest limit qr min = −rℓw; qr min is the zero-value of Eq. 2b (for maximum possible pumping). For any given value of net recharge rate r, the slope of the critical line segment for this negative-qr range is (rL)−1. The allowance of negative remaining qr after pumping implies that more fresh groundwater can be sustainably withdrawn by pumping qw at ℓw than is recharged inland of ℓw. This is so because pumping draws fresh groundwater from both a landward and a seaward zone around ℓw and can be sustained as long as the resulting coastal groundwater divide ℓdiv and the resulting intrusion toe ℓT remain seaward of ℓw. As both the upper qr limit qr max = r(L – ℓw) and the lower qr limit qr min = −rℓw depend on the net recharge rate r, the SMS may also be illustrated as function of r (Fig. 2b). 2.1 Determining a Sustainable Management Space (SMS) A5 in Online Material 1): and the critical case of complete SWI intrusion into the coastal aquifer up to its defining coastal groundwater divide (illustrated in Fig. 1b; blue solid line in Fig. 2a; eq. A5 in Online Material 1): Material 1): ℓT L ¼ ℓdiv L ¼ 1 rLqr þ ℓw L for qmin r < qr > 0⋅ ð2bÞ ð2bÞ These conditions are further quantified and illustrated in the Results section. Here, we note that Eqs. 2a-b form a general basis for delimiting an SMS for coastal groundwater, as illustrated in Fig. 2a. These conditions are further quantified and illustrated in the Results section. Here, we note that Eqs. 2a-b form a general basis for delimiting an SMS for coastal groundwater, as illustrated in Fig. 2a. For the critical case of well intrusion (Fig. 1a; Eq. 2a; red dashed line in Fig. 2a), the location of the SWI interface toe (ℓT/L) is by definition the representative well location, i.e., ℓT/L =ℓw/L. The naturally bounded upper limit (qr max) of the seaward groundwater qr lT/L qr max = r(L- lw) qr min = - r lw 0 Limiting condition: lT = lw Limiting condition: lT = ldiv Complete intrusion Well intrusion Limiting conditions lT/L = lw/L -qr (a) - qr + qr Limiting conditions Complete intrusion Well intrusion r (b) 0 Fig. 2 Schematic representation of SMS (grey). The limits of the SMS for coastal groundwater are shown for given relative well location ℓw/L, as determined by critical complete intrusion (blue line, corresponding to intrusion conditions in Fig. 1b) and critical well intrusion (red line, corresponding to intrusion conditions in Fig. 1a). 2.1 Determining a Sustainable Management Space (SMS) This r-space illustration of the SMS complements that for a fixed r (Fig. 2a). The full shaded area in Fig. 2b represents the general SMS, falling between the upper bound of qr max = r(L – ℓw) (green dashed line) and the lower bound of qr min = −rℓw (blue solid line). The total site-specific range of sustainable qr is the vertical line of qr min < qr > qr max for the given site-specific r value. The partitioning of the site-specific range of sustainable qr between the positive segment 0 ≤qr ≤qr max and the negative segment qr min < qr ≤0 represents the fresh-groundwater fraction drawn from the landside (limited by the critical case of well intrusion; Fig. 1a) and from the seaside (limited by the critical case of complete intrusion; Fig. 1b) around the pumping location ℓw, respectively. 3.1 General and Specific Sustainable Management Space In an aquifer characterized geometrically by L/Hsea, moving the representative well location further inland (increasing ℓw/L, and thereby also ℓw/Hsea) allows for more pumping and smaller sustainable qr before reaching the limit of complete intrusion. From the calculation results for different site conditions –Online Material 2, Fig. S1– follows that: (1) for any given location ℓw/L, aquifers with higher recharge rate r allow for more pumping of fresh groundwater, and thus lower remaining qr; (2) for any given recharge rate r, sites with greater hydraulic conductivity K imply greater SWI (greater ℓT/L values) before a critical condition is reached. In general, varying the K value shifts (up-down) the SWI toe location ℓT/L value under relatively small change in the range of sustainable qr (Online Material 2, Fig. S1a-b), while varying the r value shifts (left-right) the sustainable qr range under relatively small change in the resulting ℓT/L range (Fig. 3 and Online Material 2, Fig. S1c-d). Furthermore, aquifers at various sites may differ in terms of their geometrical ratio ℓw/Hsea. For any given combination of recharge r and relative well position ℓw/L, greater remaining groundwater flow after pumping qr (smaller pumping qw) is then required for sustainability in sites with smaller ℓw/ Hsea (deeper aquifer and/or higher sea-level) (Fig. 3 and Online Material 2, Fig. S2). Figure 4 (and Online Material 2, Fig. S3) show the total general SMS as function of r (spanning all possible well locations relative to aquifer depth 0 < ℓw/Hsea →∞) for a generic aquifer with parameter value ranges outlined in Table 1. The investigated ranges of relative well location 0.1 < ℓw/L < 0.9 and groundwater recharge 1 ≤r ≤1000 mm yr−1 cover most ℓw/L and r values that can realistically prevail in different aquifers and world regions. Across these ranges then, the total general SMS (grey area in Fig. 4 and Online Material 2, Fig. S3) applies also specifically, in approximately its full extent, for all site conditions of ℓw/Hsea > 40 or hydraulic conductivity K ≤1 m d−1. It is only for the complementary combination of site- specific conditions ℓw/Hsea ≤40 and K > 1 m d−1 that the specific SMS is considerably smaller (hatched in Fig. 4 and Online Material 2, Fig.S3-S4) than the total general SMS. 2.2 Exploring Specific Aquifer Vulnerability Relative to its SMS The results presented in the sequel go beyond common effect analysis: by using the above- developed general framework, we investigate how SMS results change for various combina- tions of naturally given aquifer and hydro-climatic conditions (hydraulic conductivity K, coastal aquifer extent L, slope sinφ and depth below sea level Hsea, and groundwater recharge rate r) and human-determined exploitation choices (pumping qw, and pumping location ℓw). Table 1 summarizes the parameter value ranges used in this analysis to typologize the SWI and associated SMS behaviour across a realistic range of conditions that may prevail around the world. Generic aquifer results are illustrated in terms of the SMS for some realistic fixed conditions (corresponding to Fig. 2a) and for variable conditions (corresponding to Fig. 2b) of net recharge rate r across a range of different possible pumping locations ℓw/L. Furthermore, site-specific controls and limits of SMS are also concretely quan- tified for the study aquifers ICA (Israel) and CAA (Cyprus), aiming to trace the groundwater-use and related SWI impacts in relation to the SMS of these coastal regions. 4070 K. Mazi et al. Table 1 Characteristics and parameters used in the modelling of generic aquifers Constant Range of variation Aquifer length, L (m) 5000 Inland boundary condition no-flow, qb = 0 Slope of impervious aquifer base, sinφ 0.01 Depth of sea at the coast to the aquifer base, Hsea (m) 5–600 Well location, ℓw (m) 500–4500 Recharge rate, r (mm yr−1) 1–1000 Hydraulic conductivity, K (m d−1) 1 and 30 L/Hsea: ratio of aquifer length to aquifer depth at the coast 8.3–1000 ℓw/L: ratio of pumping location to aquifer length 0.1–0.9 ℓw/Hsea: ratio of pumping location to aquifer depth at the coast 7.5–200 Table 1 Characteristics and parameters used in the modelling of generic aquifers 3.1 General and Specific Sustainable Management Space The reason for this bifurcation is that the site-specific lower qr limit (determined from ℓT = ℓdiv > 0) approaches rapidly the general lower limit qr min = −rℓw (implying ℓT = ℓdiv = 0 and ℓw/Hsea →∞) with 4071 A Sustainable Management Space for the Use of Coastal Groundwater 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Normalized location of interface toe Remaining groundwater flow from the pumping location, qr (m2 d-1) lw/L = 0.8, r = 100 mm yr-1 30 40 60 100 200 lw/Hsea qr max qr min Sustainable management space Well intrusion Complete intrusion (a) -12 -8 -4 0 4 8 12 (a) Remaining groundwater flow from the pumping location, qr (m2 d-1) qr qr 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 -12 -8 -4 0 4 8 12 Normalized location of interface toe Remaining groundwater flow from the pumping location, qr (m2 d-1) lw/L = 0.8, r = 1000 mm yr-1 20 30 40 60 100 200 qr min qr max lw/Hsea Sustainable management space Complete intrusion (b) Fig. 3 Intrusion curves revealing SMS for coastal groundwater. Results are shown for two different relations between well position and coastal aquifer depth ℓw/Hsea, under given aquifer recharge rate r and relative well location ℓw/L; in all illustrated cases, hydraulic conductivity K = 30 m d−1. In terms of groundwater flow remaining after pumping, the SMS extends from the upper-most limit qr max = r(L - ℓw) for zero-pumping to the lowest limit qr min = −rℓw (for zero-value of the line Eq. 2), which is approached asymptotically as ℓw/Hsea →∞ 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 -12 -8 -4 0 4 8 12 Normalized location of interface toe lw/L = 0.8, r = 1000 mm yr-1 20 30 40 60 100 200 q min qr max lw/Hsea Sustainable management space Complete intrusion (b) (b) Remaining groundwater flow from the pumping location, qr (m2 d-1) r Fig. 3 Intrusion curves revealing SMS for coastal groundwater. Results are shown for two different relations between well position and coastal aquifer depth ℓw/Hsea, under given aquifer recharge rate r and relative well location ℓw/L; in all illustrated cases, hydraulic conductivity K = 30 m d−1. 3.1 General and Specific Sustainable Management Space For lw/Hsea ≤ 40 and K > 1 m d-1 -15 -12 -9 -6 -3 0 3 6 9 12 15 0 200 400 600 800 1000 Remaining groundwater flow from the pumping location, qr (m2 d-1) r (mm yr-1) lw/L = 0.3 qr max qr min Series2 Series4 qr max, K = 30 m d-1 qr min, K = 30 m d-1 qr max, K = 1 m d-1 qr min, K = 1 m d-1 (a) For lw/Hsea ≤ 40 and K > 1 m d-1 -15 -12 -9 -6 -3 0 3 6 9 12 15 0 200 400 600 800 1000 Remaining groundwater flow from the pumping location, qr (m2 d-1) r (mm yr-1) lw/L = 0.9 Series2 Series1 qr max, K = 30 m d-1 qr min, K = 30 m d-1 qr max, K = 1 m d-1 qr min, K = 1 m d-1 (b) Fig. 4 SMS for use of coastal groundwater. The general SMS area (whole grey area) is quantified for all possible well location relations to aquifer depth (0 < ℓw/Hsea →∞) in terms of the general upper (qr max, dashed line) and lower (qr min, solid line) limits of groundwater flow qr remaining after pumping as function of groundwater recharge rate r. Results are shown for two different well locations relative to total aquifer extent (ℓw/L) and hydraulic conductivity values K. For site conditions of ℓw/Hsea > 40 or K ≤1 m d−1, the general space (grey) is approximately applicable also as site-specific SMS. For the complementary site conditions ℓw/ Hsea ≤40 and K > 1 m d−1, the site-specific SMS is restricted to the hatched space part, with Figs. 3.1 General and Specific Sustainable Management Space In terms of groundwater flow remaining after pumping, the SMS extends from the upper-most limit qr max = r(L - ℓw) for zero-pumping to the lowest limit qr min = −rℓw (for zero-value of the line Eq. 2), which is approached asymptotically as ℓw/Hsea →∞ increasing ℓw/Hsea and is close to it for ℓw/Hsea > 40 (Fig. 3). Figs. S4-S5 in Online Material 2 show further details of the site-specific SMS for aquifers with different ℓw/Hsea (and K) and how it approaches the total general SMS as ℓw/Hsea →∞. increasing ℓw/Hsea and is close to it for ℓw/Hsea > 40 (Fig. 3). Figs. S4-S5 in Online Material 2 show further details of the site-specific SMS for aquifers with different ℓw/Hsea (and K) and how it approaches the total general SMS as ℓw/Hsea →∞. The results in Fig. 4 thus identify a general rule of thumb for two complementary site condition combinations: (a) the total general SMS delimited by –rℓw < qr ≤r(L – ℓw) (total grey, Fig. 4) is sustainable for aquifer site conditions ℓw/Hsea > 40 or hydraulic conduc- tivity K ≤1 m d−1; (b) the more restricted SMS for 0 < qr ≤r(L – ℓw) (hatched, Fig. 4) is sustainable for aquifer site conditions ℓw/Hsea ≤40 and K > 1 m d−1. The results in Fig. 4 thus identify a general rule of thumb for two complementary site condition combinations: (a) the total general SMS delimited by –rℓw < qr ≤r(L – ℓw) (total grey, Fig. 4) is sustainable for aquifer site conditions ℓw/Hsea > 40 or hydraulic conduc- tivity K ≤1 m d−1; (b) the more restricted SMS for 0 < qr ≤r(L – ℓw) (hatched, Fig. 4) is sustainable for aquifer site conditions ℓw/Hsea ≤40 and K > 1 m d−1. 4072 K. Mazi et al. 3.1 General and Specific Sustainable Management Space S3-S5 in Online Material 2 showing further details of how the site-specific SMS is determined for different aquifer cases in terms of site-specific ℓw/Hsea and K For lw/Hsea ≤ 40 and K > 1 m d-1 -15 -12 -9 -6 -3 0 3 6 9 12 15 0 200 400 600 800 1000 Remaining groundwater flow from the pumping location, qr (m2 d-1) r (mm yr-1) lw/L = 0.3 qr max qr min Series2 Series4 qr max, K = 30 m d-1 qr min, K = 30 m d-1 qr max, K = 1 m d-1 qr min, K = 1 m d-1 (a) (a) For lw/Hsea ≤ 40 and K > 1 m d-1 For lw/Hsea ≤ 40 and K > 1 m d-1 -15 -12 -9 -6 -3 0 3 6 9 12 15 0 200 400 600 800 1000 Remaining groundwater flow from the pumping location, qr (m2 d-1) r (mm yr-1) lw/L = 0.9 Series2 Series1 qr max, K = 30 m d-1 qr min, K = 30 m d-1 qr max, K = 1 m d-1 qr min, K = 1 m d-1 (b) (b) For lw/Hsea ≤ 40 and K > 1 m d-1 Fig. 4 SMS for use of coastal groundwater. The general SMS area (whole grey area) is quantified for all possible well location relations to aquifer depth (0 < ℓw/Hsea →∞) in terms of the general upper (qr max, dashed line) and lower (qr min, solid line) limits of groundwater flow qr remaining after pumping as function of groundwater recharge rate r. Results are shown for two different well locations relative to total aquifer extent (ℓw/L) and hydraulic conductivity values K. For site conditions of ℓw/Hsea > 40 or K ≤1 m d−1, the general space (grey) is approximately applicable also as site-specific SMS. For the complementary site conditions ℓw/ Hsea ≤40 and K > 1 m d−1, the site-specific SMS is restricted to the hatched space part, with Figs. S3-S5 in Online Material 2 showing further details of how the site-specific SMS is determined for different aquifer cases in terms of site-specific ℓw/Hsea and K Fig. 4 SMS for use of coastal groundwater. 3.1 General and Specific Sustainable Management Space The general SMS area (whole grey area) is quantified for all possible well location relations to aquifer depth (0 < ℓw/Hsea →∞) in terms of the general upper (qr max, dashed line) and lower (qr min, solid line) limits of groundwater flow qr remaining after pumping as function of groundwater recharge rate r. Results are shown for two different well locations relative to total aquifer extent (ℓw/L) and hydraulic conductivity values K. For site conditions of ℓw/Hsea > 40 or K ≤1 m d−1, the general space (grey) is approximately applicable also as site-specific SMS. For the complementary site conditions ℓw/ Hsea ≤40 and K > 1 m d−1, the site-specific SMS is restricted to the hatched space part, with Figs. S3-S5 in Online Material 2 showing further details of how the site-specific SMS is determined for different aquifer cases in terms of site-specific ℓw/Hsea and K Part (a) of the identified rule of thumb reflects that even negative qr is sustainable for aquifers with either relatively low conductivity (K ≤1 m d−1) or relatively small aquifer depth below sea level (ℓw/Hsea > 40), so that fresh groundwater can be pumped from both the seaside and the landside of the representative well location ℓw/L, as long as qr remains above the general lower limit qr min = −rℓw. Part (b) of the rule reflects that qr must remain positive for aquifers with both relatively high conductivity (K > 1 m d−1) and relatively large A Sustainable Management Space for the Use of Coastal Groundwater 4073 aquifer depth below sea level (ℓw/Hsea ≤40), so that only groundwater recharged on the landside of ℓw/L can be pumped sustainably. aquifer depth below sea level (ℓw/Hsea ≤40), so that only groundwater recharged on the landside of ℓw/L can be pumped sustainably. Overall, the total grey area of the general SMS in Fig. 4 is the same, regardless of management-chosen well location ℓw/L or prevailing aquifer K-value. This is because the recharge rate r ultimately determines the available flow of fresh groundwater that can be partitioned between pumped water and remaining flow left to resist critical SWI as submarine groundwater discharge qSD (Destouni and Prieto 2003; Destouni et al. 2008; Prieto and Destouni 2011). A choice of farther inland well location (greater ℓw/L) will increase the negative relative to the positive part of the general SMS (Fig. 3.2 Concrete Site-Specific Implications of the SMS Mazi et al. (2014) have studied and modelled SWI conditions in the specific aquifer cases of ICA and CAA, considering different pumping locations and pumping rates under their current net recharge; we refer to that work for detailed site and analysis descriptions. Here, the SMS for these sites is determined for variable net recharge r (to capture possible effects of changed r due to changes in human groundwater use, hydro-climatic change and/or artificial recharge) and two alternative pumping locations. The presented analysis enables calculating and visualising the implications of the current pumping rate for current and possibly changed recharge conditions. We further determine the minimum r-value required for sustaining the current pumping rate within the site-specific SMS. In Online Material 2, Figs. S6 and S7 show, respectively for the ICA and CAA aquifers, (a) the maps with the investigated profile sections and (b) the cross- sections as schematized in the present modelling, with the main geometrical and physical characteristics, along with the aquifer recharge rates, freshwater inflows and exploitation schemes (locations of fully penetrating troughs and pumping rates). Table-T1 in Online Material 2 summarizes the parameters of the conceptual cross-sections of the ICA and the CAA. 3.1 General and Specific Sustainable Management Space 4), implying that more of the sustainable groundwater withdrawal can come from the seaside and less from the landside of ℓw/L. Furthermore, pumping more inland will make complete intrusion, rather than well intrusion, the likely limiting condition. Conversely, pumping more seaward will increase the positive relative to the negative part of the total SMS and make well intrusion more likely as limiting condition. 3.2.1 Israel Coastal Aquifer (ICA) The ICA has an area of ~1900 km2, its depth exposure to the sea is Hsea ≈200 m with L/ Hsea = 100, the inclination of its 20-km long base is 1 %, its mean hydraulic conductivity K = 30 m d−1, and the recharge rate 240 mm yr−1; pumping is qw = 3000 m2 yr−1 at ℓw = 3 km. The interface toe position for these current conditions is calculated by the analytical model to be ℓT = 2.6 km (Mazi et al. 2014). Figure 5a shows the SMS for the resulting current values ℓw/L = 0.15 and ℓw/Hsea = 15 under variable recharge r; in that figure the cross (+) indicates the current remaining ground- water flow qr after the current pumping. The positive current qr (cross, Fig. 5a) implies that, for this current pumping location, pumping is sustainable only from the area inland of the representative well location. The well intrusion condition ℓT = ℓw (Fig. 1b) applies as the critical one for the current r = 240 mm yr−1 and for all r ≤816 mm yr−1 (calculated, not shown). For r > 816 mm yr−1, the lower limit qr min falls in the negative part of the sustainable space with complete intrusion ℓT = ℓdiv (Fig. 1a) as critical condition. K. Mazi et al. 4074 -5 0 5 10 15 0 50 100 150 200 250 300 Remaining groundwater flow from the pumping locaon, qr (m2 d-1) r (mm yr-1) dash-dot lines: qr max solid lines: qr min lw/L lw/Hsea more inland well 0.15 15 0.5 50 trace of qrfor current qw qr for current qw (1) (2) (a) ICA -1 0 1 2 3 4 0 25 50 75 100 125 150 175 200 Remaining groundwater flow from the pumping locaon, qr (m2 d-1) r (mm yr-1) 0.33 20 0.67 40 lw/L more inland well lw/Hsea trace of qrfor current qw qr for current qw dash-dot lines: lines: qr max solid lines: qr min (1) (2) (b) CAA Fig. 5 SMS for a Israel Coastal Aquifer (ICA) and b Cyprus Akrotiri Aquifer (CAA). Aquifer conditions are characterized by the scaled quantities ℓw/L and ℓw/Hsea, for two pumping locations, with the black dotted lines tracing the remaining groundwater flow qr directly after pumping with the current rate under variable recharge and boundary inflows. 3.2.1 Israel Coastal Aquifer (ICA) (a) ICA: current pumping position at 3 km from the coast (1), and hypothetical pumping position moved more inland to 10 km (2) and (b) CAA: current pumping position at 1 km from the coast (1), and hypothetical pumping position moved more inland to 2 km (2). Figs. S6 for ICA and S7 for CAA in Online Material 2 show further details of the modelled characteristics of the two aquifer cases -5 0 5 10 15 0 50 100 150 200 250 300 Remaining groundwater flow from the pumping locaon, qr (m2 d-1) r (mm yr-1) dash-dot lines: qr max solid lines: qr min lw/L lw/Hsea more inland well 0.15 15 0.5 50 trace of qrfor current qw qr for current qw (1) (2) (a) ICA (a) ( y ) -1 0 1 2 3 4 0 25 50 75 100 125 150 175 200 Remaining groundwater flow from the pumping locaon, qr (m2 d-1) r (mm yr-1) 0.33 20 0.67 40 lw/L more inland well lw/Hsea trace of qrfor current qw qr for current qw dash-dot lines: lines: qr max solid lines: qr min (1) (2) (b) CAA dash-dot lines: lines: qr max solid lines: qr min r (mm yr-1) Fig. 5 SMS for a Israel Coastal Aquifer (ICA) and b Cyprus Akrotiri Aquifer (CAA). Aquifer conditions are characterized by the scaled quantities ℓw/L and ℓw/Hsea, for two pumping locations, with the black dotted lines tracing the remaining groundwater flow qr directly after pumping with the current rate under variable recharge and boundary inflows. (a) ICA: current pumping position at 3 km from the coast (1), and hypothetical pumping position moved more inland to 10 km (2) and (b) CAA: current pumping position at 1 km from the coast (1), and hypothetical pumping position moved more inland to 2 km (2). Figs. S6 for ICA and S7 for CAA in Online Material 2 show further details of the modelled characteristics of the two aquifer cases Fig. 5 SMS for a Israel Coastal Aquifer (ICA) and b Cyprus Akrotiri Aquifer (CAA). Aquifer conditions are characterized by the scaled quantities ℓw/L and ℓw/Hsea, for two pumping locations, with the black dotted lines tracing the remaining groundwater flow qr directly after pumping with the current rate under variable recharge and boundary inflows. 3.2.2 Cyprus Akrotiri Aquifer (CAA) The area of the CAA is ~40 km2, its depth at the coastline is Hsea ≈50 m, with L/Hsea = 60, the inclination of its 3-km long base is 1.7 % and mean hydraulic conductivity K = 28 m d−1, and replenishmentisr=92mmyr−1and(inthiscaseknownprevailing)boundaryinflow|qb|=549m2yr−1; pumping is qw = 500 m2 yr−1 at ℓw = 1 km (Mazi 2000; Koussis 2001; Mazi et al. 2004a, 2004b). The interface toe position for these current conditions is calculated by the analytical model to be ℓT = 640 m (Mazi et al. 2014). Figure 5b shows the SMS for the resulting current values ℓw/L = 0.33 and ℓw/Hsea = 20 under variable recharge; the cross (+) indicates the current remaining groundwater flow qr after pumping. For the current representative pumping location, the positive qr (+ mark) implies that the pumped water originates landward of the well. The qr min line, falling in the positive part of the SMS, indicates also well intrusion ℓT = ℓw (Fig. 1b) as the current critical condition, holding as long as r ≤268 mm yr.−1 and the boundary inflow is |qb| ≤406 m3 m−1 yr−1 (analysis for the latter limit not shown); if groundwater replenishment exceeds these values, the lower limit qr min falls in the negative part of the SMS and complete intrusion (ℓT = ℓdiv, Fig. 1a) becomes the critical condition. We also calculate the associated SMS for hypothetic relocation of the representative well to ℓw = 2 km, yielding ℓw/L = 0.5 and ℓw/Hsea = 40 (Fig. 5b). The positive qr-value (x mark) implies that all current pumping is then still satisfied from the area landwards of the well, as it is also for the current ℓw = 1 km. However, for ℓw = 2 km, the lower limit qr min lies in the negative part of the sustainable space for r > 22 mm yr−1 and boundary inflow |qb| > 363 m3 m−1 yr−1 (analysis for the latter limit not shown), which switches the critical condition from the former well intrusion to complete intrusion. Figure 5b shows further the trace (dotted) of the site-specific upper qr limit for different r (with the r value range also associated with a |qb| range, not shown), under the current pumping rate applied at the two investigated pumping locations. 3.2.1 Israel Coastal Aquifer (ICA) (a) ICA: current pumping position at 3 km from the coast (1), and hypothetical pumping position moved more inland to 10 km (2) and (b) CAA: current pumping position at 1 km from the coast (1), and hypothetical pumping position moved more inland to 2 km (2). Figs. S6 for ICA and S7 for CAA in Online Material 2 show further details of the modelled characteristics of the two aquifer cases We furthermore calculate the site-specific SMS after hypothetically relocating the pumping to 10 km from the coastline, yielding ℓw/L = 0.5 and ℓw/Hsea = 50. Under current pumping and recharge rate then, qr becomes negative (x-mark, Fig. 5a), which implies that the critical condition would switch from well intrusion (ℓT = ℓw, Fig. 1b) to complete A Sustainable Management Space for the Use of Coastal Groundwater 4075 intrusion (ℓT = ℓdiv, Fig. 1a). The complete intrusion condition is critical for r ≥32 mm yr−1, as the lower limit qr min falls in the negative part of the sustainable space for this r-range. intrusion (ℓT = ℓdiv, Fig. 1a). The complete intrusion condition is critical for r ≥32 mm yr−1, as the lower limit qr min falls in the negative part of the sustainable space for this r-range. Figure 5a also traces (dotted lines) the site-specific upper limit of qr for different recharge r with the current pumping qw applied to the two different representative well locations ℓw. For the current pumping location at ℓw = 3 km, the current pumping rate is unsustainable for r ≤230 mm yr−1, because the qr-trace for this pumping intersects the qr min line at that r value. For the hypothetical pumping at ℓw = 10 km, the minimum required recharge for sustainability is 204 mm yr−1 under the current pumping rate. For both pumping-location cases, and particularly for the current one, the minimum required recharge is close to the current recharge r = 240 mm yr−1. Thus, well intrusion is a real threat under expected future hydro-climatic change and/or increased human use of the coastal groundwater at this site. Moving the pumping inland to ℓw = 10 km may evade the critical well intrusion condition, but complete SWI and associated loss of the fresh groundwater resource as far inland as ℓT = ℓdiv = 5305 m is then threatening the aquifer under changed hydro-climate and/or human freshwater use. 4 Discussion Overall hydro-climatic changes influence the marine and/or inland forcing of coastal aquifers, which then respond to the changed forcing according to their specific characteristics. In contrast to the overall hydro-climatic changes and the naturally given aquifer geometry and conditions, the location and rate of groundwater pumping is a human-controlled intervention and must therefore be an essential element of groundwater-resources management under hydro-climatic regime changes. The focus is then here on providing managers of coastal aquifers with a planning tool for sustainable exploitation of the groundwater resource that can as far as possible meet freshwater demand, taking into account a changing hydro-climatic forcing and the given aquifer geometric and hydrogeological conditions, while avoiding critical tipping points. The mean macro-characteristics of a regional aquifer, Hsea, K, L, sinφ, r, qb, ℓw and qw, can be estimated reasonably well and, based on these and various scenarios for the hydro-climatic forcing, the limits for sustainability can be determined in terms of the groundwater flow qr left in the aquifer immediately after the pumping location, or equivalently the submarine discharge qSD into the sea. Performing such analysis for a regional coastal aquifer, with certain slope, K- value and key geometrical macro-characteristics (expressed as ℓw/Hsea and ℓw/L) over a relevant range of recharge r (and possible non-zero boundary flow |qb|) values, yields that aquifer’s SMS and its bounding qr(r) curves for various pumping rates qw; that analysis also informs on how the pumped groundwater qw partitions between the zone 0 < ℓ≤ℓw on the seaside and the zone ℓw ≤ℓ≤L on the landside of the representative pumping location ℓw. The theoretical, general total SMS is obtained for all possible 0 < ℓw/Hsea →∞and contains all the different specific SMS corresponding to any actual, finite site-specific ℓw/Hsea value (past, current or future). From the results for the general total SMS emerges as general rule of thumb that the total general SMS is practically applicable across the entire range of investigated recharge condi- tions, from dry (r ≤100 mm yr−1) to wet (r = 1000 mm yr−1), for sites characterized by either ℓw/Hsea > 40 or a low hydraulic conductivity K ≤1 m d−1 (and aquifer slopes ~1 %; see effects of different slopes in Mazi et al. (2013)). Furthermore, qr can be negative for any well location in the aquifer if K ≤1 m d−1; qr can also be negative for any K-value if ℓw/Hsea > 40. 3.2.2 Cyprus Akrotiri Aquifer (CAA) For the current well location ℓw = 1 km, the current pumping rate is unsustainable under a ~ 20 % decrease of the replenishment, i.e., for r < 64 mm yr−1 and |qb| < 476 m3 m−1 yr−1, as the intersection of the dotted line with the qr min line indicates. For the hypothetical pumping at ℓw = 2 km, sustaining the current pumping rate requires a minimum recharge r = 49 mm yr−1 and boundary inflow |qb| = 433 m3 m−1 yr−1, which would be a decrease of the current replenishment by ~30 %. In the CAA case, the K. Mazi et al. 4076 prevailing relatively large boundary inflow |qb| prevents the remaining flow qr from actually decreasing to zero, as it might in principle do in the ICA case. prevailing relatively large boundary inflow |qb| prevents the remaining flow qr from actually decreasing to zero, as it might in principle do in the ICA case. 4 Discussion For the complementary aquifer conditions K > 1 m d−1 and ℓw/Hsea ≤40, it is instead prudent to maintain positive qr. This restriction leaves small opportunities for use of coastal groundwater in regions of low recharge and/or for pumping locations that are already far inland from the coastline (ℓw/L close to 1, e.g., due to already progressed seawater intrusion), particularly if the aquifer does not receive much inflow from farther inland areas to enhance the flow derived from the groundwater recharge within the coastal region itself. Results thus indicate the ratios ℓw/Hsea and ℓw/L as suitable indicators for estimating the risk of SWI under various groundwater exploitation conditions; e.g., under stable recharge r, greater pumping rate qw is sustainable at sites with relatively large ℓw/Hsea-values (small aquifer depth below sea level). However, as the representative well location moves inland and ℓw/L increases, the critical aquifer condition may change from one of well intrusion (Fig. 1a) to one of complete intrusion (Fig. 1b). The interface toe location is more sensitive A Sustainable Management Space for the Use of Coastal Groundwater 4077 to ℓw/Hsea than to r/K. Therefore, given an aquifer geometry L/Hsea, decisions regarding pumping locations can be taken mainly by evaluating the ratio ℓw/Hsea for each possible pumping location ℓw/L. to ℓw/Hsea than to r/K. Therefore, given an aquifer geometry L/Hsea, decisions regarding pumping locations can be taken mainly by evaluating the ratio ℓw/Hsea for each possible pumping location ℓw/L. The specific SMS has here been concretely determined for the specific aquifer case of ICA, for possible representative well locations at ℓw = 3 (current) and 10 km (hypothetical) and for aquifer depth at the coast Hsea = 200 m. Since for ℓw = 10 km the ratio ℓw/Hsea = 50 (i.e., > 40) and K = 30 m d−1, the simple rule of thumb allows here negative qr (Fig. 5), which implies that pumping of some coastal groundwater also from seawards of the representative well location can be sustainable. The calculated specific SMS can be used to also examine aquifer management alternatives under different recharge and pumping scenarios; e.g., if recharge in the ICA decreases by more than 5 % of the current value, today’s pumping rate will not be sustainable. In contrast, if more recharge is applied, e.g., +8 % through artificial recharge, an additional rate of 12 % of the current qw could be pumped sustainably. 4 Discussion In the other concrete aquifer case investigated here, the CAA case, the specific SMS was calculated for representative well locations ℓw = 1 (current) and 2 km (hypothetical) from the coast. For ℓw = 2 km, the simple rule of thumb is on the limit of allowing pumping groundwater also from seaward of the well (i.e., of allowing qr < 0), because in that case ℓw/Hsea = 40 and K = 28 m d−1 > 1 m d−1. In this particular case, calculations show that, by disregarding the rule of thumb and allowing qr < 0, the maximum pumping rate might be increased but only by 8.6 %, taking then also the risk that the coastal aquifer could be lost by complete SWI as a result of, e.g., a small decline of the natural replenishment of fresh groundwater (recharge plus some boundary inflow in this case). Furthermore, examining the specific SMS for variable aquifer replenishment shows that the current pumping rate would not be sustainable, from either pumping location, if the replenishment were to decline, e.g., due to climate change, by ~20 % for ℓw = 1 km and by ~30 % for ℓw = 2 km. 5 Conclusions The simple analytical rules and bounds derived here quantify an SMS for human use of coastal groundwater in general terms that are applicable across different regional scales and parts of the world. Specifically, this quantification identifies and relates the SMS to the key human controls (ℓw/L, ℓw/Hsea) and the overall mostly naturally given forcing (mainly indicated by Hsea/L and less so by r/K; with higher bed slope expanding the SMS) that determine SWI under different coastal aquifer conditions. Without being global sums or averages of some single variables, these controls quantify the SMS boundaries by accounting for the wide- ranging local-regional variability that exists across the world’s coastal regions. Since in most cases of practical interest the average aquifer hydraulic conductivity may be assumed to be K > 1 m d−1, the actual site-specific SMS will be typically restricted relative to the general total SMS. To achieve a pumping rate that can as far as possible meet certain freshwater demand, a manager of a coastal aquifer should select pumping locations such that ℓw/Hsea > 40, thereby possibly allowing for sustainable drawing of groundwater also from seaward of the pumping location (qr < 0). For each human-determined pair of control variables (ℓw, qw), the proximity of the interface toe to critical SWI conditions (well intrusion or complete coastal aquifer intrusion) must be evaluated (Figs. 2a and 3) and, then, a possible increase in pumping rate must be weighed against the incurred higher intrusion risk. For a preliminary pumping selection (of ℓw, qw), the sensitivity of SWI to a possible reduction of 4078 K. Mazi et al. natural recharge r must be also investigated. If the exploitation opportunities are tight, the option of artificial recharge enhancement can be gauged by analysing the resulting SMS under variable r (Fig. 2b). By further extending the present basic deterministic analysis to a stochastic analysis, a more complete and ultimately more appropriate framework can be developed for assessing gains and risks of the various human selections for groundwater management; such an approach will be presented in a future work. In general, accounting for local-regional variability is essential for sustainable management of human freshwater use. Managers, planners and policy-makers responsible for water re- sources, environmental sustainability and climate adaptation must decide their strategies and operations in direct relation to prevailing local-regional conditions identified by the aforemen- tioned key controls. Human and Animal Rights No. Human and Animal Rights No. Informed Consent Not applicable Informed Consent Not applicable 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 repro- duction 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. Compliance with Ethical Standards Conflict of Interest None. Conflict of Interest None. Human and Animal Rights No. 5 Conclusions Regarding these practical needs, but also the related scientific perspectives, the quantification of the SMS developed here is sufficiently simple and general to enable consistent regional assessment, cross-regional comparison, and larger-scale aggregation of multiple regions (up to the global scale) for coastal groundwater resources under current conditions and future change scenarios of human water-use/demand, hydro-climate and sea level. Acknowledgments This research has been supported by the Navarino Environmental Observatory (NEO), the Nova R&D project KLIV, Stockholm University’s strategic research program Ekoklim and the Swedish Research Council Formas (Project No. 2014-43). Compliance with Ethical Standards Compliance with Ethical Standards References Bring A, Asokan SM, Jaramillo F, et al. 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The Relevance of Sexual History and Vulnerability in the Prosecution of Sexual Offences
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1 See Report by Baroness Vivien Stern CBE of an independent review into how rape complaints are handled by public authorities in England and Wales (2010, Home Office). 2 For discussion of the provisions introduced in a variety of jurisdictions see Scottish Executive Vulnerable and Intimidated Witnesses: Review of Provisions in Other Jurisdictions (2002, Scottish Executive Central Research Unit). 3 L Ellison ‘Commentary on Re A (No 2)’ in R Hunter, C McGlynn, E Rackley (eds) (2010) Feminist Judgments: From Theory to Practice (First Edition) 205-211, citing A McColgan, Women under the Law (2000). 4 See for example L Kelly, J Temkin, and S Griffiths, Section 41: An Evaluation of New Legislation Limiting Sexual History in Rape Trials Home Office, June 20, 2006; M Burman, L Jamieson, J Nicholson and O Brooks Impact of Aspects of the Law of Evidence in Sexual Offence Trials: An Evaluation Study (2007). 5 In Scotland, those who allege offences are termed ‘complainers’; in England and Wales the term is ‘complainants’. Likewise, those standing trial in Scotland are the ‘accused’, but are ‘defendants’ in England and Wales. The Relevance of Sexual History and Vulnerability in the Prosecution of Sexual Offences Liz Campbell and Sharon Cowan Introduction The investigation and prosecution of sexual offences remains one of the most fraught and problematic aspects of criminal justice. Even with the introduction of various protective measures for vulnerable witnesses and complainers, deep and justifiable concerns persist about the level of reporting to the police, the extent of attrition, and the conviction rates.1 The treatment (actual and perceived) of complainers in the court room has a significant influence on these matters. Sexual offence trials frequently involve the leading of sexual history evidence and the cross-examination of complainers about their previous sexual experience, behaviour and partners. This is despite the introduction of so-called “rape shield” provisions, which purport to restrict the use of sexual history evidence and to curb judicial discretion as regards its admission. Such provisions have been enacted in various common law jurisdictions including England and Wales, Canada, and Scotland, in an effort to improve courtroom experience and thus encourage greater reporting of sexual violence.2 The rationale for these measures is to address the pervasive culture of disbelief regarding complainers and to offset the secondary victimisation or re-traumatisation that occurs through and as a consequence of the trial process. Rape shield legislation aims to protect the complainer’s right to privacy and dignity, as well as to increase the accuracy in fact-finding. y g Although ‘rape shield’ protections have been embedded within criminal justice systems for some time, it has been suggested by commentators in various jurisdictions that these legislative efforts remain susceptible to being sidestepped either through defence trial strategies or through “judicial override”,3 such that the law in action is less protective and useful than was hoped.4 As we shall see below, this observation is borne out in Scotland. This chapter examines the ways in which laws designed to protect sexual assault complainers in Scotland fail in practice. 5 We will argue that focusing on the ‘rape shield’ provisions, as well as the measures designed to protect vulnerable witnesses, as ‘solutions’ to the problems faced by sexual assault complainers in an adversarial system, allows us to ignore more systemic questions about how those complainers become vulnerable within the criminal justice system, as well as how the criminal justice system understands and perpetuates a certain view of what it means to be vulnerable. Introduction 1 Focusing on Scotland as a case study, the first section outlines legislation limiting the use of sexual history evidence in criminal trials, and the evaluation of the impact of this legislation, before moving on to the second section, examining provisions introduced to help criminal justice agents identify vulnerable complainers, and in particular those that have alleged sexual assault. We will argue that since only a tiny minority of vulnerable sexual assault complaints ever make it to trial, such provisions cannot properly protect the vast majority of sexual assault victims who are vulnerable, and that in the absence of a more radical systemic and holistic review of criminal law, evidence and procedure relating to sexual assault, the provisions may serve as a distraction from, or an apparent panacea to, more significant, serious and entrenched challenges that vulnerable sexual assault victims face. Although in what follows we refer primarily to legislation and practice in the Scottish criminal justice system, given the widespread use of similar protections in many other jurisdictions, our conclusions have relevance for the promotion of just outcomes in sexual assault proceedings more generally. Moreover, we aim to contribute to both contemporary critical scholarship on sexual assault, and to ongoing debates about the importance of recognising vulnerability as partly produced by social institutions and structures.6 The vulnerability lens through which many feminists and other critical scholars analyse legal and policy interventions across a broad spectrum of issues (including sex work, hate crime, family law, welfare rights and so on) has yet to be applied in this context of criminal evidence and procedure, where the criminal justice system formally recognises the vulnerability of some of its subjects, but fails to properly engage with more meaningful substantive questions of access to justice for others. In this paper, we use the lens of vulnerability to examine the problematic ways in which criminal evidence and procedure in Scotland fails to protect many of the most vulnerable victims of sexual assault, despite recent reforms. We suggest that more research is urgently required to shed light on the extent of the ‘justice gap’, and that further and deeper reform is needed, at the substantive, procedural and cultural levels. 6 M Fineman ‘The vulnerable subject; anchoring equality in the human condition’ 20 (2008) Yale Journal of Law and Feminism 1-23. 7 Recorded Crime in Scotland 2014-15, http://www.gov.scot/Resource/0048/00484776.pdf p1. “Crimes recorded by the police in Scotland decreased by 5% from 270,397 to 256,350. This is the lowest level of recorded crime since 1974.” 8 Ibid. A. SEXUAL HISTORY: THE SCOTTISH CONTEXT As the rates of other sorts of crime continue to drop,7 the recording of sexual crimes in Scotland has increased and is at the highest level since 1971, the first year for which comparable crime groups are available.8 This may be explained by a growing likelihood of a report being made and the reporting of “historical” offences, rather than solely as a result of increased offending. But it is unlikely that the rise in recorded incidences can, in its entirety, be explained by increased reporting: it is reasonable to suppose that it can also be at least partially explained by an increase in offences. Regardless, the growing rate of recorded offences is not matched by a high conviction rate. The highest acquittal rate for any offence in Scotland is for rape and attempted rape, where 34% of those prosecuted in 2014-15 were acquitted on a “not guilty” verdict. 9 Sexual assault has an acquittal rate of 21%, in contrast to a general acquittal rate of 5%. In 2014-15, 19% of those tried for rape and attempted rape Ibid. Criminal Proceedings in Scotland, 2014-15 http://www.gov.scot/Resource/0049/00494474.pdf p. 13. 2 2 received a “not proven” verdict, the highest rate overall, followed by sexual assault at 11 %.10 Moreover, the conviction rate as a proportion of reported cases of rape is low. In 2009 it stood at a mere 3%,11 though as Rape Crisis Scotland highlighted, this figure may be somewhat inaccurate: the two sets of data from which the 3% rate was derived measure slightly different things, one focusing on offences, the other on offenders.12 Matters are improving somewhat: in 2014-15 there were 125 convictions for rape and attempted rape,13 with a total of 1901 reports of rape and attempted rape during the same year, 1797 of which were reports of rape.14 This represents a conviction rate of 6.6% of reported cases. The proportion of rapes prosecuted that result in a conviction has also risen to 46%.15 However, it remains the fact that the majority of sexual crimes are not reported, and of those that are the majority do not result in conviction. 10 Ibid. Scotland has three possible criminal verdicts: guilty, not guilty, and not proven. The effect of the not proven verdict is the same as that of not guilty – i.e. acquittal. The not proven verdict has long been controversial, not least for its illogicality – if the case is not proven, then surely the accused should be found not guilty. See for example P Duff, ‘The not proven verdict: jury mythology and “moral panics”’ 41 (1) (1996) Jurid. Rev. 1-12. The Scottish Parliament’s Justice Committee has recently reexamined the verdict in the 2016 Criminal Verdicts Bill, and are recommending it be abandoned: see http://www.bbc.co.uk/news/uk-scotland- scotland-politics-35527022, last accessed 1 September 2016. A. SEXUAL HISTORY: THE SCOTTISH CONTEXT According to the Scottish Crime and Justice Survey 2014/15, most (87.4%) of adults who have experienced serious sexual assault16 in Scotland said that that they knew the offender in some way, while 54.8% said that the perpetuator was their partner.17 Amongst those who had reported more than one form of serious sexual assault since the age of 16, 95.2% said that they knew the offender, and 76.8% said the offender was their partner. This underlines the fact that so many allegations of rape and other sexual offences hinge on the presence or otherwise of consent, rather than identity. A claim of lack of consent can be corroborated by, for example, evidence of physical injury; however, as is often said, consent frequently comes down to which of the competing testimonies the jury believes. Thus, the cross examination of the complainer about her sexual history, and ultimately character, becomes central in assessing whether or not her complaint is credible, meaning that finding corroboration of the lack of consent is extremely challenging. A pre-existing relationship or connection with the accused compounds the complexity of sexual offence trials in which 11 This is similar to, if a little lower than, the conviction rate in other jurisdictions. See M Burman, L Lovett, and L Kelly ‘Different systems, similar outcomes? Tracking attrition in reported rape cases in eleven countries. accessed 1 September. Of course conviction rates are not by themselves an indicator that sexual assault is being taken seriously; nor, as Larcombe has argued, are they necessarily the sole or even main objective that feminists ought to be pursuing. See W Larcombe ‘Falling rape conviction rates: (some) feminist aims and measures for rape law’ 19(1) (2011) Feminist Legal Studies 27-45. See also C McGlynn, ‘Feminism, rape and the search for justice’ 31 (2011) OJLS 825, pp. 825-826; S Cowan ‘Taking a break from the legal to transform the social’ in D Cowan, and D Wincott (eds) (2015) Exploring the Legal in Socio-Legal Studies (Palgrave MacMillan). 12 h // i i l d k/bl / h d 3 i i f i l d h i h accessed 1 September. Of course conviction rates are not by themselves an indicator that sexual assault is being taken seriously; nor, as Larcombe has argued, are they necessarily the sole or even main objective that feminists ought to be pursuing. 13 Criminal Proceedings in Scotland, n9, p. 4. 14 Criminal Proceedings in Scotland, n9, p. 4. 14 http://www.gov.scot/Publications/2015/09/5338/318214, last accessed 1 September. 10 Ibid. Scotland has three possible criminal verdicts: guilty, not guilty, and not proven. The effect of the not proven verdict is the same as that of not guilty – i.e. acquittal. The not proven verdict has long been controversial, not least for its illogicality – if the case is not proven, then surely the accused should be found not guilty. See for example P Duff, ‘The not proven verdict: jury mythology and “moral panics”’ 41 (1) (1996) Jurid. Rev. 1-12. The Scottish Parliament’s Justice Committee has recently reexamined the verdict in the 2016 Criminal Verdicts Bill, and are recommending it be abandoned: see http://www.bbc.co.uk/news/uk-scotland- scotland-politics-35527022, last accessed 1 September 2016. 11 This is similar to, if a little lower than, the conviction rate in other jurisdictions. See M Burman, L Lovett, and L Kelly ‘Different systems, similar outcomes? Tracking attrition in reported rape cases in eleven countries. Country briefing: Scotland’ (2009) http://www.sccjr.ac.uk/wp- content/uploads/2012/11/Daphne_Scotland_Briefing-__Different_Systems,_similar_outcomes(3).pdf, last accessed 1 September. Of course conviction rates are not by themselves an indicator that sexual assault is being taken seriously; nor, as Larcombe has argued, are they necessarily the sole or even main objective that feminists ought to be pursuing. See W Larcombe ‘Falling rape conviction rates: (some) feminist aims and measures for rape law’ 19(1) (2011) Feminist Legal Studies 27-45. See also C McGlynn, ‘Feminism, rape and the search for justice’ 31 (2011) OJLS 825, pp. 825-826; S Cowan ‘Taking a break from the legal to transform the social’ in D Cowan, and D Wincott (eds) (2015) Exploring the Legal in Socio-Legal Studies (Palgrave MacMillan). 12 http://www.rapecrisisscotland.org.uk/blog/the-contested-3-conviction-rate-for-rape-in-scotland-what-is-the- real-story/ last accessed 1 September 2016. 13 Criminal Proceedings in Scotland, n9, p. 4. 14 http://www.gov.scot/Publications/2015/09/5338/318214, last accessed 1 September. 15 http://www.rapecrisisscotland.org.uk/news/rape-crisis-scotland-welcomes-a-40-increase-in-the-number-of- people-convicted-for-rape-and-attempted-rape-in-scotland/ last accessed 1September. 16 Serious sexual assault is defined as forcing or attempting to force someone to have sexual intercourse or other sexual activity (The Scottish Crime and Justice Survey 2014/15: Sexual Victimisation & Stalking p. 10). Less serious sexual offences are indecent exposure; sexual threats or being touched sexually when it was not wanted (e.g. groping or unwanted kissing) (p. 11). 17 Ibid p. 36 A. SEXUAL HISTORY: THE SCOTTISH CONTEXT See W Larcombe ‘Falling rape conviction rates: (some) feminist aims and measures for rape law’ 19(1) (2011) Feminist Legal Studies 27-45. See also C McGlynn, ‘Feminism, rape and the search for justice’ 31 (2011) OJLS 825, pp. 825-826; S Cowan ‘Taking a break from the legal to transform the social’ in D Cowan, and D Wincott (eds) (2015) Exploring the Legal in Socio-Legal Studies (Palgrave MacMillan). 12 p g p 15 http://www.rapecrisisscotland.org.uk/news/rape-crisis-scotland-welcomes-a-40-increase-in-the-num people-convicted-for-rape-and-attempted-rape-in-scotland/ last accessed 1September. 16 Serious sexual assault is defined as forcing or attempting to force someone to have sexual intercourse or other sexual activity (The Scottish Crime and Justice Survey 2014/15: Sexual Victimisation & Stalking p. 10). Less serious sexual offences are indecent exposure; sexual threats or being touched sexually when it was not wanted (e.g. groping or unwanted kissing) (p. 11). 17 16 Serious sexual assault is defined as forcing or attempting to force someone to have sexual intercourse or other sexual activity (The Scottish Crime and Justice Survey 2014/15: Sexual Victimisation & Stalking p. 10). Less serious sexual offences are indecent exposure; sexual threats or being touched sexually when it was not wanted (e.g. groping or unwanted kissing) (p. 11). 17 Ibid p 36 3 sexual history evidence is used.18 As Michele Burman et al note, in practice the complainer’s sexual history evidence is regarded as relevant to establishing the guilt of the accused, particularly when it concerns a past history between the complainer and accused.19 Many have commented that old common law rules of evidence about corroboration and inferences of credibility from those have left a legacy of disbelief where the complainer (historically of course a woman, until the changes brought about in section 1 of Sexual Offences Act 2009 allowed men to be recognised legally as potential victims of rape) was ‘unchaste’. These “myths” 20 about rape and complainers endure maintain that “unchaste women” are more likely to consent to sex, and that such women are “less worthy of belief”.21 As Temkin and Krahe have shown, such beliefs impact on judicial reasoning, the views of barristers, and the determinations of juries. A. SEXUAL HISTORY: THE SCOTTISH CONTEXT For instance, some degree of victim blaming is evident in a recent survey on Scottish social attitudes, and this is closely linked with judgment about behaviour and character.22 Just over half (58%) of those surveyed said that a woman who wore revealing clothing on a night out was “not at all to blame” for being raped, and 60% said the same of a woman who was very drunk. But this still leaves around 40 % who believed that the woman was to some degree to blame for the assault. What is more, 23% agreed that “women often lie about being raped”, with women, older people, and less educated people more likely to agree to this. There are further gender differences in this context. In evaluations of sexual assault, other studies suggest that women are harsher in their verdicts towards the accused and more believing of the complainant’s claim compared to men.23 18 Even where physical injury is present, a previous/current partner might still argue consent – see the Canadian case of R v JA 2011 SCC 28, and for comment, K Busby ‘Every breath you take: erotic asphyxiation, vengeful wives, and other enduring myths in spousal sexual assault prosecutions’ 24 (2) (2012) Canadian Journal of Women and the Law, 328-358; J Koshan ‘Consciousness and consent in sexual assault cases’ (2011) available at http://ablawg.ca/2011/06/17/consciousness-and-consent-in-sexual-assault-cases/ last accessed 28 July 2016. 19 M Burman, L Jamieson, J Nicholson and O Brooks Impact of Aspects of the Law of Evidence in Sexual Offence Trials: An Evaluation Study (2007, Scottish Government) p. 4. 20 J Temkin, ‘Prosecuting and defending rape: perspectives from the bar’ 27 (2) (2000) Journal of Law and Society 219-248. The notion of ‘rape myths’ is not an uncontroversial one. Some argue that rape myths are themselves myths - see H Reece ‘Rape myths: is elite opinion right and popular opinion wrong?’ 33 (3) (2013) Oxford Journal of Legal Studies 445-473. 23 R Schuller and P Hastings, ‘Complainant sexual history evidence: its impact on mock jurors’ decisions’ 26 (2002) Psychology of Women Quarterly 252–261, p. 254 citing Bell, Kuriloff, & Lottes, 1994, and Quackenbush, 1989; see also R Schuller and A Wall ‘The Effects of Defendant and Complainant Intoxication on Mock Jurors' Judgments of Sexual Assault’ 22 (1998) Psychology of Women Quarterly 555-573. 22 Scottish Social Attitudes Survey 2014: Attitudes to violence against women in Scotland (Scottish Gove 2015). y y g 21 R v Seaboyer, McLachlin J at (1991) 83 D.L.R. (4th), 193, p. 258. 18 Even where physical injury is present, a previous/current partner might still argue consent – see the Canadian case of R v JA 2011 SCC 28, and for comment, K Busby ‘Every breath you take: erotic asphyxiation, vengeful wives, and other enduring myths in spousal sexual assault prosecutions’ 24 (2) (2012) Canadian Journal of Women and the Law, 328-358; J Koshan ‘Consciousness and consent in sexual assault cases’ (2011) available at http://ablawg.ca/2011/06/17/consciousness-and-consent-in-sexual-assault-cases/ last accessed 28 July 2016. 19 M Burman, L Jamieson, J Nicholson and O Brooks Impact of Aspects of the Law of Evidence in Sexual Offence Trials: An Evaluation Study (2007, Scottish Government) p. 4. A. SEXUAL HISTORY: THE SCOTTISH CONTEXT Others suggest that myths (and stereotypes), particularly about what the behaviour, dress or demeanour of the complainer signifies, deeply permeate the ways in which sexual offences are perceived in public opinion and dealt with at all stages of the criminal justice system (see eg E Finch and V Munro ‘Of bodies, boundaries and borders: intoxicated sexual consent under the law of Scotland and England’ 1 (2005) Juridical Review 53-72; L Ellison and V Munro ‘A stranger in the bushes, or an elephant in the room? critical reflections upon received rape myth wisdom in the context of a mock jury study’ 13(4) (2010) New Criminal Law Review 781-801; Ellison LE, Munro, V, ‘Of ‘normal sex’ and ‘real rape’: exploring the use of socio-sexual scripts in (mock) jury deliberation’, 18(3) (2009) Social and legal Studies 1-22; J Temkin and B Krahé Sexual Assault and the Justice Gap: A question of Attitude (2009, Hart); B Krahé ‘Myths about Rape Myths? Let the Evidence Speak: A Comment on Reece’ (2013) available at: http://www.unipotsdam.de/sozialpsychologie/fileadmin/projects/sozialpsychologie/assets/Comment_Reece_Pap er.pdf, last accessed 1 September 2016; J Conaghan and Y Russell ‘Rape myths, law, and feminist research: 'myths about myths?'’ 22(1) (2014) Feminist Legal Studies 25-48. http://www.unipotsdam.de/sozialpsychologie/fileadmin/projects/sozialpsychologie/assets/Comment_Reece_Pap er.pdf, last accessed 1 September 2016; J Conaghan and Y Russell ‘Rape myths, law, and feminist research: 'myths about myths?'’ 22(1) (2014) Feminist Legal Studies 25-48. y , ( ) ( ), , p 22 Scottish Social Attitudes Survey 2014: Attitudes to violence against women in Scotland (Scottish Government 2015). 23 R Schuller and P Hastings, ‘Complainant sexual history evidence: its impact on mock jurors’ decisions’ 26 (2002) Psychology of Women Quarterly 252–261, p. 254 citing Bell, Kuriloff, & Lottes, 1994, and Quackenbush, 1989; see also R Schuller and A Wall ‘The Effects of Defendant and Complainant Intoxication on Mock Jurors' Judgments of Sexual Assault’ 22 (1998) Psychology of Women Quarterly 555-573. 4 Sexual history evidence can impact negatively on complainer credibility in that juries may perceive particular witnesses as more credible or trustworthy than others. Previous apparent or actual promiscuity can be conflated with likelihood of consenting to any subsequent sexual act. Of course, there is limited capacity to ascertain the workings and deliberations of juries, since section 8 of the Contempt of Court Act 1981 prevents research with ‘live’ juries. A. SEXUAL HISTORY: THE SCOTTISH CONTEXT That being said, rigorous research producing robust findings about the perceptions and decisions of mock juries allow us to draw some conclusions about the likely impact of sexual history evidence on actual juries. p y j In this respect Regina Schuller and Patricia Hastings’ study on the impact of complainant sexual history evidence on mock jurors’ decisions is noteworthy. In their study, prior history evidence influenced participants’ judgements of cases, with the impact of this information most pronounced when the sexual history information involved sexual intercourse.24 Sexual history evidence was used by the mock jurors to assess the complainant’s credibility and likelihood that she consented and these perceptions were related directly to their judgements of guilt. The study showed that the introduction of complainant sexual history evidence was not used to support the defendant’s defence of an honest but mistaken belief in consent, which was in the context of the study a legally permissible inference but rather was used in a legally inappropriate manner to assess the complainant’s credibility and likelihood that she consented.25 This study has significant implications for rape shield legislation in its findings regarding the improper use of sexual history evidence by jurors. S. 288C. 28 As amended by the Sexual Offences (Procedure and Evidence) (Scotland) Act 2002. pp See Redressing the Balance: Cross-Examination in Rape and Sexual Offence Trials (Scottish Executive, 000). 24 Schuller and Hastings, ‘Complainant Sexual History’ p. 257. 25 Ibid 258 9 pp 26 See Redressing the Balance: Cross-Examination in Rape and Sexual Offence Trials (Scottish Executiv 2000). 27 S 288C Ibid. pp. 258 9. See Redressing the Balance: Cross-Examination in Rape and Sexual Offence Trials (Scottish Executive, 24 Schuller and Hastings, ‘Complainant Sexual History’ p. 257. 25 Ibid. pp. 258-9. 26 See Redressing the Balance: Cross-Examination in Rape and Sexual Offence Trials (Scottish Executive, 2000). 27 S. 288C. 28 As amended by the Sexual Offences (Procedure and Evidence) (Scotland) Act 2002. 1. Law on the books “Rape shield” legislation has been enacted in various jurisdictions since the early 1970s in an effort to limit the use of sexual history and character evidence in criminal proceedings. In Scotland, this first took the form of section 36 of the Law Reform (Miscellaneous Provisions) (Scotland) Act 1985, which amended the Criminal Procedure (Scotland) Act 1975 to introduce restrictions on the use of sexual history and sexual character evidence of complainers in sexual offence trials. This section was repealed in 1995 and replaced by s. 274 and s. 275 of the Criminal Procedure (Scotland) Act 1995, which essentially replicated its predecessor but also extended the protection’s scope to a broader range of sexual offences. The Scottish Executive sought views in 2000 regarding the alteration of the law,26 leading to the enactment of the Sexual Offences (Procedure and Evidence) (Scotland) Act 2002, which amended the 1995 Act. This altered the landscape radically, introducing new provisions to limit the scope of questioning relating to a complainer’s character and sexual history in sexual offence trials.27 The relevant provisions with respect to introducing sexual history evidence remain sections 274 and 275 of the Criminal Procedure (Scotland) Act 1995.28 These provisions relate to sexual offences and indecent assaults, and other forms of intimate interpersonal (or domestic) violence is not included within their scope. 5 Section 274 has four distinct and alternative subsections. Section 274(1)(a) prohibits the leading of evidence or questioning that would show or tend to show the complainer is not of “good character (whether in relation to sexual matters or otherwise)”. Section 274(1)(b) prevents the complainer from being questioned, or evidence being led, about any “sexual behaviour not forming part of the subject matter of the charge”. Section 274(1)(c) prohibits evidence that the complainer has at any time “other than shortly before, at the same time as, or shortly after” the alleged offence “engaged in behaviour, not being sexual behaviour” which might found an inference that she consented or is not a credible or reliable witness. Section 274(1)(d) restricts evidence of “any condition or predisposition” to which the complainer is subject which might lead to the inference being drawn that the complainer consented or is not a credible or reliable witness. 1. Law on the books Together these provisions were intended to draw a tight net around the scope of questioning related to previous sexual behaviour, particularly where evidence related to sexual history was being used tactically as a defence strategy to undermine the credibility of the complainer. In order to protect the fair trial rights of the accused, however, crucially, s. 275 contains an exception to these restrictions, which allows the defence to make an application to introduce sexual history evidence notwithstanding s. 274. It sets out a three-stage cumulative test which must be satisfied before the trial judge can allow questioning or evidence to be led about sexual history or character. First, the evidence must relate to a specific occurrence or occurrences of behaviour, or to specific facts regarding the character, condition or predisposition of the complainer. Second, the behaviour or facts must be relevant to establishing the accused’s guilt. Third, the probative value of the material must be significant and outweigh any risk of prejudice to the administration of justice, which includes the appropriate protection of the complainer’s dignity or privacy. The compatibility of this legislative framework with the European Convention on Human Rights was upheld in Moir v HM Advocate.29 Moir had been convicted of rape and sexual assault, and argued that Art 6 of the ECHR was breached by the excessive extent of the s. 274 prohibition and the restricted extent to which the prohibition could be over-ridden. The Court refused the appeal, holding that s. 275 was a reasonable and flexible response to the problem of the “embarrassment and humiliation of a complainer in a rape trial”30 and a legitimate means of achieving the legislative objective, and the legislation did not have a disproportionate effect per se.31 However, it was observed by the Lord Justice-Clerk that a prior course of cohabitation by a complainer with an accused would not constitute engaging in sexual behaviour not forming part of the subject-matter of a charge (s. 274(1)(b)), and therefore such cohabitation would be beyond the scope of s. 274’s protections.32 In other words, evidence of cohabitation may be admitted. Such matters were considered further by the Privy Council in DS v HM Advocate, examining the admissibility of evidence regarding the behaviour of the complainer.33 The Privy Council also held that the power to exclude evidence of “sexual behaviour” in s. 29 Moir v HM Adv 2005 1 JC 102 30 Ibid. para 29. 31 Ibid. paras 36-38. 32 Ibid. para 29. 33 DS v HM Adv 2007 SC (PC) 1 34 Ibid. para 27. 35 Doorson v Netherlands (1996) 22 EHHR 330 at para 70. 36 Judge v United Kingdom 2011 SCCR 241. 37 See Scottish Executive Vulnerable and Intimidated Witnesses section 5 for a discussion of similar ‘tensions’ in a number of international jurisdictions, relating to the balancing of the fair trial rights of the accused with the privacy rights of the witness. 38 M Burman et al ‘Impact of Aspects’, p. 4. 39 B Brown, M Burman and J Jamieson Sexual History and Sexual Character Evidence in Scottish Sexual Offence Trials (1992, Edinburgh: Scottish Office Central Research Unit); B Brown, M Burman and J Jamieson Sex Crimes on Trial: Sexual History and Sexual Character Evidence in Scottish Sexual Offence Trials (1993, Edinburgh: Edinburgh University Press; M Burman et al ‘Impact of Aspects’. 40 M Burman et al ‘Impact of Aspects’, p. 39. 41 Ibid. p. 2. 1. Law on the books 274(1)(b) did not extend to a prior course of cohabitation between the accused and the complainer. What is more, the exclusion of evidence of non-sexual behavior under s. 274(1)(c) did not extend to evidence that is directed simply to words that the complainer may have said to a third party which bear on her credibility or reliability. So, while the protection afforded to complainers by s. 274 is “very 6 wide”,34 according to the Privy Council, it does not apply to words the complainer might have said to a third party, nor to cohabitation. The European Court of Human Rights has held that the "principles of fair trial also require that in appropriate cases the interests of the defence are balanced against those of witnesses or victims called upon to testify”.35 Thus, it is not surprising that the Court approved of the Scottish scheme in Judge v United Kingdom, dismissing Judge’s claim that the UK had breached his Art 6 rights.36 The Court emphasised that it was for domestic courts to decide whether it was appropriate to call a witness, and an issue would arise under Art.6(3)(d), which guarantees the right of the accused to examine or have examined witnesses against him, only if restrictions placed on the right to examine witnesses were so restrictive as to deprive that provision of its effect. The European Court remarked that the Scottish Parliament had introduced these provisions on the basis that, in criminal trials for sexual offences, evidence as to the complainer’s sexual history and character was rarely relevant and, even where it was, its probative value was usually weak when compared with its prejudicial effect. Accordingly, the Parliament was entitled to take action to protect the rights of complainers and to generally prohibit the introduction of bad character evidence against them, whilst providing for an exception where such evidence was relevant or probative. Therefore, s. 274 and s. 275 were regarded as a reasonable and flexible response to the problem of questioning of complainers in cases concerning sexual offences and a legitimate means of achieving the objectives pursued by the legislature.37 2. Law in practice So what happens in practice when defence counsel wants to adduce evidence about the sexual behaviour of the complainer in a sexual offences trial? First of all, it is necessary that a written application be submitted to the court, in advance of trial. This requirement applies to both the prosecution and the defence. This is regarded as resulting in greater transparency as to the reasoning behind applications, but not as resulting in discussion of the relevance of the evidence by the Court.38 The different formulations of Scottish rape shield legislation have been independently evaluated.39 The most recent of these studies, published in 2007, indicated that 72% of all High Court sexual offence trials from 2004-05 included a s275 application,40 with 76% of rape trials involving such applications. These figures represented an increase in the use of sexual history and character evidence since the introduction of the amendments in the 2002 Act. Just 7% of the s275 applications were disallowed, and in all but a small number of cases, all evidence allowed in the application was introduced in the trial, usually through cross-examination of the complainer.41 Several of the interviewed practitioners considered it 7 7 relatively easy to demonstrate that sexual history/character evidence is relevant.42 These findings are both surprising and worrying – and yet to date there has been no further research in Scotland indicating whether the figures have changed or whether the success of s275 applications has remained at such a high level, demonstrating a real and urgent need for more scrutiny and data on this issue. In addition, case law on these provisions is rather chequered, with some dubious decisions. For instance, in Kinnin v HM Advocate the appeal court held that evidence of comments made by the complainer to K’s son in the weeks prior to the alleged incident indicating that she wanted a sexual relationship with K’s son was an essential part of K’s defence and had been wrongly excluded.43 Remarkably, the Crown offered no objection to the admission of the evidence and admitted that it appeared relevant and might have a bearing on the issues. The Court accepted that the evidence was not too remote in time or in its relationship to the issue of whether the physical contact had been consensual. 42 Ibid. p. 133. 43 Kinnin v HM Advocate 2003 SCCR 295. 44 Cumming v HM Advocate 2003 SCCR 261. 45 M Burman et al ‘Impact of Aspects’, p. 50. 46 Ibid. p. 3. 47 HM Advocate v Ronald 2007 SCCR 451. 48 M v HM Advocate (No.2) [2013] HCJAC 22. 2. Law in practice Nonetheless, the issue was deemed to be 8 8 collateral and “removed in time and character to the charges on the indictment” and so inadmissible under common law.49 On appeal, this ruling was upheld, though there was a divergence of views as to the admissibility of the making of false complaints and the propensity to do so. Thus, the case was remitted to a Full Bench.50 As Pete Duff states, however, the matters for “consideration and determination”51 were not resolved,52 leaving the law somewhat uncertain. The Lord Justice General, giving the leading opinion, repeated that the common law is not circumvented by s. 275 to admit evidence that would otherwise be excluded as collateral. As for whether a relevant prior false complaint would always be inadmissible as a collateral issue, he stressed that such a complaint could only go to witness credibility.53 He considered the proven dishonesty of a witness as an exception to this rule, where a prior allegation could be admitted only if its falsity is proved “by reference to established fact in the form of a previous conviction”.54 Critically, there was no conviction here, and so the matter was deemed to be inadmissible. Lord Clarke expressed a different opinion, though agreeing with the ultimate conclusion regarding admissibility in this instance. He rejected a “prescriptive regime” predicated on conviction, preferring instead a proportionate approach which would involve careful scrutiny of any claims.55 As Duff outlines, while Lord Clarke’s “nuanced” approach could lead to lengthy “satellite litigation”, it is more likely to avoid injustice.56 pp g y g , y j Furthermore, the Lord Justice General distinguished the facts of Ronald on the basis that there the complainer had an “objectively diagnosed medical condition” which could contribute to the making of false complaints whereas in M, she did not. 2. Law in practice It seems remarkable that the complainer’s willingness to have sex with the accused’s son was deemed to be relevant to consent to sex with Kinnin himself. Some of the legal practitioners interviewed in Burman’s study viewed this decision, along with similar decisions (in Cumming for example44), as a reason for a subsequent increase in s.275 applications.45 Evidence or questioning concerning the character of the complainer featured in approximately 24% of cases in Burman et al’s 2007 study, often concerning the complainer’s use of alcohol or drugs.46 g The admissibility of such evidence, regarding former inconsistent complaints, borderline personality disorder and alcohol dependency syndrome, was considered and upheld in HM Advocate v Ronald.47 Ronald was charged with rape; he claimed that he and the complainer had had consensual sexual intercourse, but that following a disagreement she made an allegation of rape. The defence sought to bring evidence of a previous allegation which they said was false. The Court held that where, as here, a complainer was diagnosed with a borderline personality disorder and an alcohol dependency syndrome, evidence about not only the disorder and the syndrome but also other aspects of a person’s behaviour would be relevant to the existence of a “predisposition” in the sense in which the word was used in s. 275(1)(a). Moreover, the Court held that this complainer’s recorded accounts of the incidents were relevant to the defence both as an example of impulsiveness and lack of self control relevant to her diagnosis and also as a means of challenging her credibility. Nonetheless, it was held that it would have been inappropriate to allow the accused to lead evidence or cross examine the complainer in an attempt to prove that she had not been raped or otherwise abused on other occasions. Such issues were considered further in M v HM Advocate,48 where M was convicted of the historic sexual abuse of three children. He had applied under s. 275 to question one of the complainers about a false claim she had made to the police of sexual assault by a third party when in her teens. The complainer had been cautioned and charged with wasting police time but not prosecuted. The defence argued that this matter was relevant as it went to the credibility of her complaint of abuse by M. 49 Ibid. para 6. 50 M v HM Advocate [2012] HCJAC 83, para 23. 51 Ibid. 52 P Duff, ‘The admissibility of previous false allegations of sexual assault: CJM (No.2) v HM Advocate’ 17(3) (2013) Edinburgh Law Review 381-387. 53 M v HM Advocate (No 2) n. 48, para 27. 54 Ibid. para 32. 55 Ibid. para 51. 56 P Duff, ‘The admissibility of previous false allegations’, p. 384. 57 M v HM Advocate (No 2), para 39. 58 Ibid. 59 Ibid. 60 Ibid. para 53. 61 P Duff, ‘The admissibility of previous false allegations’, p. 387. 62 M Burman et al ‘Impact of Aspects’, p82. 63 M v HM Advocate (No 2) n48. p 61 P Duff, ‘The admissibility of previous false allegations’, p. 387. 2. Law in practice 57 He observed that “[e]vidence that a complainer suffers from an objectively diagnosed medical condition and that such a condition may, as a generality, have a bearing on a person’s ability to know or tell the truth is admissible, but the matter stops there as a matter of expediency”.58 So, he disapproved of the admission in Ronald of expert evidence beyond that concerning the complainer’s psychiatric conditions which could affect her ability to know or tell the truth.59 The Lord Justice General invited the court to disapprove of Ronald, though this was not necessary for the disposal of the present case. Lord Clarke preferred to postpone such matters.60 Duff has disagreed with such disapprobation of Ronald, suggesting that this would lead to a rigid (and arbitrary) rule limiting the factors which a psychiatrist can cite.61 However, his objection overlooks the degree to which discretion in this context in the past has facilitated the introduction of speculation and stereotyping (see below). Overall, concern was raised by some of the judges interviewed in Burman’s study about the intricacy of law in this area,62 and the provisions themselves were described as an “elaborate code” in M v HMA.63 Taken together, these cases do seem to overly constrain the 9 ‘rape shield’, and could indicate that the protections can be undermined somewhat indiscriminately, thereby circumventing the intentions of the legislators, and impeding certainty in the law. However, Peter Duff suggests that “[o]ne must be cautious about reading a pattern or trend into these cases because there are examples from the previous shield regime where judges took a very robust approach to the issue of relevance and, thus, individual decisions may reflect little more than the attitudes of different judges rather than any underlying change as a result of the new legislation”.64 This focus on individual judges calls to mind Louise Ellison’s observation regarding the all-male composition of the House of Lords in R v A in 2001.65 Similarly, there is striking gender imbalance in the Scottish courts. There are nine female judges in Scotland, out of 34 Senators of the College of Justice. The Lord Justice Clerk is now, for the first time, a woman, Lady Dorrian. 64 P Duff, ‘The Scottish "rape shield": as good as it gets?’ 15 (2011) Edin LR 218-242, p. 236. 65 L Ellison, ‘Commentary on Re A (No. 2), p206. 66 See, for example, Lady Hale, Deputy President of the UK Supreme Court, “Appointments to the Supreme Court”, Conference to mark the tenth anniversary of the Judicial Appointments Commission, University of Birmingham, 6 November 2015, https://www.supremecourt.uk/docs/speech-151106.pdf. 67 Note about the relative infrequency of sexual assault appeal cases in Scotland. 68 M Burman et al ‘Impact of Aspects’, p3. 69 Ibid. p. 7. 70 P Duff, ‘The admissibility of previous false allegations’ p. 238. 71 L Bain, ‘The failures of ‘shield legislation’: sexual history evidence, feminism and the law’ 96 (2010) Aberdeen Student Law Review 96-110, p. 101. 72 s275A. 73 M Burman et al ‘Impact of Aspects’, p. 97. 64 P Duff, ‘The Scottish "rape shield": as good as it gets?’ 15 (2011) Edin LR 218-242, p. 236. 65 L Ellison, ‘Commentary on Re A (No. 2), p206. 66 See, for example, Lady Hale, Deputy President of the UK Supreme Court, “Appointments to the Supreme Court”, Conference to mark the tenth anniversary of the Judicial Appointments Commission, University of Birmingham, 6 November 2015, https://www.supremecourt.uk/docs/speech-151106.pdf. 67 Note about the relative infrequency of sexual assault appeal cases in Scotland. 68 M Burman et al ‘Impact of Aspects’, p3. 69 Ibid. p. 7. 2. Law in practice Yet, as Burman et al stress, prior convictions relating to domestic abuse 10 could be relevant in a sexual offence case in demonstrating a previous history of violence against a woman, even more so where the same woman is involved.74 Reform is needed here so as to permit the inclusion of such previous convictions alongside previous sexual assault convictions. Having set out these provisions and problems relating to the use of sexual history evidence in Scottish sexual assault trials, it is worth examining other measures that have been considered to ameliorate the difficulties faced by complainers in sexual assault trials. One possible way forward is independent legal representation, via legal aid, for complainers. This issue has been discussed in some detail in England and Wales by Ellison, and in Scotland by Raitt, Duff (this volume) and Chalmers.75 It was also debated recently by the Scottish Parliament’s Justice Committee at their 26th meeting, Session 4, on 22 September 2015, with respect to an amendment to the Criminal Justice Scotland Bill that would allow legal aid funding for representation of sexual assault complainers who want to challenge the defence’s use of ‘private’ information, including medical records, in sexual assault trials.76 We will not rehearse further the arguments made by Duff and others on the merits or otherwise of legal representation for those alleging sexual assault. However, it is worth noting that, while the Justice Committee failed to agree to the amendment on legal aid for representation, their discussion sparked a request, from the Cabinet Secretary for Justice, to the Crown Office and Procurator Fiscal Service (COPFS), and Scottish Courts and Tribunal Service (SCTS), for a short monitoring exercise regarding the number of s. 275 applications made and granted. g g g pp g The resulting data, published in a letter from the Cabinet Secretary for Justice on 26 June 2016,77 shows that from 11 January – 11 April 2016, 57 applications were made under s. 275 (52 in the High Court and 5 in the Sheriff Courts).78 Of these, 51 were unopposed by the Crown (48 in the High Court and 3 in the Sheriff Courts), and 6 were opposed (4 in the High Court and 2 in the Sheriff Courts). Of the 52 High Court applications, 42 were granted in full, 5 were granted in part, and 5 refused. 2. Law in practice While Baroness Hale emphasises the difference that having female judges can make more generally in the legal system,66 it remains to be seen what, if any, the difference will be in terms of attitude towards, or outcomes, in sexual assault cases.67 Burman et al’s study indicated that Crown applications to introduce sexual history evidence usually related to evidence required to enable a jury to make sense of subsequent evidence or to contextualise the alleged events.68 But the reforms in 2002 had the “largely unanticipated and unintended consequences of the introduction of more sexual history and character evidence than occurred under the 1995 legislation”.69 As Duff has stated: “It is perhaps significant that the 2007 study indicates that judges have difficulty imagining circumstances where they would rule out otherwise relevant evidence in order to protect the complainer”.70 This lack of judicial imagination speaks to a lack of understanding of the secondary victimisation often suffered by rape complainers and their potential vulnerability when giving evidence, or regarding their treatment in the criminal justice system more generally (see below). As Leanne Bain concludes, referring to Burman’s study: “it is clear that the legislation has failed to achieve its goals… the more formalised procedure means that evidence sought is 'far more detailed and extensive' than under verbal procedures, and 'greater emphasis on early preparation' means that the defence has become even more skilled at ensuring its introduction, often through the use of multiple applications.”71 Finally, and notably, where the court allows questioning or evidence under s. 275, this triggers disclosure of the accused’s previous convictions for sexual offences or any offence where a substantial sexual element was present in its commission.72 This provision, which instantiates a quid pro quo, is unique to Scotland. Burman et al provide the most up to date figures we have on this, again highlighting a pressing need for more research in this area. They highlight that in eight rape cases in their study, the accused had a previous conviction for assault, assault to injury, or assault to severe injury in the context of domestic abuse;73 these convictions would not be disclosed under s.275A as they do not involve a substantial sexual element. 74 Ibid. 75 Put in references. 76 Official Report of the Justice Committee, Tuesday 22 September 2015, available at http://www.parliament.scot/parliamentarybusiness/report.aspx?r=10100&mode=pdf, last accessed 1 September 2016. 77 Letter from Michael Matheson to Margaret Mitchell, MSP, available at http://www.parliament.scot/General%20Documents/20160624CSfJtoConvenerILR.pdf, last accesses 1 September 2016. 78 The Sheriff court is a court of first instance in Scotland, usually with one sitting Sheriff. There is a right of appeal to the High Court of Justiciary, which also is a court of first instance for more serious cases, hence the higher number of sexual assault cases coming before the High Court than the Sheriff court. 79 http://www.parliament.scot/parliamentarybusiness/report.aspx?r=10100&mode=pdf at Column 32. 80 See for example, S Lees Carnal Knowledge: Rape on Trial (2002, Hamish Hamilton); L Ellison and V Munro ‘Taking Trauma Seriously: Critical Reflections on the Criminal Justice Process’ (2016) The International Journal of Evidence & Proof 1-26 81 P Richards, S Morris and E Richards Turning up the Volume: The Vulnerable Witnesses Scotland Act 2004 (Scottish Government, 2008) 82 Ibid. para 1. 2. Law in practice In other words, 47 of 52 applications - 90% - were at least partially granted at the High Court. At the Sheriff Courts, only 1 of the 5 applications was granted and the other 4 were refused. Without more detailed information, it is difficult to offer any nuanced analysis of these figures. We cannot say whether this is higher or lower than previous years, because we have no comparative statistics, and we do not know whether a 3-month sample is necessarily indicative of any particular trend or pattern. While we can say that 47 of 52 applications were granted in full or in part, and that this seems like a high success rate, without more data, such as the number of sexual assault cases during that period, it is impossible to know whether this demonstrates the same kind of worryingly high rate of successful applications as seen in the Burman et al study. There is a pressing need for more sustained data generation in this area in order to be able to understand the experiences of those complainers who go through sexual assault trials, the practices of those who make, challenge and assess applications, and the question of whether further interventions, such as independent representation, are needed. q , p p , What we can say, interestingly, is that at the High Court, 4 of the 5 rejected 11 applications were not challenged by the Crown; at the Sheriff Courts, 2 of the rejected applications were similarly not challenged. Although these numbers are small, and it is difficult, as suggested above, to reach any robust conclusions, the data does draw attention to the claim made by Margaret Mitchell, who introduced the independent legal representation amendment to the Criminal Justice Scotland Bill. She argued that based on the evidence of victim support groups, the Crown is not proactive enough in its challenges to s. 275 applications.79 Clearly, more research is needed here. Leaving aside independent representation, there are a number of other provisions designed to support victims and witnesses who give evidence at trial, including those who claim to have been sexually assaulted. Here we turn to the issue of “special measures”. C. SPECIAL MEASURES FOR VULNERABLE WITNESS AND VICTIMS Although the complainer in a sexual assault trial may be subject to humiliating and discrediting questions about their sexual (or medical) history, they do have access to mechanisms within the criminal process that may help to minimise the detrimental effects of having to speak about intimate details of the offence, and of their lives more generally, in a public court room. If deemed a ‘vulnerable witness’ the complainer may be able to at least partially shield themselves from some traumatic aspects of giving evidence (though not from cross examination, as discussed below), through the use of what are commonly termed ‘special measures’ such as screens or live video links. Since it has been acknowledged that giving evidence in a sexual assault trial may be experienced as a secondary form of victimisation that causes further trauma,80 special measures are not only an attempt to achieve the ‘best evidence’ possible in criminal proceedings81 but also go some way to recognising that the process itself can be a significant disincentive for victims of sexual assault to report offences. But who is defined as a vulnerable witness? To what extent are these attempts to protect complainers realised, and what is the impact of such protections on sexual victims? 1. Who is a ‘vulnerable’ witness and how are they identified? a. Who is vulnerable? a. Who is vulnerable? Richards et al82 suggest that, although the 2004 Act largely reproduced existing provisions, it was felt by the Scottish 12 government that a firm, clear legislative statement with a more inclusive definition of vulnerability and specific details of special measures was needed, as those who were vulnerable were not always getting the appropriate necessary support.83 The category of ‘vulnerable’ was then further extended by the Victims and Witnesses (Scotland) Act 2014, which raised the age threshold for children to 18.84 In addition, witnesses who are complainers in trials related to human trafficking, domestic abuse, stalking, and – importantly for our purposes – sexual assault, are also now deemed vulnerable.85 When deciding whether a person is vulnerable, s271(2) of the 1995 Act (as amended by the 2004 Act), states that the court must take into account the following factors: When deciding whether a person is vulnerable, s271(2) of the 1995 Act (as amended by the 2004 Act), states that the court must take into account the following factors: (a) the nature and circumstances of the alleged offence to which the proceedings relate, (b) the nature of the evidence which the person is likely to give, p y g (c) the relationship (if any) between the person and the accused, (d) the person’s age and maturity, (e) any behaviour towards the person on the part of— (i) the accused, (ii) members of the family or associates of the accused, (iii) any other person who is likely to be an accused or a witness in the proceedings, and (i) the accused, (ii) members of the family or associates of the accused, (iii) any other person who is likely to be an accused or a witness in the proceedings, and (f) such other matters, including86 (i) the social and cultural background and ethnic origins of the person, (ii) the person’s sexual orientation, (iii) the domestic and employment circumstances of the person, (iv) any religious beliefs or political opinions of the person, and (v) any physical disability or other physical impairment which the person has (iii) the domestic and employment circumstances of the person, (iv) any religious beliefs or political opinions of the person, and (v) any physical disability or other physical impairment which the person has. 88 P Richards et al, Turning Up the Volume. 89 Ibid. para 27. 83 For discussion of the international context in which the 2004 Act came about see Richards et al, ibid, chapter 2). 84 S. 10(a). 85 S. 10(c). 86 I.e. this is a non-exhaustive list - other relevant issues can be taken into consideration according to the Act’s Explanatory Notes: available at http://www.legislation.gov.uk/asp/2004/3/notes/division/3/1/1/1last accessed 1 September 2016. 87 S. 10(e), Victims and Witnesses (Scotland) Act 2014. A witness can also be considered vulnerable if “there is considered to be a significant risk of harm to the person by reason only of the fact that the person is giving or is to give evidence in the proceedings” (s. 10(d)). a. Who is vulnerable? In Scotland, provisions on the treatment of vulnerable witnesses and special measures were first introduced by the Criminal Procedure (Scotland) Act 1995. Under the 1995 Act, only children were deemed to be vulnerable witnesses. This was extended to adults with mental disorders by the Crime and Punishment (Scotland) Act 1997, and again by the Vulnerable Witnesses (Scotland) Act 2004: s. 1 of the 2004 Act amends s. 271 of the 1995 Act to include those who are under 16; and those for whom there is a “significant risk that the quality of the evidence to be given by the person will be diminished” either because of a mental disorder, (as defined by s. 328 of the Mental Health (Care and Treatment) (Scotland) Act 2003), or because of fear or distress about giving evidence at trial. 88 P Richards et al, Turning Up the Volume. 89 p 87 S. 10(e), Victims and Witnesses (Scotland) Act 2014. A witness can also be considered vulnerable if “there is considered to be a significant risk of harm to the person by reason only of the fact that the person is giving or is to give evidence in the proceedings” (s. 10(d)). a. Who is vulnerable? The best interests (and any views) of the witness (or their parents/guardians) as to special measures also have to be taken into account.87 The best interests (and any views) of the witness (or their parents/guardians) as to special measures also have to be taken into account.87 There is as yet no evaluation of the most recent 2014 expansion of the measures. But in their evaluation of the 2004 framework, Richards et al interviewed 74 justice professionals and 11 vulnerable witnesses or their representatives about their experiences.88 They found that many welcomed the new Act, despite some reservations by the judiciary;89 however, they highlight particularly strong concerns about how criminal justice practitioners identity vulnerable adults, especially since, as they point out, many of the factors detailed in subsection (f) above are not immediately discernible: “Many examples were given of adults who may well have been vulnerable witnesses but received no offer of special measures”.90 p 87 S. 10(e), Victims and Witnesses (Scotland) Act 2014. A witness can also be considered vulnerable if “there is considered to be a significant risk of harm to the person by reason only of the fact that the person is giving or is to give evidence in the proceedings” (s. 10(d)). 13 This finding raises the question, first, of what is meant by vulnerability, and secondly, how criminal justice practitioners might recognise it. When debating the Policing and Crime Bill in the UK parliament in 2009, Jacqui Smith stated in relation to prostitution that: “The mark of any civilised society is how it protects the most vulnerable”.91 As Vanessa Munro and Jane Scoular have documented, vulnerability discourses proliferate in contemporary criminal justice debates.92 But this proliferation does not help with the question of what constitutes vulnerability, and whether we can ever reach a common understanding of vulnerability, so that we can ‘know it when we see it’. 97 V Munro ‘Title’ (2016) Social and Legal Studies Page no. needed here – article based on her paper is in press and will be out later in 2016. 98 N Haas and A García ‘Encounters with vulnerability: the victim, the fragile, the monster, the queer, the abject, the nomadic, the feminine, the shameful, and the rest….’ 11(1) (2015) Graduate Journal of Social Science 151- 161, p. 152. 94 Ibid. p. 2; see also J Butler Precarious life: The powers of mourning and violence (2006, Verso) pp. 29–31; R Braidotti ‘Affirmation versus Vulnerability: On contemporary ethical debates’ 10 (2006) Canadian Journal of Continental Philosophy 235–254. 96 M Fineman ‘The Vulnerable Subject’ p 9 88 Ibid. para 41. 91 Available at http://www.publications.parliament.uk/pa/cm200809/cmhansrd/cm090119/debtext/90119-0010.htm, column 524, last accessed 1 September 2016. 92 V Munro and J Scoular ‘Abusing vulnerability? Contemporary law and policy responses to sex work and sex trafficking in the UK’ 20(3) (2012) Feminist Legal Studies 189-206. 93 M Fineman, ‘The Vulnerable Subject’, p. 1. 94 Ibid p 10 a. Who is vulnerable? According to vulnerability’s contemporary academic champion, Martha Fineman, vulnerability is “universal and constant, inherent in the human condition”, and that as such we all share “common vulnerabilities”93 even while specific vulnerabilities are “particular”.94 Fineman argues that rather than press for state recognition of the liberal autonomous choosing subject, we should embrace the notion of the ‘‘vulnerable subject’’ as ‘‘far more representative of actual lived experience and the human condition’’.95 In contrast to negative interpretations of vulnerability as coterminous with victimhood, Fineman urges us to ‘‘reclaim’’ vulnerability as a ‘‘heuristic’’ concept.96 This would, she seems to suggest, allow us to examine how factors that are commonly folded into vulnerability are socio-politically and institutionally constructed. Other commentators are more sceptical of the progressive potential of vulnerability, however. Munro suggests that using vulnerability to particularly mark out specific groups of people “risks ‘othering’ those individuals or groups in ways that further entrench their difference and stigma, whilst leaving unchallenged the residual norm of unbounded, empowered, and capable human agency”.97 Similarly, Haas and García have suggested that “Whenever the deployment of vulnerability is only applied to ‘marginal’ subjectivities and exceptional situations, ideologies about the body as a naturally-given are reified, effacing the deeply political, exclusionary, and gendered and cultural affiliations…”.98 Munro argues that vulnerability might also replicate some of the problems faced in more traditional rights-based claims related to the ranking of hierarchising of harms and hence vulnerability claims. Her concerns are echoed by Elaine Craig, who highlights the potential for group and community based markers of vulnerability to simply perpetuate “ entrenched social hierarchies” 88 Ibid. para 41. 91 Available at http://www.publications.parliament.uk/pa/cm200809/cmhansrd/cm090119/debtext/90119-0010.htm, column 524, last accessed 1 September 2016. 92 V Munro and J Scoular ‘Abusing vulnerability? Contemporary law and policy responses to sex work and sex trafficking in the UK’ 20(3) (2012) Feminist Legal Studies 189-206. 93 M Fineman, ‘The Vulnerable Subject’, p. 1. 94 Ibid p 10 96 M Fineman, ‘The Vulnerable Subject’, p. a. Who is vulnerable? 9 14 including but not confined to those related to moralistic views about sex, gender stereotypes and “the individualized rather than systemic response structure of the criminal justice system”.99 In short, anxieties about relying on the concept of vulnerability seems to centre around a worry that, as conceptualised by for example Fineman, vulnerability runs the risk of being overinclusive; or that the potential for any practical or political application of it is mitigated by a tendency to over-individualise vulnerability in a way that focuses on individual solutions without addressing the the question of how and why people end up vulnerable. In concentrating on how a particular person is vulnerable, we might paper over more systemic fractures that perpetuate existing social inequalities and hierarchies. In the present context, this translates into a potential neglect, both of the ways in which the criminal justice system itself creates vulnerabilities, and the current political and legal failure to take seriously the significant and structural vulnerabilities of many sexual assault victims. y g y The use of vulnerability in the Scottish legislation, which focuses on particular groups of people, arguably risks prompting all of the concerns mentioned here. For example, do the characteristics listed in s. 271 (2) (f) (set out above) indicate inherent vulnerability? Notwithstanding the explicit statement of the Scottish Executive that the use of the term vulnerability in the legislation was not intended to denote any inherent personal factor or deficit,100 it is hard to see how sexual orientation or any of the other factors do not relate to something personal and inherent to the individual, even if not strictly seen as a ‘deficit’. And what makes these characteristics the correct ones to include in such a list? At one level it is easy to see how this list was generated – it corresponds closely with the list of characteristics protected in hate crimes legislation (ethnicity, sexual ‘orientation’, religion, disability), though it does not mention transgender identity (which was added as a hate crime via the Aggravated by Prejudice (Scotland) Act 2009); and it includes a broader range of potentially vulnerable people since it includes consideration of a person’s social and cultural background, their domestic and employment circumstances, and their political opinions. 99 E Craig ‘Capacity to consent to sexual risk’ 17(1) New Criminal Law Review: An International and Interdisciplinary Journal (2014) 103-134, p. 127. 100 Scottish Executive Vulnerable and Intimidated Witnesses, p. 4. 101 L Piggott ‘Prosecuting disability hate crime: a disabling solution?’ 5(1) (2011) Place and Policy Online 25- 34. a. Who is vulnerable? Some of these characteristics are protected in other sorts of rights frameworks such as the Convention for the Status of Refugees 1951 (ethnicity, sexual orientation, social and cultural background, political opinion), or equality-based anti-discrimination laws. The relevance and meaning of ‘domestic and employment circumstances of a person’ is more opaque but it seems that we might interpret this as circumstances of duress or where there is a heavily unbalanced power relationship, such that the witness might feel significant pressure not to give certain kinds of evidence. In any case, despite the fact that these listed factors are all commonly recognised vectors of vulnerability, reliance upon these as ways of recognising and marking out vulnerability may at first glance merely perpetuate a problematic, individualistic and essentialised understanding of what it is to be vulnerable. For example, Piggott suggests that focusing on the characteristic of the victim of hate crime, rather than the social circumstances that make hate crime possible “depends on the identification of a person as different, thereby reinforcing culturally embedded ideas of normality”.101 The individualising tendencies of the legislation are especially noticeable when we take the factors listed in the 2004 Act as relevant to establishing vulnerability as independent of each other; i.e., there is no recognition of the complex ways in which different vectors of vulnerability intersect and are compounded in complex ways. For example, research in 15 England and Wales has shown that sexual assault victims who have mental health problems are less likely to report, less likely to have the incident recorded by police, and are less likely to be believed by criminal justice agents.102 Indeed, Ellison et al emphasise that although many studies point to the high rate of prevalence of sexual violence against those with mental ill health or learning disabilities, due to massive underreporting, the scale of this problem is not known103 (though of course this is true of sexual offences more generally). This is a pattern replicated at the international level, where research has shown that those who experience mental ill health are especially at risk of sexual assault.104 Betsy Stanko has described this as the de facto decriminalisation of sexual assault of those who suffer from mental ill health or have learning difficulties: one report of her research suggests that “Those with learning difficulties were 67% less likely to have their case referred by police for prosecution than those without. 102 L Ellison, V Munro, K Hohl, and P Wallang ‘Challenging Criminal Justice? Psychosocial disability and rape victimisation’ 15(2) (2015) Criminology and Criminal Justice 225-244. 103 L Ellison et al, ‘Challenging Criminal Justice’. 104 L Ellison ‘The Use and Abuse of Psychiatric Evidence in Rape Trials’ (2009) 13(1) The International Journal of Evidence and Proof 28-49, p. 48. 105 https://www.theguardian.com/commentisfree/2014/mar/08/rape-consent-vulnerability-barfield-stanko. 104 See for example S Lees, Carnal Knowledge; J Temkin and B Krahé, Sexual Assault; J Temkin, ‘Prosecuting and Defending Rape’. 107 L Ellison,’The Use and Abuse’. 108 F Raitt ‘Disclosure of Records and Privacy Rights in Rape Cases’ 15(1) (2011) Edinburgh Law Review 33- 56, p. 40. Raitt points out that there are few formal rights afforded to the complainant, with respect to privacy, in the criminal justice context. She suggests that only Articles 2,6 and 8 of the ECHR give the complainer any formal recourse, but that these are so broad that they may be difficult to pin down. However, see the recent 2016 ruling where the Court of Session in Scotland said that a complainer’s Article 8 rights would be infringed by the disclosure of private medical records, and that she was entitled to legal aid to challenge the application for disclosure: http://www.bbc.co.uk/news/uk-scotland-35562009. For discussion of the history of the reform of disclosure rules, see Duff (this volume). 109 See for example: E McDonald ‘Resisting defence access to counselling records in cases of sexual offending: does the law effectively protect clinician and client rights?’ 5(2) (2013) Sexual Abuse in Australia and New Zealand 12-20; L Gotell ‘Colonization through disclosure: confidential records, sexual assault complainants and canadian law’ 10(3) (2001) Social Legal Studies September 315-346. 108 F Raitt ‘Disclosure of Records and Privacy Rights in Rape Cases’ 15(1) (2011) Edinburgh Law Review 33- 56, p. 40. Raitt points out that there are few formal rights afforded to the complainant, with respect to privacy, in the criminal justice context. She suggests that only Articles 2,6 and 8 of the ECHR give the complainer any formal recourse, but that these are so broad that they may be difficult to pin down. However, see the recent 2016 ruling where the Court of Session in Scotland said that a complainer’s Article 8 rights would be infringed by the disclosure of private medical records, and that she was entitled to legal aid to challenge the application for disclosure: http://www.bbc.co.uk/news/uk-scotland-35562009. For discussion of the history of the reform of disclosure rules, see Duff (this volume). 107 L Ellison,’The Use and Abuse’. a. Who is vulnerable? Mental illness reduced the chance by 40%.”105 The complicated ways in which various factors related to for example, drug or alcohol use, mental health, a previous relationship with the accused, and a previous allegation of sexual assault, as well as difficulty in narrating a coherent account, interact with the long documented existing gendered ‘blind spots’ in a sexual assault trial106 means that an atomistic approach to vulnerability in the court room does not adequately capture or reflect the reality of victims’ experiences. Indeed, many if not most sexual assault victims are unlikely to get anywhere near the criminal trial in the first place, particularly where they experience one or more of these other factors, such as drug use and / or poor mental health (see also Cairns, this volume). When they do, Ellison has highlighted the frequent inappropriate use of psychiatric and confidential mental health records in sexual assault cases in England and Wales, despite procedures being put in place for complainants to challenge applications for the disclosure of such records.107 Similarly, Fiona Raitt has argued that recent amendments to rules of disclosure in Scotland have widened the duty to disclose to the point where they fail to sufficiently protect the privacy rights of complainers, in particular those of sexual assault complainers who have a history of mental illness.108 Similar arguments have been made in other jurisdictions regarding medical records, and even simply records of previous counselling. 109 There may also be a culture of victim blaming and scepticism around those with mental ill health who allege sexual assault, particularly where they have been multiply , 108 F Raitt ‘Disclosure of Records and Privacy Rights in Rape Cases’ 15(1) (2011) Edinburgh Law Review 33- 56, p. 40. Raitt points out that there are few formal rights afforded to the complainant, with respect to privacy, in the criminal justice context. She suggests that only Articles 2,6 and 8 of the ECHR give the complainer any formal recourse, but that these are so broad that they may be difficult to pin down. However, see the recent 2016 ruling where the Court of Session in Scotland said that a complainer’s Article 8 rights would be infringed by the disclosure of private medical records, and that she was entitled to legal aid to challenge the application for disclosure: http://www.bbc.co.uk/news/uk-scotland-35562009. For discussion of the history of the reform of disclosure rules, see Duff (this volume). 102 L Ellison, V Munro, K Hohl, and P Wallang ‘Challenging Criminal Justice? Psychosocial disability and rape victimisation’ 15(2) (2015) Criminology and Criminal Justice 225-244. 103 L Ellison et al, ‘Challenging Criminal Justice’. 104 L Ellison ‘The Use and Abuse of Psychiatric Evidence in Rape Trials’ (2009) 13(1) The International Journal of Evidence and Proof 28-49, p. 48. 105 https://www.theguardian.com/commentisfree/2014/mar/08/rape-consent-vulnerability-barfield-stanko. 104 See for example S Lees, Carnal Knowledge; J Temkin and B Krahé, Sexual Assault; J Temkin, ‘Prosecuting and Defending Rape’. L Ellison et al, Challenging Criminal Justice . 111 See Cairns (this volume) for a critical discussion of what is meant by ‘access to justice’; see also W Larcombe ‘Falling Rape Conviction Rates’, critiquing the way in which feminist interactions with the criminal justice system’s treatment of rape too often end in an unreflective call for more prosecutions. 112 M Burton, R Evans and A Sanders Are Special Measures for Vulnerable and Intimidated Witnesses Working? Evidence from the criminal justice agencies Home Office Online Report 01/06 (2006, Home Office), summarised in M Burton, R Evans and A Sanders, An evaluation of the use of special measures for vulnerable and intimidated witnesses Home Office Research Findings 270 (2006, Home Office). See also M Burton, R Evans and A Sanders ‘Vulnerable and intimidated witnesses and the adversarial process in England and Wales’ (2007) The International Journal of Evidence and Proof 1-23; M Burton, R Evans and A Sanders ‘Implementing special measures for vulnerable and intimidated witnesses: the problem of identification’ (2006) Criminal Law Review 229-240. 113 a. Who is vulnerable? ( ) 109 See for example: E McDonald ‘Resisting defence access to counselling records in cases of sexual offending: does the law effectively protect clinician and client rights?’ 5(2) (2013) Sexual Abuse in Australia and New Zealand 12-20; L Gotell ‘Colonization through disclosure: confidential records, sexual assault complainants and canadian law’ 10(3) (2001) Social Legal Studies September 315-346. 109 See for example: E McDonald ‘Resisting defence access to counselling records in cases of sexual offending: does the law effectively protect clinician and client rights?’ 5(2) (2013) Sexual Abuse in Australia and New Zealand 12-20; L Gotell ‘Colonization through disclosure: confidential records, sexual assault complainants and canadian law’ 10(3) (2001) Social Legal Studies September 315-346. 109 See for example: E McDonald ‘Resisting defence access to counselling records in cases of sexual offending: does the law effectively protect clinician and client rights?’ 5(2) (2013) Sexual Abuse in Australia and New Zealand 12-20; L Gotell ‘Colonization through disclosure: confidential records, sexual assault complainants and canadian law’ 10(3) (2001) Social Legal Studies September 315-346. 16 victimised.110 The ameliorative effects of the vulnerable witness provisions are unlikely in practice then to make much of a dent in the problem of ‘access to justice’ in the sense of complainers having their claim – and their personhood – treated with dignity and respect.111 victimised.110 The ameliorative effects of the vulnerable witness provisions are unlikely in practice then to make much of a dent in the problem of ‘access to justice’ in the sense of complainers having their claim – and their personhood – treated with dignity and respect.111 113 Report by Baroness Vivien Stern. 110 L Ellison et al, ‘Challenging Criminal Justice’. b. Identifying vulnerable witnesses So how is vulnerability, as complex as it is, recognised and acted upon by criminal justice agents? Burton et al conducted research in England and Wales, evaluating the effectiveness of special measures, introduced by the Youth and Criminal Evidence Act 1999, in real life cases, though not specifically sexual assault cases.112 Their findings are similar to that of Richards et al in Scotland, discussed above: generally the treatment of vulnerable and intimidated witnesses (VIW) has improved but implementation is inconsistent. Having interviewed criminal justice agents and victims, tracked prosecution cases, and observed court practice, they concluded that the processes for identifying VIW significantly underestimated the number of those who were truly vulnerable: on a ‘very conservative estimate’ they concluded that 24% as opposed to the official figures of 7-10% were probably VIW. Like Richards et al, they found that police officers had problems identifying VIW, particularly those with mental disorders, or learning disabilities, and those who were intimidated, and also were not always communicating effectively about the cases where someone was identified as VIW. They also found that the CPS rarely identified individuals as VIW, with some being identified for the first time by Victim Support at court, often too late for measures to be implemented. And although interviewed complainants reported that special measures enabled them to give evidence that they might otherwise not have given, Burton et al 2006 concluded that there was a significant unmet need. Importantly, and chiming with concerns raised above, they also found an informal hierarchy in VIW identification (presumably for pragmatic reasons) where children and victims of sexual offences were more easily and therefore more readily identified than others, and were more likely to benefit from special measures. b. Identifying vulnerable witnesses updated in September 2015.114 This document aims to allow agencies to “identify best practice and obtain consistency of approach to improve victims and witness engagement and support… to understand and meet victim and witness needs, treating them appropriately, professionally and with respect at all times”.115 Those deemed vulnerable by way of status (children, those with mental disorders and victims of specified offences) will be identified automatically, and a special arm of COPFS called Victim Information and Advice (VIA) contacts these people to discuss the most appropriate special measures, and make applications to courts, amongst other things. For ‘other’ vulnerable witnesses, the identification process is not so straightforward. VIA contacts those identified as potentially vulnerable by investigating police officers,116 or they can be identified, by Victim Support, other agencies, h b f CO S h h h i i l j i h l lf f It is not clear then that the statutory protections offered through special measures, have in themselves given us an adequate understanding of what constitutes vulnerability, and how we recognise it, in the context of sexual offences. And alongside these concerns about the meaning of vulnerability and how it is applied in practice, is the question of the impact of the measures themselves. 114 Working together for Victims and Witnesses: Protocol Between Crown Office and Procurator Fiscal Service (COPFS), Scottish Courts and Tribunal Service (SCTS), Police Scotland and Victim Support Scotland (VSS) (2015, Scottish Courts and Tribunal Service). 115 Ibid. para 3. 116 Ibid. para 23. 117 Ibid. para 94. 118 Part 2 of the 2004 Act deals with witnesses in the civil context – for the purposes of this paper we will refer only to Part 1 of the 2004 Act which amends existing criminal law. b. Identifying vulnerable witnesses This resonates with the findings of the 2010 Stern review – that prosecutors were generally reluctant to apply to use video evidence with adults.113 In response to the Victims and Witnesses (Scotland) Act 2014, and, apparently, the European Directive on the rights, support and protection of victims of crime, there is now a joint protocol between COPFS, SCTS, Police Scotland and Victim Support Scotland, last 17 updated in September 2015.114 This document aims to allow agencies to “identify best practice and obtain consistency of approach to improve victims and witness engagement and support… to understand and meet victim and witness needs, treating them appropriately, professionally and with respect at all times”.115 Those deemed vulnerable by way of status (children, those with mental disorders and victims of specified offences) will be identified automatically, and a special arm of COPFS called Victim Information and Advice (VIA) contacts these people to discuss the most appropriate special measures, and make applications to courts, amongst other things. For ‘other’ vulnerable witnesses, the identification process is not so straightforward. VIA contacts those identified as potentially vulnerable by investigating police officers,116 or they can be identified, by Victim Support, other agencies, or other members of COPFS throughout the criminal justice process; they can also self-refer or be referred by a representative at the point of citation. 117 However, a true understanding of how this process works in practice requires evaluation, not only of the extent to which the criminal justice process allows for identification and communication amongst it agents of potentially vulnerable witnesses, but also the extent of, for example, mental health training received by criminal justice agents such as the police and the Crown Office, as well as defence solicitors and even judges. The views and experiences of those who are (and are not) categorised as vulnerable are of course also central to such an evaluation. 2. What impact do special measures have? If a witness is found to be vulnerable, the 2004 Act sets out a range of special measures designed to protect them from the harsh realities of engaging with criminal justice processes.118 These are: the use of a screen, a live tv link, use of a video recorded prior statement, use of evidence taken by a court appointed ‘commissioner’, the opportunity for the witness to have a person supporting them as they give evidence, and ‘any other measure’ the the Scottish Ministers may prescribe (1995 Act s. 275, as amended by s1 of the 2004 Act). The 2014 Act also added provision for closed courts (s. 21). Witnesses deemed vulnerable are entitled to ‘standard’ special measures, i.e. use of a live tv link, a screen, and a supporter, and are entitled to apply for non-standard measures, i.e. for evidence in chief to be given in the form of a recorded prior statement, evidence to be taken by a commissioner, or to exclude the public from the court while the witness gives evidence (closed court). Those who are not automatically deemed vulnerable can apply for any of these special measures, to be granted at the court’s discretion.119 118 Part 2 of the 2004 Act deals with witnesses in the civil context – for the purposes of this paper we will refer only to Part 1 of the 2004 Act which amends existing criminal law. 18 How effective are these special measure and what is their impact on a sexual assault trial? No evaluative study has been conducted in Scotland since Richards et al’s study was published in 2008, as discussed above. However, in England and Wales, Ellison and Munro investigated the impact of three special measures upon mock juror evaluations of adult rape testimony: (1) live-links; (2) video recorded evidence-in-chief followed by live-link cross- examination; and (3) protective screens.120 Their findings are illustrative since they refer to the same sorts of special measures as introduced in Scotland and elsewhere in the last decades. The authors found that with respect to verdicts, jurors’ evaluations of responsibility often had more to do with prior expectations regarding ‘appropriate’ responses to rape and ‘normal’ socio-sexual behaviour than they did with the mode by which the complainant’s testimony was delivered. 124 N Westera, M Kebbell and B Milne ‘Want a better criminal justice response to rape? Improve police interviews with complainants and suspects’ (2016) Violence Against Women 1-22; N Westera, M Powell and B Milne, B ‘Lost in the detail: Prosecutors' perceptions of the utility of video recorded police interviews as rape complainant evidence’ (2015) Australian and New Zealand Journal of Criminology 1-17. 125 N Westera, M Kebbell and B Milne ‘Losing two thirds of the story: a comparison of the video-recorded police interview and live evidence of rape complainants 4 (2013) Criminal Law Review 290-308. 2. What impact do special measures have? 121 Their findings also “provide little support for the suggestion that the emotional impact of testimony will be reduced when a witness appears on a screen, translating into a loss of juror empathy.”122 Overall they found that special measures themselves did not seem to have any discernible impact on jurors’ assessments of the complainant’s credibility, and that they could equally work ‘in favour’ as much as ‘against’ the complainant. As mentioned above, the 2010 Stern review found that prosecutors in England and Wales were generally reluctant to make special measures applications for pre-recorded video interviewing with adults.123 More recently, Westera et al in Queensland, Australia, tried to explain this prosecutorial reluctance in their own criminal justice system.124 They evaluated police video interviews as evidence in chief in sexual assault trials, and found that police officers were often ‘lost in the detail’ such that the interviews were not seen by prosecutors as the best form of evidence to present to the court. However, they found in their 2013 New Zealand study that when compared with recorded police interviews, live evidence in chief at trial lost over two thirds of the detail of evidence relevant to establishing whether or not the alleged offence had occurred.125 So while the use of prerecorded interviews might be 119 See Scottish Court and Tribunal Service, Evidence and Procedure Review Report (2015), available at http://www.scotcourts.gov.uk/docs/default-source/aboutscs/reports-and-data/reports-data/evidence-and- procedure-full-report---publication-version-pdf.pdf?sfvrsn=2 and Scottish Court and Tribunal Service Evidence and Procedure Review – Next Steps (2016), available at https://www.scotcourts.gov.uk/about-the-scottish-court- service/scs-news/2016/02/26/evidence-and-procedure-review---next-steps. It may well be that the Scottish criminal justice system is generally moving in the direction of updating much of its evidential process through wider use of electronic and digital technology (SCTS) 2015, 2016), for reasons of cost efficiency, as well as to better ‘ascertain the truth’ (see Duff this volume). The SCTS 2015 report makes recommendations, such as taking all vulnerable witness statements and cross examinations by video as a matter of course. Whether or not this would bring us closer to an inquisitorial system is beyond the scope of this chapter. 119 See Scottish Court and Tribunal Service, Evidence and Procedure Review Report (2015), available at http://www.scotcourts.gov.uk/docs/default-source/aboutscs/reports-and-data/reports-data/evidence-and- procedure-full-report---publication-version-pdf.pdf?sfvrsn=2 and Scottish Court and Tribunal Service Evidence and Procedure Review – Next Steps (2016), available at https://www.scotcourts.gov.uk/about-the-scottish-court- service/scs-news/2016/02/26/evidence-and-procedure-review---next-steps. 2. What impact do special measures have? It may well be that the Scottish criminal justice system is generally moving in the direction of updating much of its evidential process through wider use of electronic and digital technology (SCTS) 2015, 2016), for reasons of cost efficiency, as well as to better ‘ascertain the truth’ (see Duff this volume). The SCTS 2015 report makes recommendations, such as taking all vulnerable witness statements and cross examinations by video as a matter of course. Whether or not this would bring us closer to an inquisitorial system is beyond the scope of this chapter. 120 L Ellison and V Munro ‘A ‘special’delivery? exploring the impact of screens, live-links and video-recorded evidence on mock juror deliberation in rape trials’ 23(1) (2013) Social & Legal Studies 3-29; L Ellison and V Munro ‘Special measures in rape trials: exploring the impact of screens, live links and video-recorded evidence on mock juror deliberation’ (2012, ESRC Briefing Report). j ( g p 121 However, as they point out, “verdict outcome alone offers a limited indicator of influence, and myriad other variables can play a part in its framing. It is necessary, therefore, to delve further into the substantive content of the deliberations to explore more subtle signs of influence.” L Ellison and V Munro, ‘Special Measures’, p. 3. 122 Ibid 123 Report by Baroness Vivien Stern. 123 Report by Baroness Vivien Stern. p y 124 N Westera, M Kebbell and B Milne ‘Want a better criminal justice response to rape? Improve police interviews with complainants and suspects’ (2016) Violence Against Women 1-22; N Westera, M Powell and B Milne, B ‘Lost in the detail: Prosecutors' perceptions of the utility of video recorded police interviews as rape complainant evidence’ (2015) Australian and New Zealand Journal of Criminology 1-17. 125 N Westera, M Kebbell and B Milne ‘Losing two thirds of the story: a comparison of the video-recorded police interview and live evidence of rape complainants 4 (2013) Criminal Law Review 290-308. 19 essential to protect complainers and produce ‘best evidence’, they must be conducted in a way that is useful to prosecutors if the special measure is to be fully effective. Special measures are obviously essential, not “to provide (witnesses) with an unfair advantage, but to allow them to participate in a meaningful way”.126 Sexual assault is clearly a traumatic experience, and having to recount the assault at trial often compounds the trauma for many complainers. 2. What impact do special measures have? This is to some extent recognised through explicit inclusion of sexual assault claimants as vulnerable within the terms of the Vulnerable Witnesses (Scotland) Act 2014. However, Hardy et al have shown in their study that ‘trauma memory’, account incoherence and dissociation all affect not only the ways in which sexual assault is recounted to the police, and the quality of evidence available to the prosecution, but the way in which a complainer is treated by criminal justice agents, and therefore, whether or not complainers decide to take the allegation forward in the first instance.127 In other words, vulnerability arising from the trauma of the assault, or from some other characteristic such as drug or alcohol use, mental ill health, or some combination of these issues, as well as the treatment of complainants by criminal justice agents, has an impact on whether sexual assault cases drop out of the criminal justice system, or even enter the criminal justice system at all. As Ellison and Munro have recently argued, the impact of trauma on victims of crime at every stage in the criminal justice process, and in particular on sexual assault victims, has barely been acknowledged either by policy makers or practitioners.128 Legislation relating to the treatment of evidence in sexual assault trials does not address these issues; treating special measures as a solution ignores the extent to which witnesses are made more vulnerable by the criminal justice system itself. It also ignores the ways in which trauma and vulnerability can affect both the quality of a complainer’s testimony and the credibility it is afforded by the judge and jury.129 That is not to detract from the importance of protecting witnesses in sexual offences trials. But an overly narrow focus of our energy on refining provisions that deal only with the way in which a complainer’s evidence is presented in court can distract us from addressing questions about the deep seated vulnerabilities that lead certain complainers to be both more vulnerable to sexual assault, and yet less likely to have access to criminal justice redress, or indeed to be retraumatised by the adversarial process, for example through cross examination. 126 Scottish Executive Vulnerable and Intimidated Witnesses, p. 99. 127 A Hardy, K Young, and E Holmes ‘Does trauma memory play a role in the experience of reporting sexual assault during police interviews? an exploratory study’ 17(8) (2009) Memory 783-8. See also G Gudjonsson ‘Psychological vulnerabilities during police interviews: why are they important?’ 15(2) (2010) Legal and Criminological Psychology 161–175. 128 L Ellison and V Munro, ‘Taking Trauma Seriously’. 129 Ibid; see also J Herlihy and S Turner ‘Untested assumptions: psychological research and credibility assessment in legal decision-making’ 6 (2015) European Journal of Psychotraumatology 27380. g y 129 Ibid; see also J Herlihy and S Turner ‘Untested assumptions: psychological research and credibility assessment in legal decision-making’ 6 (2015) European Journal of Psychotraumatology 27380. g y gy 128 L Ellison and V Munro, ‘Taking Trauma Seriously’. 126 Scottish Executive Vulnerable and Intimidated Witnesses, p. 99. 7 A Hardy, K Young, and E Holmes ‘Does trauma memory play a role in the experience of reporting sexu sault during police interviews? an exploratory study’ 17(8) (2009) Memory 783-8. See also G Gudjonsson Psychological vulnerabilities during police interviews: why are they important?’ 15(2) (2010) Legal and riminological Psychology 161–175. D. CONCLUSION It is frequently lamented that law ‘in action’ often does not reflect law ‘in the books’. This is perhaps especially so in the area of sexual assault where feminist commentators, amongst others, have noted the obduracy of assumptions and stereotypes about ideal victim behaviour, despite repeated attempts at substantive and procedural law reform. The spectre of sexual history looms over every individual who considers reporting a sexual offence. Laws introduced to limit defence lawyers’ reliance upon sexual history evidence to cast doubt on a complainer’s behaviour have been shown to be ineffective in practice, and in some cases can exacerbate the most problematic aspects of referring to a complainer’s sexual history. This renders complainers especially ‘vulnerable’ to being humiliated and disempowered in the criminal justice process. Although it appears that the majority of sexual assault trials 20 ultimately do include evidence of sexual history, special measures can protect sexual assault complainers from some of the embarrassment and trauma of having to give evidence and have their credibility and sexual integrity challenged in a public court room. But we do not know enough about vulnerability in sexual offences; there is dearth of data on these issues, particularly in Scotland, where little is known – excepting anecdotal accounts and reports from NGOS – about sexual assault complainers’ contemporary experiences in the criminal justice system, not only of their experience leading up to and of the trial process, including applications to introduce their sexual history and other private information, but also if, when, and how the vulnerable witness procedures impact upon them. We do not know how vulnerability is currently identified or communicated by police officers, or by COPFS or other agencies, or whether there is reluctance to apply certain kinds of special measures to certain kinds of complainers. We do not know the experiences of those who are deemed vulnerable – and those who may need to be, but are not. Neither do we have data on whether or not particular measures such as police interviews provide the best kind of court room evidence, or the impact that any of the special measures have on the court room dynamics, or outcomes. It might well also be true that there is an informal hierarchy of identification. Scotland deems certain categories of people vulnerable, who are automatically identified and contacted (children, those with mental disorders and those who have alleged particular offences). 130 Though in England and Wales, Jacobsen, and Jacobsen and Seden, have examined the experiences of people with learning disabilities and learning difficulties within the criminal justice system: J Jacobsen Police Responses to Suspects with Learning Disabilities and Learning Difficulties: A Review of Policy and Practice (2008, Prison Reform Trust); J Jacobson and R Seden A Review of Court Provision for Defendants with Learning Disabilities and Learning Difficulties (2009, Prison Reform Trust). In addition, Poltnikoff and Woolfson have evaluated the government’s commitment to young witnesses: J Plotnikoff and R Woolfson Evaluation of young witness support: examining the impact on witnesses and the criminal justice system (2007, Home Office); J Plotnikoff and R Woolfson Measuring up? Evaluating Implementation of Government Commitment to Young Witnesses in the Criminal Justice system (2009, NSPCC). 131 2013, p. 1073, cited in L Ellison and V Munro ‘Taking Trauma Seriously’. D. CONCLUSION However, those who do not fall within those categories may not be easily identifiable as vulnerable. Again, given that neither sexual history evidence applications nor the vulnerable witnesses scheme has been evaluated for around a decade,130 there is an urgent need for more research in Scotland - and in England and Wales - to better understand the reliance on sexual history evidence, the gaps in provision of special measures, the impact that the use of sexual history and other private information, as well as the special measures themselves, have on witnesses and other trial participants, and if possible, trial outcomes. Nonetheless, a better understanding of the processes currently in place should not distract us from important questions about the socially embedded structures that prompt vulnerability. While protecting individual complainers and allowing them to have a voice in the criminal justice process are laudable aims, this cannot come at the expense of a proper inquiry as to what is meant by vulnerability, and the ways in which the justice process itself is implicated in compounding vulnerability and trauma. Peroni and Timmer have recently called for judicial and legislative authorities to justify why a group is considered especially vulnerable and why an individual should be treated as a member of that group. 131 Going further, we have argued that what is needed is a more systemic focus on the ways in which certain kinds of victims become, by virtue of their circumstances, especially vulnerable, both to being sexually assault in the first instance, and to being denigrated and disbelieved in the criminal justice process, with little likelihood of being treated with respect, dignity or indeed receiving just outcomes, however that might be understood. j g Cardwell and Hervey recently observed that critical analysis of the use of legal techniques can highlight the way that law itself “sustains certain assumptions that support structures of 21 power as ‘background rules of the game’, essentially by hiding them from scrutiny”.132 We have argued that the laws of evidence and criminal procedures around sexual assault must be held up to closer scrutiny. 132 P Cardwell and T Hervey ‘Bringing the technical into the socio-legal: the metaphors of law and legal scholarship of a 21st Century European Union’ in D Cowan and D Wincott (eds) Exploring the Legal in Socio- Legal Studies (2015, Palgrave MacMillan). 133 A Riles 'A New Agenda for the Cultural Study of Law: Taking on the Technicalities' 53 (2005) Buffalo Law Review 973-1033, p. 1008, fn 9. D. CONCLUSION There has also been a proliferation of rules, exceptions, policies, protocols and case law relating to the use of sexual history evidence, and to the proper treatment of vulnerable victims and witness; as Annalisa Riles has suggested, "when controversies flare up the literature becomes technical".133 We have seen the policy and academic landscape become arguably overly technical in this legal area; what we have not seen is any real engagement with what it means to be vulnerable regarding sexual assault. A concerted attempt must be made by academics, policy and lawmakers, and practitioners to properly understand the underlying dynamics, and the reality of the depth and breadth of lived vulnerability in this context. 22
https://openalex.org/W2998220497
https://hal.archives-ouvertes.fr/hal-02439978/file/2020_Ghini_Nutrients.pdf
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DHA-Induced Perturbation of Human Serum Metabolome. Role of the Food Matrix and Co-Administration of Oat β-glucan and Anthocyanins
Nutrients
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DHA-Induced Perturbation of Human Serum Metabolome. Role of the Food Matrix and Co-Administration of Oat β-glucan and Anthocyanins Bucci 336, 47521 Cesena (FC), Italy 5 Department of Chemistry, University of Florence, via della Lastruccia, 3-50019 Sesto Fiorentino (FI), Italy 6 GIOTTO Biotech S.r.l., Via Madonna del Piano, 6-50019 Sesto Fiorentino (FI), Italy Department of Chemistry, University of Florence, via della Lastruccia, 3 50019 Sesto Fiorentino (FI), Italy 6 GIOTTO Biotech S.r.l., Via Madonna del Piano, 6-50019 Sesto Fiorentino (FI), Italy 6 GIOTTO Biotech S.r.l., Via Madonna del Piano, 6-50019 Sesto Fiorentino (FI), Italy 7 Max Rubner-Institute, Federal Research Centre for Nutrition and Food, Haid-und-NeuStrasse 9, DE-76131 Karlsruhe, Germany; achim.bub@mri.bund.de 8 Université Clermont Auvergne, INRA, UNH, Unité de Nutrition Humaine, CRNH Auvergne, F63000 Clermont-Ferrand, France; corinne.malpuech-brugere@uca.fr p g 9 School of Food Science and Nutrition, University of Leeds, Woodhouse Ln, Leeds LS2 9JT, UK; C.Orfila@leeds.ac.uk 10 Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna via Massarenti 9 40138 Bologna (BO) Italy; luigi ricciardiello@unibo it 10 Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, 10 Department of Medical and Surgical Sciences, Alma Mater Studiorum University of B via Massarenti 9, 40138 Bologna (BO), Italy; luigi.ricciardiello@unibo.it Department of Medical and Surgical Sciences, Alma Mater Studiorum University via Massarenti 9, 40138 Bologna (BO), Italy; luigi.ricciardiello@unibo.it * Correspondence: francesco.capozzi@unibo.it; Tel.: +39-0547-338106 Received: 25 November 2019; Accepted: 23 December 2019; Published: 27 December 2019 Received: 25 November 2019; Accepted: 23 December 2019; Published: 27 December 2019 Abstract: Docosahexaenoic acid (DHA) has been reported to have a positive impact on many diet-related disease risks, including metabolic syndrome. Although many DHA-enriched foods have been marketed, the impact of different food matrices on the effect of DHA is unknown. As well, the possibility to enhance DHA effectiveness through the co-administration of other bioactives has seldom been considered. We evaluated DHA effects on the serum metabolome administered to volunteers at risk of metabolic syndrome as an ingredient of three different foods. Foods were enriched with DHA alone or in combination with oat beta-glucan or anthocyanins and were administered to volunteers for 4 weeks. Serum samples collected at the beginning and end of the trial were analysed by NMR-based metabolomics. Multivariate and univariate statistical analyses were used to characterize modifications in the serum metabolome and to evaluate bioactive-bioactive and bioactive-food matrix interactions. DHA administration induces metabolome perturbation that is influenced by the food matrix and the co-presence of other bioactives. DHA-Induced Perturbation of Human Serum Metabolome. Role of the Food Matrix and Co-Administration of Oat β-glucan and Anthocyanins Veronica Ghini 1, Leonardo Tenori 2 , Francesco Capozzi 3,4,*, Claudio Luchinat 1,5,6 , Achim Bub 7 , Corinne Malpuech-Brugere 8, Caroline Orfila 9, Luigi Ricciardiello 10 and Alessandra Bordoni 3,4 1 Center of Magnetic Resonance (CERM), University of Florence, via Luigi Sacconi, 6-50019 Sesto Fiorentino (FI), Italy; ghini@cerm.unifi.it (V.G.); luchinat@cerm.unifi.it (C.L.) 1 Center of Magnetic Resonance (CERM), University of Florence, via Luigi Sacconi, 6 50019 S t Fi ti (FI) It l hi i@ ifiit (VG ) l hi t@ ifiit (C L ) Center of Magnetic Resonance (CERM), University of Florence, via Luigi Sacconi, 6-50019 Sesto Fiorentino (FI), Italy; ghini@cerm.unifi.it (V.G.); luchinat@cerm.unifi.it (C.L.) 2 Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla, 3-50134 Florence (FI), Italy; tenori@cerm.unifi.it y g 2 Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla, 3-50134 Florence (FI), Italy; tenori@cerm.unifi.it y 3 Department of Agricultural and Food Sciences, Alma Mater Studiorum University of Bologna, Piazza G. Goidanich, 60, 47521 Cesena (FC), Italy; alessandra.bordoni@unibo.it y 4 Interdepartmental Centre for Industrial Agrofood Research, Alma Mater Studiorum University of Bologna Via Q. nutrients nutrients nutrients nutrients 1. Introduction Bioactives or nutraceuticals are any substances that are foods, or parts of a food, and provide health benefits, including the prevention and treatment of diseases [1]. Bioactives are usually present in common foods at a low concentration, typically far from the optimally effective dose range. To overcome this, the food and drink industry is developing new products containing high concentrations of selected bioactives, so-called “functional foods”. Functional foods are foods that provide benefits to the body, additional to adequate nutrition, either improving health and well-being or reducing the risk of disease, or both [2]. The majority of them is generated starting from a specific functional ingredient. Thus, the term “functional food” mainly refers to food that is fortified or enriched with bioactive compounds [3]. Bioactives can be considered an integral part of tailored nutrition prescription, therefore, representing a promising approach for both prevention and management of metabolic disorders. Regrettably, meta-analyses evaluating the effects of nutraceuticals seldom discriminate the source of the active compounds, which can be delivered (i) as a dietary supplement, (ii) as a specific dietary treatment (i.e., the increased consumption of food naturally containing the bioactive) or (iii) as a bioactive-enriched food (BEF). In the latter case, the food matrix in which the bioactive is embedded could have a role in the final effect. As an example, other constituents in a food matrix could aid or hinder the bio-accessibility and bioavailability of the bioactives. In the present study, we investigated by NMR the effects of supplementation with docosahexaenoic acid (C22:6 n-3, DHA) on the plasma metabolome of human volunteers at risk of metabolic syndrome (MetS). DHA is a well-known bioactive that has been reported to have a positive impact on many diet-related disease risks, including MetS [4]. DHA was administered as a functional ingredient of three foods. Each food was enriched with DHA alone or in combination with other bioactives that have also been associated with a reduced risk of MetS: Oat beta-glucan (OBG), and anthocyanins (AC) [5–7]. The serum metabolome was evaluated by NMR-based metabolomics to investigate the perturbation induced by the dietary treatment, critically evaluating bioactive-food matrix interactions and the extent of possible synergy/antagonism between DHA and the other bioactives. It has been suggested that metabolomics could play a role in dietary assessment and identification of novel biomarkers of dietary intake [8–14]. 1. Introduction NMR-based metabolomics [15–17] are an efficient and highly reproducible platform for the analysis of biofluids. The type and abundance of metabolites detected in a biological sample can be viewed as a global fingerprint that unambiguously describes the current metabolic status of an individual [18–21]. In the context of the new approach of combining traditional methods with novel metabolomic techniques for well-being and optimal nutrition [22], NMR-metabolomics have shown immense potential for individual monitoring in response to clinical and dietary intervention [23–27]. DHA-Induced Perturbation of Human Serum Metabolome. Role of the Food Matrix and Co-Administration of Oat β-glucan and Anthocyanins In particular, when co-administered with oat beta-glucan, DHA induces a strong rearrangement in the lipoprotein profile of the subjects. The observed modifications are consistent with clinical results and indicate that metabolomics represents a possible strategy to choose the most appropriate food matrices for bioactive enrichment. Keywords: anthocyanins; bioactive enriched food; docosahexaenoic acid; NMR-based metabolomics; oat beta glucans www.mdpi.com/journal/nutrients Nutrients 2020, 12, 86; doi:10.3390/nu12010086 Nutrients 2020, 12, 86 2 of 16 2.1. Bioactive Enriched Food Three different foods (milkshake, biscuits and pancake), belonging to different food matrices (dairy, bakery and egg-based food), were enriched with DHA, AC, OBG, DHA + AC or DHA + OBG. Each BEF category was produced using the same recipe, with small modifications due to the addition of the active ingredient(s). DHA, AC and OBG enrichment was obtained by adding OVO-DHA® (Applications Sante des Lipides Sarl, Marseille, France), Eminol® (ABRO BIOTEC SL, Zaragoza, Spain) and SweOat® bran BG28 XF (Swedish Oat Fiber, Väröbacka, Sweden), respectively. Pancake and milkshake were manufactured by production plants coordinated by ADEXGO Kft (Lapostelki utca, Hungary), and biscuits by Desarrollos Panaderos Levantinos SLL (Valencia, Spain). BEF were formulated in order to supply similar daily amounts of bioactives within and among the different food matrices (about 250 mg DHA, 3 g OBG and 50 mg AC). The different matrices appeared not equally compliant to the enrichment with 3 of 16 Nutrients 2020, 12, 86 AC due to the sensory and physico-chemical characteristics of the polyphenols, and to their different interactions with the food matrix/food processing. Thus, it was decided to enrich each matrix with the highest possible amount of AC, taking care to maintain good sensory characteristics and consumers’ acceptance of the product. Due to the shelf life and retention of bioactives during storage, BEF were produced in different batches. Bioactive concentrations were measured in at least 3 products in each batch, on the day of production and on the last day of shelf life, and the average amount of bioactives delivered with one portion of BEF is reported in Table 1. DHA and BG content were comparable and not statistically different in BEF from the different matrices, while AC content was different in the order pancake > milkshake > biscuits. Anyway, the amount of AC delivered with BEF within the same matrix (and in the same pilot) was almost the same. Table 1. The average amount of bioactives delivered with one portion of bioactive-enriched foods (BEF). 1. 2.1. Bioactive Enriched Food The average amount of bioactives delivered with one portion of bioactive-enriched foods DHA (mg) AC (mg) OBG (g) Biscuits DHA 292 0 0 Biscuits DHA + AC 302 19 0 Biscuits DHA + OBG 329 0 2.9 Biscuits AC 0 17 0 Biscuits OBG 0 0 3.6 Pancake DHA 225 0 0 Pancake DHA + AC 208 57 0 Pancake DHA + OBG 215 0 4.3 Pancake AC 0 58 0 Pancake OBG 0 0 3.7 Milkshake DHA 261 0 0 Milkshake DHA + AC 228 12 0 Milkshake DHA + OBG 226 0 3.8 Milkshake AC 0 10 0 Milkshake OBG 0 0 4.2 Table 1. The average amount of bioactives delivered with one portion of bioactive-enriched foods (BEF). DHA (mg) AC (mg) OBG (g) 2.2. Subjects The study was conducted by running 3 separate trials, each of them testing one of the 3 BEF categories (milkshake, biscuits or pancake), as reported in Bub et al. [7]. The trials were conducted: (i) Max Rubner-Institut, Karlsruhe, Germany, MRI, (milkshake); (ii) Centre de Recherche en Nutrition Humaine Auvergne, Clermont-Ferrand, France, CRNH (biscuits); (iii) School of Food Science and Nutrition, University of Leeds, Leeds, UK, ULE (pancake). Enrolled subjects were healthy men and women aged 18 to 80 years presenting 2, 3 or 4 of the criteria for MetS diagnosis [elevated waist circumference ≥102 cm (men) or ≥88 cm (women); fasting triglycerides ≥150 mg/dL; fasting HDL-cholesterol ≤40 mg/dL (men) or ≤50 mg/dL (women); systolic blood pressure ≥130 mmHg and/or diastolic blood pressure ≥85 mmHg or hypotensive treatment; fasting glucose ≥110 mg/dL], with at least one of them being elevated fasting triglycerides (TG) or low high-density lipoprotein cholesterol (HDL-C) [7]. In each trial, volunteers received a BEF belonging to the same matrix (milkshake, biscuits or pancake) for 28 days. Trials were randomized, double-blind, parallel dietary intervention without a placebo. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki. Ethical approval for the study protocol was obtained from national authorities: MRI ethical committee approval reference number: F-2014-062 (State Medical Chamber Baden-Wuerttemberg); CRNH Regional Committee ethics reference number: 2014-A01290-47; ULE MEEC-Ethics reference number: MEEC 13-027, amended reference number MEEC 14-017. In each trial, participants were divided into 5 subgroups, each one receiving a specific enrichment, and were required to consume one portion of the allocated BEF daily, for a total of 4 weeks. Volunteers were on a free diet, apart from the indication to limit the consumption of food naturally containing high 4 of 16 Nutrients 2020, 12, 86 amounts of the bioactives (i.e., fish for DHA) to one portion per day and were required to maintain their usual lifestyle during the intervention. At baseline (T0) and after 4 weeks of intervention (T1), fasting blood samples were collected for NMR analysis. Since haemolysis has been reported to interfere with NMR analysis contributing to erroneous results [28], visually haemolysed samples were excluded. The number of participants having suitable samples for analysis at both T0 and T1 is reported in Table 2. Table 2. Number of study participants according to treatment. Table 2. 2.3. NMR Sample Preparation 2.3. NMR Sample Preparation NMR samples were prepared according to standard procedures [29,30]. Frozen serum samples were thawed at room temperature and shaken before use. A total of 350 µL of each sample was added to 350 µL of a phosphate sodium buffer (70 mM Na2HPO4; 20% (v/v) D2O; 0.025% (v/v) NaN3; 0.8% (w/v) sodium trimethylsilyl (2,2,3,3-2H4) propionate (TMSP), pH 7.4). The mixtures were homogenized by vortexing for 30 s, and a total of 600 µL of each mixture was transferred into a 5.00 mm NMR tube (Bruker BioSpin Gmbh, Rheinstetten, Germany) for analysis. 2.4. NMR Experiments 1H-NMR spectra for all samples were acquired using a Bruker 600 MHz spectrometer (Bruker BioSpin Gmbh, Rheinstetten, Germany) operating at 600.13 MHz proton Larmor frequency and equipped with a 5mm PATXI 1H-13C-15N and 2H-decoupling probe including a z-axis gradient coil, an automatic tuning-matching (ATM) and an automatic and refrigerate sample changer (SampleJet, Bruker BioSpin Gmbh, Rheinstetten, Germany). A BTO 2000 thermocouple served for temperature stabilization at the level of approximately 0.1 K at the sample. Before measurement, samples were kept for 5 min inside the NMR probe head for temperature equilibration at 310 K. 1 For each serum sample, 2 monodimensional 1H NMR spectra were acquired with water peak suppression and different pulse sequences that allowed the selective observation of different molecular components: (i) a standard NOESY (Nuclear Overhauser Effect Spectroscopy) [31] 1Dpresat (noesygppr1d.comp; Bruker BioSpin Gmbh, Germany) pulse sequence, using 32 scans, 98,304 data points, a spectral width of 18,028 Hz, an acquisition time of 2.7 s, a relaxation delay of 4 s and a mixing time of 0.01 s. This pulse sequence was designed to obtain a spectrum in which both signals of metabolites and high molecular weight molecules (lipids and lipoproteins) were visible. (ii) a standard CPMG (Carr-Purcell-Meiboom-Gill) [32] (cpmgpr1d.comp; Bruker BioSpin Gmbh, Rheinstetten, Germany) pulse sequence, using 32 scans, 73,728 data points, a spectral width of 12,019 Hz and a relaxation delay of 4 s. This pulse sequence was designed for the selective observation of small molecule components in solutions containing macromolecules. 2.2. Subjects Number of study participants according to treatment. Matrix Subjects (TOT) DHA AC OBG DHA + AC DHA + OBG Milkshake 66 14 15 12 12 13 Biscuits 37 7 7 9 8 6 Pancake 14 5 3 1 2 3 Total 117 26 25 22 22 22 2 3 NMR Sample Preparation 2.5. NMR Spectra Processing and Spectral Analysis Free induction decays were multiplied by an exponential function equivalent to a 0.3 Hz line-broadening factor before applying a Fourier transform. Transformed spectra were automatically corrected for phase and baseline distortions and calibrated (glucose doublet at δ 5.24 ppm) using TopSpin 3.5 (Bruker BioSpin Gmbh, Rheinstetten, Germany). Each spectrum, in the region of 10.00–0.2 ppm, was segmented into 0.02 ppm chemical shift buckets, and the corresponding spectral areas were integrated using the AMIX software (Bruker BioSpin Gmbh, Rheinstetten, Germany). Bucketing was a method to decrease the data dimensionality 5 of 16 Nutrients 2020, 12, 86 and to compensate for a small shift in the signals, making the analyses more robust and reproducible. The area of each bin was normalized to the total spectral area, calculated with the exclusion of the water region (4.40–5.00 ppm). 2.6. Statistical Analysis Various kinds of multivariate statistical techniques were applied on the obtained buckets using R 3.0.2 in house scripts. Unsupervised Principal Component Analysis (PCA) was used to obtain a preliminary overview of the data (visualization in a reduced space, clusters detection, screening for outliers). Partial Least Squares (PLS) and Multilevel PLS (M-PLS) was employed to perform supervised data reduction and classification. Canonical analysis (CA) was used in combination with PCA to increase supervised data reduction and classification. The global accuracy for classification was assessed by means of a Monte Carlo validation scheme. Accordingly, each dataset was randomly divided into a training and a test set, including the 90% and 10% of the data, respectively. The training set was used to build the model, whereas the test set was used to validate its discriminant and predictive power; this operation was repeated 500 times. The resultant confusion matrix was reported, and discrimination accuracy, specificity and sensitivity were estimated according to standard definitions. The metabolites, whose peaks in the spectra were well defined and resolved, were assigned, and their levels analysed. The assignment procedure was made up using an NMR spectra library of pure organic compounds, public databases (e.g., Human Metabolome Database [33]), storing reference NMR spectra of metabolites, spiking NMR experiments and using literature data [34]. The relative concentrations of the various metabolites were calculated by integrating the corresponding signals in the spectra [35]. The pairwise Wilcoxon signed-rank test was used for the determination of the meaningful metabolites; false discovery rate correction was applied using the Benjamini and Hochberg method (FDR): A p-value of 0.05 was deemed significant. 3. Results 0, 12, x FOR PEER REVIEW sess the quality of the overall spectral dataset, PCA was applied on the 2 ere 234 was the total number of sera analysed, and 397 was the number of ts in which each spectrum was segmented, as described in the Material and core plot (Figure 2) evidenced that all samples, although collected in three d in three different recruiting centres, each one testing a specific food matr us to each other and no outliers were detected. The uniform distribution o ly visualized by colouring the scores according to T0/T1 (Figure 2A), supple atrix/centres (Figure 2C). 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are Figure 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are labelled. , 12, x FOR PEER REVIEW ess the quality of the overall spectral dataset, PCA was applied on the 2 ere 234 was the total number of sera analysed, and 397 was the number of ts in which each spectrum was segmented, as described in the Material and core plot (Figure 2) evidenced that all samples, although collected in three in three different recruiting centres, each one testing a specific food mat us to each other and no outliers were detected. The uniform distribution o y visualized by colouring the scores according to T0/T1 (Figure 2A), supple atrix/centres (Figure 2C). Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites ar Figure 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are labelled. rix/centres (Figure 2C). ure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and ectively, of the total variance. In the score plot, each dot represents a different serum samp h colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. (B n dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahex (DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = sample Figure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and 7.9%, respectively, of the total variance. In the score plot, each dot represents a different serum sample, and each colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. 3. Results 0, 12, x FOR PEER REVIEW sess the quality of the overall spectral dataset, PCA was applied on the 234 ere 234 was the total number of sera analysed, and 397 was the number of 0.0 ts in which each spectrum was segmented, as described in the Material and M core plot (Figure 2) evidenced that all samples, although collected in three dif in three different recruiting centres, each one testing a specific food matrix us to each other and no outliers were detected. The uniform distribution of t ly visualized by colouring the scores according to T0/T1 (Figure 2A), suppleme atrix/centres (Figure 2C). 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% an tively, of the total variance. In the score plot, each dot represents a different serum samp olour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. (B dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahe DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = sample ny; magenta dots = samples from the UK; light green dots = samples from France. Figure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and 7.9%, respectively, of the total variance. In the score plot, each dot represents a different serum sample, and each colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. (B) Dark green dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahexaenoic acid (DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = samples from Germany; magenta dots = samples from the UK; light green dots = samples from France. A i d (b f ft t t t) M PLS l i d t id th ibl 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are Figure 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are labelled. 3. Results Untargeted NMR-based metabolomic analysis of serum samples was used to analyse the metabolomics effect of DHA supplementation, alone or in combination with OBG and AC, as an ingredient of the three different BEF. 1H CPMG NMR spectra were acquired verifying their high reproducibility. Figure 1 shows a typical CPMG spectrum of a serum sample. To assess the quality of the overall spectral dataset, PCA was applied on the 234 × 397 data matrix where 234 was the total number of sera analysed, and 397 was the number of 0.02 chemical shift buckets in which each spectrum was segmented, as described in the Material and Methods. The resulting score plot (Figure 2) evidenced that all samples, although collected in three different trials performed in three different recruiting centres, each one testing a specific food matrix, were well homogenous to each other and no outliers were detected. The uniform distribution of the samples can be easily visualized by colouring the scores according to T0/T1 (Figure 2A), supplements (Figure 2B), and matrix/centres (Figure 2C). By using supervised PCA—CA analysis to discriminate the five different supplements, separately at T0 and T1, and by considering the prediction accuracy of the models obtained, it turned out that the recruitment protocol selected a very homogenous population, without introducing any meaningful source of variation in the sera metabolome. As expected, considering all the samples collected before the treatment (T0), the confusion matrix was consistent with a random distribution among the different treatments (Figure S1). After the administration of BEFs (T1), discrimination among groups raised up to 30% (Figure S1), indicating the presence of some modifications of the metabolomic profiles due to the treatments. 6 of 16 rifying Nutrients 2020, 12, 86 t of the three d bility Figure 1 s ents 2020, 12, 86 6 o the three different BEF. H CPMG NMR spectra were acquired verifyi y. Figure 1 shows a typical CPMG spectrum of a serum sample. 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are la Figure 1. Typical 1H-NMRCPMG spectrum of serum. Most abundant metabolites are labelled. 3. Results (B) Dark green dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahexaenoic acid (DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = samples from Germany; magenta dots = samples from the UK; light green dots = samples from France. 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% an Figure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and 7.9%, e 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% an tively, of the total variance. In the score plot, each dot represents a different serum sam olour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. ( dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahe DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = sampl Figure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and 7.9%, respectively, of the total variance. In the score plot, each dot represents a different serum sample, and each colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. (B) Dark green dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahexaenoic acid (DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = samples from Germany; magenta dots = samples from the UK; light green dots = samples from France. e 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% an ctively, of the total variance. In the score plot, each dot represents a different serum sam colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. ( dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahe DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = sampl Figure 2. Principal component analysis (PCA) score plot. PC1 and PC2 account for 79.9% and 7.9%, respectively, of the total variance. In the score plot, each dot represents a different serum sample, and each colour represents a different group: (A) Blue dots = T0 samples; red dots = T1 samples. 3. Results Accuracies significantly above the chance level of 50% (binomial test) are marked with: * 0.001 < p-value <0.05; ** p-value <0.001. Considering all food matrices together, supplementation with DHA gave a moderately high discrimination accuracy (74%) between T0 and T1. In contrast, AC and OBG alone did not significantly alter the individual metabolome. Interestingly, co-administration of DHA and AC reduced the metabolomic effects of the fatty acid, while a strong cooperative and synergic effect was detected in volunteers assuming DHA + OBG. In fact, the DHA + OBG enrichment provided the best classification accuracy (86%) that was further improved considering the milkshake group only (94%). The strong effect of DHA + OBG observed after consumption of the milkshake could not be unequivocally explained by a deeper impact exerted by this specific enrichment-food matrix interaction on the metabolome, because the same analysis could not be significantly repeated on enriched pancake and biscuits due to the very low number of available samples. Nevertheless, even though not reliable, the results obtained for the subjects allocated to enriched biscuits were reported in Table S1 and they were in line with those obtained for the enriched milkshake. It could be argued that the higher accuracy obtained considering only the milkshake may be simply due to the reduction of noise and variability that were detected when all matrices were included in the analysis. Thus, to validate the observed strong effect of DHA + OBG supplementation, the M-PLS discrimination model (T0 vs. T1) built on samples from volunteers consuming the milkshake was used to predict the collection time (T0 or T1) of samples from volunteers receiving the same enrichment (DHA + OBG) embedded in the other food matrices (Figure 3A). Notably, most of the samples (89%) were correctly classified as T0 or T1 (Figure 3B), indicating that the spectral features able to capture the metabolic changes occurring during administration of DHA + OBG were independent of the matrix used as a vehicle. All the six samples from the three subjects allocated to the enriched pancake were perfectly classified (100%), whereas in the case of enriched biscuits, the two samples of one out of the six subjects were mixed up (subject 7 in Figure 3B). 3. Results (B) Dark green dots = anthocyanins (AC); cyan dots = oat beta-glucan (OBG); orange dots = docosahexaenoic acid (DHA); yellow dots = DHA + AC; purple dots = DHA + OBG. (C) grey dots = samples from Germany; magenta dots = samples from the UK; light green dots = samples from France. ermany; magenta dots = samples from the UK; light green dots = samples from France. y using supervised PCA—CA analysis to discriminate the five different supp tely at T0 and T1, and by considering the prediction accuracy of the models obtained, A paired (before vs. after treatment) M-PLS analysis was used to consider the possible presence of a strong individual variability in response to the treatments. In this kind of approach, the effects of the treatments can be evaluated within each subject (considering only intra-individual variability), thus eliminating the noise introduced by the inter-individual variability. at the recruitment protocol selected a very homogenous population, without introduc ngful source of variation in the sera metabolome. As expected, considering all the ed before the treatment (T0), the confusion matrix was consistent with a random dist The use of M-PLS allowed us to obtain a good classification accuracy (78%) considering the whole dataset. Due to the uneven recruitment in the three trials, it was possible to significantly explore the presence of different food matrix effects only in volunteers allocated to the enriched milkshake. Acting 7 of 16 Nutrients 2020, 12, 86 this way, the discrimination accuracy rose up to 89%. Therefore, M-PLS clearly indicated that daily BEF consumption, particularly milkshake, significantly affected the individual metabolome. To assess whether the observed metabolic changes were merely an unspecific phenomena related to consumption of the BEF matrix, or whether they were specifically related to the increased intake of bioactives, M-PLS was applied to each specific enrichment, considering either all BEF or milkshake only. The resulting classification performances are reported in Table 3. Table 3. Multilevel partial least squares (M-PLS) discrimination accuracy values. Table 3. Multilevel partial least squares (M-PLS) discrimination accuracy values. All BEF Enriched Milkshake DHA 74% ** 71% * AC 63% * 54% OBG 55% 55% DHA + AC 53% 56% DHA + OBG 86% ** 94% ** Accuracies significantly above the chance level of 50% (binomial test) are marked with: * 0.001 < p-value <0.05; ** p-value <0.001. 3. Results (C) M-PLS loading plot of the first component (PC1); the significance threshold (blue line was calculated considering “buckets” with a value beyond two standard deviations of their average 1: 0.85 ppm—2: 0.89 ppm (CH3 VLDL-LDL); 3–4: 1.17–1.19 ppm—5–7: 1.27–1.31 ppm—8: 1.35 pp ((-CH2-)n VLDL-LDL); 9: 3.23 ppm; 10–11: 3.65–3.67 ppm. Figure 3. M-PLS analysis of DHA + OBG supplementation. (A) M-PLS score plot. Milkshake samples are used as a training set to discriminate T0 (blue dots) vs. T1 (red dots). Biscuit and pancake samples are used as a test set (crosses coloured according to the prediction). (B) Table reporting the prediction results. (C) M-PLS loading plot of the first component (PC1); the significance threshold (blue lines) was calculated considering “buckets” with a value beyond two standard deviations of their averages; 1: 0.85 ppm—2: 0.89 ppm (CH3 VLDL-LDL); 3–4: 1.17–1.19 ppm—5–7: 1.27–1.31 ppm—8: 1.35 ppm ((-CH2-)n VLDL-LDL); 9: 3.23 ppm; 10–11: 3.65–3.67 ppm. The significant decrement (p = 0.004) of the signal attributable to triglycerides (TG) resonance T1 1H-NOESY spectra of DHA + OBG group compared to baseline (T0) confirmed the reduction chylomicrons and VLDL. M-PLS analysis applied only on the small part of DHA + OBG spec containing the broad TG signal (4.34–4.22 ppm), which resulted in a very high (80%) discriminat Accordingly, M-PLS analysis applied on the small region of 1H-NOESY spectra containing the broad signal attributable to the resonances of lipoprotein methyl groups (0.92–0.71 ppm) resulted in a discrimination accuracy of 78% (DHA + OBG group, T0 vs. T1) (Figure S2). This result confirms that DHA + OBG administration significantly changes the lipoprotein profiles. accuracy between T0 and T1 (Figure S3). 1H-NOESY spectra were also analysed using the Bruker IVdr Lipoprotein subclass analysis (B -LISA) platform [39]. The results for all BEF samples together are reported in Tables 4 and S2 and a in agreement with previously reported data. Considering single bioactives, no modification in t lipoprotein profile was observed in volunteers receiving AC or OBG. On the contrary, DH The broad signals of methyl and methylene groups arise from the sum of partial overlapping peaks of the different fractions (HDL, LDL, IDL, VLDL and chylomicrons). Due to the different density of the lipoprotein fractions, their chemical shift differences are quite small but significantly different. 3. Results The M-PLS analysis (Figure 3A) was then explored to identify the signals in the spectra that were mainly related to the DHA + OBG effects on the serum metabolome, as depicted in the loading plot of the first component (Figure 3C). The threshold (blue lines) used to select significant signals was calculated considering bins with values beyond two standard deviations of their averages. Interestingly, most of these bins belonged to the broad signals in the spectra arising from methyl (-CH3) and methylene (-CH2-) groups of serum lipoproteins, centred at 0.86 and 1.33 ppm, respectively (Figure 1). 8 of 16 and VLD ectral ar Nutrients 2020, 12, 86 fraction reson Figure 3. M-PLS analysis of DHA + OBG supplementation. (A) M-PLS score plot. Milkshake sampl are used as a training set to discriminate T0 (blue dots) vs. T1 (red dots). Biscuit and pancake samp are used as a test set (crosses coloured according to the prediction). (B) Table reporting the predicti results. (C) M-PLS loading plot of the first component (PC1); the significance threshold (blue line was calculated considering “buckets” with a value beyond two standard deviations of their averag 1: 0.85 ppm—2: 0.89 ppm (CH3 VLDL-LDL); 3–4: 1.17–1.19 ppm—5–7: 1.27–1.31 ppm—8: 1.35 pp ((-CH2-)n VLDL-LDL); 9: 3.23 ppm; 10–11: 3.65–3.67 ppm. Figure 3. M-PLS analysis of DHA + OBG supplementation. (A) M-PLS score plot. Milkshake samples are used as a training set to discriminate T0 (blue dots) vs. T1 (red dots). Biscuit and pancake samples are used as a test set (crosses coloured according to the prediction). (B) Table reporting the prediction results. (C) M-PLS loading plot of the first component (PC1); the significance threshold (blue lines) was calculated considering “buckets” with a value beyond two standard deviations of their averages; 1: 0.85 ppm—2: 0.89 ppm (CH3 VLDL-LDL); 3–4: 1.17–1.19 ppm—5–7: 1.27–1.31 ppm—8: 1.35 ppm ((-CH2-)n VLDL-LDL); 9: 3.23 ppm; 10–11: 3.65–3.67 ppm. Figure 3. M-PLS analysis of DHA + OBG supplementation. (A) M-PLS score plot. Milkshake sampl are used as a training set to discriminate T0 (blue dots) vs. T1 (red dots). Biscuit and pancake sampl are used as a test set (crosses coloured according to the prediction). (B) Table reporting the predicti results. 3. Results Thus, in each of the two broad signals, there is a direct correlation between chemical shift and lipoprotein particle size and density, with the smaller and denser particles (HDL) contributing to a farther upfield part of the signals. In contrast, chylomicrons and VLDL, the bigger and less dense fractions, contribute to the farther downfield part of the signals [36–38]. To investigate the observed changes more in depth, an unsupervised PCA model was built on the small spectral region selected above (0.92–0.71 ppm) (Figure 4). As an effect of DHA + OBG treatment, most of the subjects (15 out of 22) moved towards more positive values along with both principal component PC1 and PC2, going from T0 to T1 (Figure 4A). In all subjects, the variation along PC1 was 9 of 16 Nutrients 2020, 12, 86 much stronger than the variation along PC2. Principal component loadings were then explored to identify in detail which portions of the broad methyl signal were mainly responsible for the changes observed in the score plot for the subjects shifting towards more positive values. The PC1 loading plot (Figure 4B,D) clearly showed a significant decrement of the spectral area between 0.905–0.862 ppm, centred at 0.884 ppm, attributable to chylomicrons and VLDL fraction resonances. The PC2 loading plot, instead, showed an increment of the spectral area between 0.882 and 0.829 ppm, centred at 0.858 ppm, attributable to LDL resonances (Figure 4C,D). Nutrients 2020, 12, x FOR PEER REVIEW 9 of 15 administration was characterised by a significant increase of total ApoB100 and LDL particle number; the observed LDL increase was mainly due to the increase of small, dense LDL (LDL 4+5). Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group. (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares). Red arrows: Subjects (15 out of 22) that, going from T0 to T1, move towards more positive value along both PC1 and PC2; blue arrows: Subject that moves towards more negative value along PC1. (B) PC1 loading plot. (C) PC2 loading plot. In both loading plots, the threshold (black horizontal lines) used to select significant parts of the methyl signal was calculated considering the spectral area with values beyond two standard deviations of their averages. (D) 1H-NOESY spectral area containing lipoprotein methyl signal. 3. Results Green square: Significant spectral area in PC1 loading plot; orange square significant spectral area in PC2 loading plot. Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group. (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares). Red arrows: Subjects (15 out of 22) that, going from T0 to T1, move towards more positive value along both PC1 and PC2; blue arrows: Subject that moves towards more negative value along PC1. (B) PC1 loading plot. (C) PC2 loading plot. In both loading plots, the threshold (black horizontal lines) used to select significant parts of the methyl signal was calculated considering the spectral area with values beyond two standard deviations of their averages. (D) 1H-NOESY spectral area containing lipoprotein methyl signal. Green square: Significant spectral area in PC1 loading plot; orange square significant spectral area in PC2 loading plot. Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares) Red Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group. (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares). Red arrows: Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group. (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares). Red arrows: Subjects (15 out of 22) that, going from T0 to T1, move towards more positive value along both PC1 and PC2; blue arrows: Subject that moves towards more negative value along PC1. (B) PC1 loading plot. (C) PC2 loading plot. In both loading plots, the threshold (black horizontal lines) used to select significant parts of the methyl signal was calculated considering the spectral area with values beyond two standard deviations of their averages. (D) 1H-NOESY spectral area containing lipoprotein methyl signal. Green square: Significant spectral area in PC1 loading plot; orange square significant spectral area in PC2 loading plot. Figure 4. PCA analysis of lipoprotein methyl group signal (CH3) (0.92–0.71 ppm) of DHA + OBG group. (A) Score plots; each colour represents a different subject at TO (dots) and T1 (squares). 3. Results Red arrows: Subjects (15 out of 22) that, going from T0 to T1, move towards more positive value along both PC1 and PC2; blue arrows: Subject that moves towards more negative value along PC1. (B) PC1 loading plot. (C) PC2 loading plot. In both loading plots, the threshold (black horizontal lines) used to select significant parts of the methyl signal was calculated considering the spectral area with values beyond two standard deviations of their averages. (D) 1H-NOESY spectral area containing lipoprotein methyl signal. Green square: Significant spectral area in PC1 loading plot; orange square significant spectral area in PC2 loading plot. 10 of 16 Nutrients 2020, 12, 86 The significant decrement (p = 0.004) of the signal attributable to triglycerides (TG) resonance, in T1 1H-NOESY spectra of DHA + OBG group compared to baseline (T0) confirmed the reduction of chylomicrons and VLDL. M-PLS analysis applied only on the small part of DHA + OBG spectra containing the broad TG signal (4.34–4.22 ppm), which resulted in a very high (80%) discrimination accuracy between T0 and T1 (Figure S3). 1H-NOESY spectra were also analysed using the Bruker IVdr Lipoprotein subclass analysis (B.I. -LISA) platform [39]. The results for all BEF samples together are reported in Tables 4 and S2 and are in agreement with previously reported data. Considering single bioactives, no modification in the lipoprotein profile was observed in volunteers receiving AC or OBG. On the contrary, DHA administration was characterised by a significant increase of total ApoB100 and LDL particle number; the observed LDL increase was mainly due to the increase of small, dense LDL (LDL 4+5). Volunteers receiving DHA + OBG showed a different and strong rearrangement in the lipoprotein profile after the dietary intervention. This group was characterized by a significant decrement of VLDL particle number and TG. The decrement of TG was associated with TG decrease in VLDL, IDL, HDL and LDL1, without any modification in LDL4 and 5 subparticles. No modification in the lipoprotein profile was also observed in volunteers receiving DHA + AC No modification in the lipoprotein profile was also observed in volunteers receiving DHA + AC. No modification in the lipoprotein profile was also observed in volunteers receiving DHA + AC. Table 4. Bruker IVdr Lipoprotein subclass analysis. 3. Results DHA DHA + OBG T0 T1 T0 T1 TG 156.48 138.83 193.56 153.63 * Chol 226.25 240.43 * 252.82 250.90 LDL-Chol 122.91 129.37 136.43 147.09 * Apo B100 100.41 108.63 * 114.74 113.23 Apo A2 32.71 32.84 35.42 34.98 * Calculated Figures Apo B100/ Apo A1 1.42 1.26 * 1.27 1.29 Total Apo B100 Particle Number 1825.71 1975.17 * 2086.31 2058.91 VLDL Particle Number 192.87 175.21 236.63 208.71 * LDL Particle Number 1496.1 1589.47 * 1703.64 1726.34 Lipoprotein Main Fractions TG-VLDL 105.3 92.49 130.67 112.03 * TG-IDL 17.61 14.07 23.67 16.52 * TG-LDL 22.05 25.26 * 24.55 22.80 TG-HDL 10.69 10.97 11.69 10.10 * Chol-VLDL 27.59 22.44 34.54 28.24 * Chol-IDL 15.97 16.93 19.76 15.57 * Chol-LDL 122.91 129.37 136.43 147.09 * Free Chol-VLDL 12.65 11.27 15.23 12.54 * Free Chol-IDL 4.48 4.95 5.48 4.47 * Phospholipids-VLDL 28.25 24.50 33.71 28.06 * Phospholipids-IDL 8.67 8.925 12.03 10.23 * Phospholipids-LDL 69.93 72.97 * 75.48 81.04 Apo A2-HDL 33.53 33.99 36.26 35.63 * Apo B-VLDL 10.61 9.63 13.01 11.48 * Apo B-LDL 82.28 87.42 * 93.7 94.94 Table 4. Bruker IVdr Lipoprotein subclass analysis. 11 of 16 Nutrients 2020, 12, 86 Table 4. Cont. 3. Results DHA DHA + OBG T0 T1 T0 T1 VLDL Subfractions TG-VLDL 1 49.54 42.19 62.56 55.9 * TG-VLDL 2 19.73 13.31 23.62 19.14 * TG-VLDL 3 15.46 12.54 18.71 16.97 * TG-VLDL 4 10.82 9.63 13.48 12.31 * TG-VLDL 5 3.50 3.4 3.59 3.31 * Chol-VLDL 1 9.93 8.89 11.59 9.13 * Chol-VLDL 2 4.68 3.73 5.67 4.87 * Free Chol-VLDL 1 3.51 3.32 4.11 3.98 * Free Chol-VLDL 2 1.93 1.67 2.31 1.93 * Free Chol-VLDL 3 2.07 1.69 2.55 2.42 * Phospholipids-VLDL 1 8.43 6.92 10.17 9.39 * Phospholipids-VLDL 2 4.81 3.71 5.86 4.905 * Phospholipids-VLDL 3 4.83 4.09 5.91 5.74 * LDL Subfractions TG-LDL 1 6.57 6.42 6.84 6.1 * TG-LDL 4 2.76 2.88 * 3.00 3.03 TG-LDL 5 3.13 3.79 * 3.94 4.01 Apo A2-HDL 2 2.98 3.19 3.74 3.57 * Apo A2-HDL 3 6.49 6.68 7.43 6.95 * HDL Subfractions Free Chol-HDL 2 1.73 1.68 1.8 1.69 * Free Chol-HDL 3 2.33 2.47 2.70 2.29 * Free Chol-HDL 4 4.57 4.475 4.97 4.41 * Phospholipids-HDL 3 15.59 15.15 15.94 16.25 * Apo A1-HDL 2 16.43 17.87 * 18.35 17.33 Apo A1-HDL 3 25.77 26.76 26.60 26.46 * TG-HDL 2 1.69 1.79 1.67 1.415 * TG-HDL 3 2.33 2.5 2.7 2.11 * TG-HDL 4 4.24 4.105 4.61 4.01 * Only parameters which resulted statistically significant in the comparison T0 vs. T1, in DHA and DHA + OBG groups, are reported. * for p < 0.05. Since DHA + OBG administration was by far the treatment inducing the most relevant modifications, changes in other metabolite levels were also investigated. The signals of 29 metabolites were unambiguously assigned and integrated with the 1H-NMR spectra of the sera. All the assigned metabolites are listed in Table S3, with the respective median concentration at T0 and T1. Despite significant changes in the concentration of some metabolites, the modification in the sera metabolomic profile observed after the consumption of food enriched with DHA + OBG remains mainly attributable to lipoprotein rearrangement. 4. Discussion In this study, the metabolic effects of DHA supplementation, alone or in combination with OBG and AC, as an ingredient of the three different BEF, were characterized by using NMR-based metabolomics. We clearly evidenced that AC or OBG alone did not cause significant changes in the serum metabolome, which was significantly modified by DHA. Interestingly, co-administration of AC or OBG influenced DHA-induced modifications (Table 3). Using the same analytical approach, we have recently evidenced similar effects in the lipidome and metabolome of cultured human hepatocytes [40]. Dietary intervention with DHA was characterised by the increase of total ApoB100 and LDL particle number, mainly the smaller and denser sub-particles LDL 4 and LDL 5 (Table 4). Omega-3 fatty 12 of 16 12 of 16 Nutrients 2020, 12, 86 acid is highly effective and generally well-tolerated TG lowering agents. However, they are reported to increase LDL [41]. A modest increase of LDL level after DHA administration is also reported in clinical trials, but since the clinical relevance of such findings is uncertain [42] and given the advocated benefits, it is relevant to determine whether the increase in LDL is offset by the improvement of LDL particle size. Although DHA supplementation induced a shift toward smaller LDL particles having greater atherogenicity [43], it also decreased the apolipoprotein B100 (ApoB100)/apolipoprotein A1 (ApoA1) ratio. Apo B100 is present in atherogenic lipoproteins VDL, IDL and LDL, while ApoA1 is a major constituent of HDL. Thus, the ApoB100/ApoA1 ratio represents the balance between atherogenic and anti-atherogenic lipoproteins. Since several studies have reported that this ratio is associated with MetS risk independently of conventional risk factors [44,45], we argue that the 4-week consumption of DHA-enriched BEF decreased the risk of MetS. Interestingly, AC co-administration completely annulled the DHA effect (Tables 3 and 4). On the contrary, when co-administered with oat beta-glucan, DHA induces a different and very strong rearrangement of the lipoprotein profile (Figure 3, Tables 3 and 4). These metabolomic perturbations are consistent with the clinical effects observed in the trial [7] and with the already reported effect of DHA, which is supposed to decrease TG concentrations by reducing TG synthesis, incorporation into VLDL, and secretion, and by enhancing TG clearance from VLDL particles [46]. To the authors’ knowledge, this is the first report indicating a synergism of DHA and OBG. 4. Discussion The combined effect of these two bioactives has been only tested in the CONFIDENCE study [47], which results are not available yet. The uneven recruitment in the three trials did not permit to explore the presence of different food matrix effects fully. Notwithstanding, considering only samples from the milkshake trial a higher M-PLS discrimination accuracy was evidenced (Table 3), letting us speculate that the food matrix in which DHA is embedded also has a role in the final effect. This could be related to the modification of DHA bioavailability. Supplemented DHA must be absorbed and distributed into tissues to exert its effect, and the food matrix, including the co-enrichment with AC or OBG, could have a role. The differential modification of DHA serum level after the consumption of the different BEF [7] confirms this hypothesis and highlights that the concentration of DHA in food cannot be considered as a univocal index of functionality since many other factors contribute to the final effect. Nevertheless, the M-PLS discrimination model (T0 vs. T1) built on samples from volunteers consuming the DHA + OBG milkshake allowed us the correct prediction of the collection time (T0 or T1) of test samples from volunteers receiving the same enrichment embedded in the other food matrices (Figure 3), indicating the coherence of the detected changes, as the robustness of the model built on the DHA + OBG milkshake consumption overcomes the “noise effect” generated by the other matrixes. 5. Concluding Remarks Nutritional factors contribute to the movements of an individual in the metabolic space [19], and with only considering this tuning in a multidimensional space, we will have the possibility of understanding the complex changes due to diet in a foodomic vision [48]. Results herein reported evidence that NMR-based metabolomics can characterise modifications in the metabolome consequent to the administration of bioactives as ingredients of BEF. Using this approach, we demonstrated that the metabolome perturbation induced by DHA administration is attributable to a rearrangement in the lipoprotein profile and it is influenced by the food matrix and the co-presence of other bioactives. In particular, the co-administration of DHA + AC reduced the metabolomic effects of the fatty acid alone, while a strong cooperative and synergic effect was monitored in the case of co-administration of DHA + OBG. Modifications observed in the metabolome were consistent with clinical results of the trial that evidenced a significant decrease in serum TG in volunteers receiving DHA + OBG-enriched milkshake [7]. Metabolomics allowed relating TG decrease to the modification in lipoprotein profile, which could better predict modification in disease risk. Notably, regardless of the source (fish, fish Nutrients 2020, 12, 86 13 of 16 13 of 16 oil, algae, supplements, etc.) the median dose required to obtain a clinical effect is reported to be above 1.5 g DHA/day [49,50]. In the present study, the daily dose of DHA was lower than 0.3 g; notwithstanding its administration as an ingredient of BEF, particularly when in combination with OBG, modified the serum metabolome in human volunteers at risk of MetS. The absence of a placebo group receiving not enriched food should not be considered as a limitation of this study since all BEF within the same matrix had the same composition apart from the addition of the different bioactives. BEF containing AC or OBG alone did not significantly alter the individual metabolome and can be considered as controls, thus excluding that the observed effects were simply due to the consumption of the foods even in the absence of supplementation. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/1/86/s1. Figure S1: 5-Group discrimination. Score plot of PCA-CA analysis at T0 (a) and T1 (b). The values of the discrimination accuracy and the confusion matrices are also reported. 5. Concluding Remarks Conflicts of Interest: The authors declare that they have no conflict of interest. References 1. Alissa, E.M.; Ferns, G.A. Functional foods and nutraceuticals in the primary prevention of cardiovascular diseases. J. 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The value of the discrimination accuracy is also reported. In the score plots, each dot represents a different serum sample of DHA+OBG group, and each colour represents a time point: blue dots= T0 samples; red dots= T1 samples; Figure S3. Score plot of M-PLS analysis (T0 vs. T1) of the spectral region (4.34–4.22 ppm) containing the signal of triglyceride resonances. The value of the discrimination accuracy is also reported. In the score plots, each dot represents a different serum sample of DHA+OBG group, and each colour represents a time point: blue dots= T0 samples; red dots= T1 samples; Table S1. M-PLS discrimination accuracy values; Table S2: Bruker IVdr Lipoprotein subclass analysis. Each significant comparison (T0 vs T1) is indicated with * for p < 0.05 and ** p < 0.01; Table S3: List of metabolites whose signals were assigned and integrated in the NMR spectra. DHA+OBG discrimination between T0 and T1 in all samples and only in milkshake; each significant comparison (T0 vs T1) is indicated with * for p < 0.05 and ** p < 0.01. Author Contributions: V.G. and L.T. performed the analysis, and equally contributed to data interpretation and manuscript writing; F.C. and C.L. supervised and contributed to data interpretation and manuscript writing; A.B. (Achim Bub), C.M.-B., and C.O. were the principal investigators in the recruiting centres; L.R. contributed to the study design; A.B. (Alessandra Bordoni) coordinated the PATHWAY-27 project, designed the study and contributed to the manuscript writing. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by European Union Seventh Framework programme (FP7/2007-2013) under Grant Agreement no. 311876: Pathway-27. The Authors thank Applications Sante des Lipides Sarl (France), ABRO BIOTEC SL (Spain), Swedish Oat Fiber (Sweden), ADEXGO Kft. 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The effect of haemolysis on the metabolomic profile of umbilical cord blood. Clin. Biochem. 2015, 48, 534–537. [CrossRef] 29. Bernini, P.; Bertini, I.; Luchinat, C.; Nincheri, P.; Staderini, S.; Turano, P. References Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks. J. Biomol. NMR 2011, 49, 231–243. [CrossRef] 30. Ghini, V.; Quaglio, D.; Luchinat, C.; Turano, P. NMR for sample quality assessment in metabolomics. New Biotechnol. 2019, 52, 25–34. [CrossRef] 31. Mckay, R.T. How the 1D-NOESY suppresses solvent signal in metabonomics NMR spectroscopy: An examination of the pulse sequence components and evolution. Concepts Magn. Reson. 2011, 38A, 197–220. [CrossRef] 32. Carr, H.Y.; Purcell, E.M. Effects of Diffusion on Free Precession in Nuclear Magnetic Resonance Experiments. Phys. Rev. 1954, 94, 630–638. [CrossRef] 33. 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Lipids Health Dis. 2016, 15, 118. [CrossRef] © 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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Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses.: 1. Desempenho e Parâmetros Sangüíneos
Revista Brasileira de Zootecnia
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João Ricardo Vieira Jorge2, Lúcia Maria Zeoula3, Ivanor Nunes do Prado3, Luiz Juliano Valério Geron4 RESUMO - O objetivo deste trabalho foi avaliar o efeito de diferentes níveis de substituição do milho pela farinha de varredura de mandioca (0, 25, 50, 75 e 100%, base da matéria seca), sobre o consumo e conversão alimentar, ganho médio diário e níveis de hematócrito, glicose e uréia sanguíneos. Utilizaram-se 35 bezerros holandeses puros de origem ou puros por cruzamento, não castrados, com idade aproximada de 80 dias e peso vivo médio de 80 kg, distribuídos em delineamento de blocos casualizados. Os animais foram alimentados à vontade, até atingir o consumo de 4 kg/dia de concentrado. Como volumoso, foi utilizado feno inteiro de capim tifton 85. Os animais terminaram o experimento, com peso médio de 164 kg. A elevação dos níveis de substituição ocasionou uma resposta linear decrescente no consumo de matéria seca. A conversão alimentar não diferiu entre os tratamentos. Os ganhos diários para os dois primeiros períodos de 28 dias reduziram-se linearmente, com a elevação dos níveis de substituição, mas não diferiram no último período de 28 dias, apresentando valores estimados, variando de 0,93 a 0,68, 1,10 a 0,89 e 1,09 kg/dia, respectivamente. Os níveis de hematócrito, glicose e uréia não foram influenciados pelos níveis de substituição do milho pela farinha de varredura. Palavras-chave: bezerros, consumo, conversão alimentar, ganho de peso, mandioca, plasma1 R. Bras. Zootec., v.31, n.1, p.192-204, 2002 R. Bras. Zootec., v.31, n.1, p.192-204, 2002 1 Parte da dissertação de mestrado em Zootecnia, apresentada pelo primeiro autor à UEM. 2 Mestre em Zootecnia, PPZ, UEM – Rua Tomé de Sousa, 195, zona 2, CEP 87010-380 Maringá, PR. E.mail: jricardo@wnet.com.br 3 Professores do PPZ/UEM, Bolsistas pesquisadores, CNPq - UEM - Av. Colombo, 5790 - CEP 87020-900. E.mail: lmzeoula@uem.br; inprado@uem.br. 4 Bolsista de iniciação científica - CNPq. E.mail: ljgeron@yahoo.com.br Replacement of Corn for Cassava Meal (Manihot esculenta, Crantz) in the Holstein Calves Diets. 1. Performance and Blood Parameters ABSTRACT - The objective of this project was to evaluate the effects of different replacement levels of corn for cassava meal (0, 25, 50, 75, and 100%, dry matter basis) on the dry matter intake and feed:gain ratio, daily weight gain and levels of blood hematocrit, glucose and urea. Thirty five intact Holstein calves, averaging 80 days old and initial live weight of 80 kg, were allotted to a randomized block design and fed concentrate, ad libitum, to reach the intake of 4 kg/day. Tifton 85 bermudagrass was fed as roughage. The final average weight was of 164 kg. The dry matter intake showed a linear behavior as the replacement levels increased. The feed:gain ratio was not affected by the replacement levels. The daily weight gain for the two first periods of 28 days (period 1 and 2, respectively) reduced as the replacement levels increased, but they were not affected in the last 28 days period (period 3), and the respective values ranged from 0.93 to 0.68, 1.10 to 0.89, and 1.09 kg/day. Levels of hematocrit, glucose and urea were not affected by the replacement of corn for cassava meal. Key Words: calves, cassava, feed:gain ratio, intake, plasma, weight gain Key Words: calves, cassava, feed:gain ratio, intake, plasma, weight gain Introdução plantéis leiteiros, é preciso direcionar estudos para os aspectos nutricionais, para que esses animais utilizem o mínimo de leite, sendo desaleitados precocemente e, também, utilizem alimentos de baixo custo, disponíveis na região, levando-se em consideração, ainda, os as- pectos de manejo e condições sanitárias. A criação dos machos de plantéis leiteiros para abate precoce é viável, mas depende dos preços de mercado. Todavia, esta deverá ser tanto mais inten- siva, quanto maior for a proporção de sangue europeu dos animais (Lucci, 1989). Deve-se salientar que a produtividade desses animais é conseqüência da com- plexa interação entre a dieta, sua preparação e seu valor nutritivo, que determinam o consumo de nutri- entes e a eficiência com que tais nutrientes são utilizados nos processos metabólicos. A principal vantagem de novilhos holandeses sobre outras raças tradicionais para o corte está no seu desempenho uniforme. A raça holandesa tem sido selecionada, principalmente, para altas produções de leite. Esta seleção resultou em uma base genética relativamente estreita, comparada com raças especializadas para corte, que acarretou para os Para que se consiga êxito na criação de machos de JORGE et al. 193 indústria, qualidade da mão-de-obra e variedades de mandioca. novilhos holandeses, em um consumo alimentar e ganho diário mais consistentes e previsíveis ao longo do período alimentar, tornando mais fácil o manejo nutricional (Grant et al., 1993). A substituição de milho por farinha de mandioca, na proporção de 50 e 100%, foi fornecida na alimen- tação de bezerros da raça Holandesa (Peixoto & Warner, 1993). O tratamento com 100% de substitui- ção apresentou ganhos de peso menores e, segundo os autores, o menor desempenho dos animais foi devido ao menor consumo de ração, pois a farinha de mandioca é quase sem gosto, muito pulverulenta e seca. Desta forma, os animais não mastigam a fari- nha da mesma forma como fariam com o milho grosseiro. Reduções no consumo também foi obser- vado por Marques et al. (2000) para novilhas, rece- bendo rações com farinha de varredura, em substitui- ção total ao milho. A literatura mostra que, se os animais tiverem acesso a um manejo correto quanto aos aspectos nutricionais e sanitários, eles podem atingir 200 kg de PV, aos seis meses de idade, com ganhos diários de até 1,40 kg/dia. Lucci et al. Introdução (1974), citados por Lucci (1989), forneceram uma mistura concentrada com 20% de proteína e feno de capim gordura de baixa qualidade, à vontade e verificaram ganho diário de 0,70 kg/dia, atingindo-se 143 kg aos 180 dias. Salles & Lucci (1998) avaliaram o desempenho de bezerros holandese submetidos a níveis de monensina sódica, visando altos ganhos e encontraram resposta quadrática para o ganho de peso (1,1 a 1,4 kg/dia). Araújo et al. (1998) alimentaram bezerros holande- ses com feno de capim coastcross (de 10 a 55% de inclusão) e concentrado e obtiveram ganho diário variando de 1,0 a 0,7 kg/dia, abatendo estes animais com 180kg de peso vivo. Campos & Lizieire (2000), utilizando um sistema intensivo de alimentação, for- necendo, em média, 5,5 kg de concentrado/animal/dia no final do período experimental, obtiveram bezerros holandeses com seis meses de idade e peso vivo médio de 196 kg. Devido à escassez de informações sobre o desem- penho ou a criação de machos provenientes de reba- nhos leiteiros e do uso de resíduos das farinheiras, este trabalho teve por objetivo avaliar o desempenho, con- sumo e conversão alimentar e níveis de hematócrito, glicose e uréia sanguíneos de bezerros holandeses, alimentados com dietas, contendo níveis crescentes de farinha de varredura, em substituição ao milho. Material e Métodos A composição química dos alimentos utilizados é mostrada na Tabela 1. receberam 4 litros diários de leite integral e uma alimentação ad libitum, a partir do 7o dia de vida, constituída por um concentrado comercial peletizado, que continha 20,7% de proteína bruta, 1,7% de cálcio e 0,57% de fósforo e feno integral de capim Tifton 85, um híbrido de Cynodon dactylum (L) Pers. As rações foram isoenergéticas, isoprotéicas, isocálcicas e isofosfóricas, seguindo as exigências nutricionais, recomendados pela Embrapa - CNPGL, segundo Campos & Lizieire (2000), para bezerros desmamados, até 180 dias de idade. As rações foram formuladas, substituindo o milho pela farinha de var- redura (FV), em níveis crescentes de 0, 25, 50, 75 e 100%. As rações foram peletizadas e fornecidas duas vezes ao dia, às 8 e 16 h. e ajustadas para o fornecimento de 10% acima do consumo voluntário. Os animais, quando atingiram 120 dias de idade, passaram a receber rações experimentais não peletizadas e ajustadas para seus requerimentos nutricionais (Tabela 2), duas vezes ao dia, restringindo-se a um máximo de 4 kg de concentrado/animal/dia. O feno foi fornecido ad libitum, sendo trocado a cada três dias, ou quando da falta deste. O leite integral fornecido aos bezerros foi prove- niente do rebanho da propriedade e composto por leite recentemente ordenhado, leite de animais com mastite, leite de animais tratados com antibióticos e colostro excedente, dependendo da disponibilidade destes. Todas as fontes foram homogeneizadas, para depois ser o leite fornecido aos animais, entre 36 e 38ºC. A partir dos sete dias de idade, foi fornecida água de boa qualidade, advinda de poço artesiano. O fornecimento foi à vontade, em baldes plásticos, fixados no lado de fora dos bezerreiros, sempre duas horas após o aleitamento. Os baldes foram vistoria- dos de duas em duas horas, para o seu abastecimento. Material e Métodos O experimento foi realizado em uma propriedade particular, denominada Fazenda Clicie, situada no município de São João de Caiuá, no noroeste do Paraná, e no Laboratório de Nutrição Animal do Departamento de Zootecnia (DZO) da Universidade Estadual de Maringá. A mandioca e seus resíduos podem ser fontes alternativas de energia, visto que os grãos mais nobres são utilizados na alimentação humana e de animais não ruminantes, que apresentam melhor res- posta à utilização deste tipo de alimento. Todavia, os dados referentes ao uso de mandioca e seus resíduos, em substituição parcial ou total dos alimentos tradici- onalmente usados na alimentação de bovinos, são escassos e pouco conclusivos (Marques et al., 2000). O experimento foi desenvolvido no período de abril a novembro de 1999. Do nascimento até 164 dias de idade, os bezerros foram mantidos em bezerreiros individuais, construídos em madeira e teto de zinco, com dimensões de 1,10 x 1,50 metros. Cada animal dispunha de cocho para o fornecimento de ração, fenil e balde para a água e leite. p ( q ) A farinha de varredura é um resíduo que se origina da limpeza das farinheiras, contendo, princi- palmente, farinha suja (imprópria para o consumo humano), e apresenta elevado teor de amido (80%) e de matéria seca (90%). De acordo com estas infor- mações, a mandioca e seus resíduos podem ser classificados como fontes energéticas (Caldas Neto et al., 2000). Todavia, os valores da composição química da raiz de mandioca e seus resíduos não são homogêneos e padronizados, como para os alimen- tos clássicos utilizados na alimentação animal (Melotti, 1972; De Bem, 1996; Martins et al., 1999). Segundo Cereda (1994), esta variação ocorre devi- do a diversos fatores, como nível tecnológico da Foram utilizados 35 bezerros, machos, puros de origem (PO) ou puros por cruzamento (PC), da raça Holandesa P&B, recém-nascidos, com peso médio inicial de 40 kg, provenientes da propriedade ou de rebanhos em bom estado sanitário, de granjas leitei- ras da região de Maringá. Esses animais receberam dieta e manejo semelhantes, do nascimento ao desaleitamento (período pré-experimental), quando após iniciou-se o período experimental. Durante o período de aleitamento, os animais R. Bras. Zootec., v.31, n.1, p.192-204, 2002 o Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Beze 194 centrados. 2 NEm - net energy for maintenance (kcal/kg) and NEg - net energy for gain (kcal/kg) according to Kearl (1982). p g ELm - energia líquida de mantença (kcal/kg) e ELg - energia líquida de ganho (kcal/kg), segundo Kearl (1982). % dry matter. oteína bruta e FDN - fibra em detergente neutro. % de matéria seca; MS - matéria seca, PB - proteína bruta e FDN - fibra em detergente neutro. % dry matter. 1 DM - dry matter, CP - crude protein and NDF - neutral detergent fiber. 2 ote a b uta e b a e dete ge te eut o mantença (kcal/kg) e ELg - energia líquida de ganho (kcal/kg), segundo Kearl (1982). % dry matter. 1 DM - dry matter, CP - crude protein and NDF - neutral detergent fiber. 2 NEm - net energy for maintenance (kcal/kg) and NEg - net energy for gain (kcal/kg) according to Kearl (1982). % de até a seca; 1 MS - matéria seca, PB - proteína bruta e FDN - fibra em detergente neutro. 2 ELm - energia líquida de mantença (kcal/kg) e ELg - energia líquida de ganho (kcal/kg), segundo Kearl (1982). Material e Métodos Após a desmama, os bezerros receberam dietas distintas, constituídas de farelo de soja (FS), milho integral triturado (M), farinha de varredura de man- dioca (FM), farinha de peixe (FP), feno de Tifton 85 triturado (FT) e sal suplementado com minerais e vitaminas (para cada kg, Ca - 230 g, P - 90 g, Mg - 20 g, S - 15 g, F - 0,9 g, Cu - 700 mg, Zn - 2.700 mg, Mn - 1.250 mg, Fe - 2.000 mg, I - 80 mg, Co - 100 mg, Se - 20 mg, vitamina A - 200.000 UI, vitamina D3 - 60.000 UI e vitamina E - 60 UI) . O feno de capim tifton 85 foi utilizado como volumoso, na forma integral, e triturado, fazendo parte dos con- Nas Tabelas 2 e 3, encontram-se as composições percentual e química das rações experimentais, correspondentes ao período da desmama, até 120 dias de idade e de 121 até 180 dias de idade, respectivamente. Os animais nascidos na propriedade foram pesa- dos por ocasião do nascimento e receberam um brinco de identificação. O manejo até ao desaleitamento foi: 1. Separação das mães, 24 horas após o nascimento; 2. Do 2o ao 3o dia, quatro litros de colostro, em baldes, duas vezes ao dia, às 8 e 16 h; 1. Separação das mães, 24 horas após o nascimento; 2. Do 2o ao 3o dia, quatro litros de colostro, em baldes, duas vezes ao dia, às 8 e 16 h; 3. Do 4o dia até ao desaleitamento, 4 litros de leite, 3. Do 4o dia até ao desaleitamento, 4 litros de leite, R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Tabela 1 - Composição química dos alimentos Table 1 - Chemical composition of the ingredients Alimentos % MS1 % PB1* % FDN1* % AMIDO* ELm2 ELg2 Feeds % DM1 % CP* % NDF1* % STARCH* Nem2 Neg2 Milho 88,0 9,4 11,4 73,5 2.013 1.357 Corn Farinha de varredura 90,7 1,5 10,4 85,7 1.974 1.328 Cassava meal Farelo de soja 88,4 49,4 15,1 3,5 2.001 1.348 Soybean meal Feno de tifton 85 90,0 10,0 76,2 4,2 900 350 Tifton 85 hay Farinha de peixe 90,5 60,0 - - 1.730 1.110 Fish meal * % de matéria seca; 1 MS - matéria seca, PB - proteína bruta e FDN - fibra em detergente neutro. Material e Métodos 2 ELm - energia líquida de mantença (kcal/kg) e ELg - energia líquida de ganho (kcal/kg), segundo Kearl (1982). * % dry matter. 1 DM - dry matter, CP - crude protein and NDF - neutral detergent fiber. 2 NEm - net energy for maintenance (kcal/kg) and NEg - net energy for gain (kcal/kg) according to Kearl (1982). Tabela 1 - Composição química dos alimentos Table 1 - Chemical composition of the ingredients Alimentos % MS1 % PB1* % FDN1* % AMIDO* ELm2 ELg2 Feeds % DM1 % CP* % NDF1* % STARCH* Nem2 Neg2 Milho 88,0 9,4 11,4 73,5 2.013 1.357 Corn Farinha de varredura 90,7 1,5 10,4 85,7 1.974 1.328 Cassava meal Farelo de soja 88,4 49,4 15,1 3,5 2.001 1.348 Soybean meal Feno de tifton 85 90,0 10,0 76,2 4,2 900 350 Tifton 85 hay Farinha de peixe 90,5 60,0 - - 1.730 1.110 Fish meal * % d é i Tabela 1 - Composição química dos alimentos Table 1 - Chemical composition of the ingredients R. Bras. Zootec., v.31, n.1, p.192-204, 2002 % y 1 DM - dry matter, CP - crude protein and NDF - neutral detergent fiber. 2 NEm - net energy for maintenance (kcal/kg) and NEg - net energy for gain (kcal/kg) according to Kearl (1982) atéria seca, PB - proteína bruta e FDN - fibra em detergente neutro. nergia líquida de mantença (kcal/kg) e ELg - energia líquida de ganho (kcal/kg), segundo Kearl (1982). tter R. Bras. Zootec., v.31, n.1, p.192-204, 2002 CP - crude protein and NDF - neutral detergent fiber. for maintenance (kcal/kg) and NEg - net energy for gain (kcal/kg) according to Kearl (1982). R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. 195 abela 2 - Composição percentual e química dos concentrados experimentais para o período do desaleitamento até 120 dias de idade able 2 Percentual and chemical composition of the experimental concentrates for the period from weaning to 120 days old Tabela 2 - Composição percentual e química dos concentrados experimentais para o período do desaleitamento até 120 dias de idade Table 2 - Percentual and chemical composition of the experimental concentrates for the period from weaning to 120 days old Variáveis Nível de substituição (%) Variables Replacement level (%) 0 25 50 75 100 Milho 75,40 56,55 37,70 18,85 - Corn Farinha de varredura - 18,85 37,70 55,91 72,17 Cassava meal Farelo de soja 11,90 13,11 14,33 16,04 19,26 Soybean meal Feno de tifton 85 7,71 6,13 4,55 3,21 2,57 Tifton 85 bermudagrass hay Farinha de peixe 1,50 1,50 1,50 1,50 1,50 Fish meal Suplemento mineral e vit. 2,00 2,00 2,00 2,00 2,00 Mineral and vit. suppl. Uréia - 0,36 0,73 1,00 1,00 Urea Calcário 0,75 0,75 0,75 0,75 0,75 Limestone Fosfato bicálcico 0,45 0,45 0,45 0,45 0,45 Dicalcium phosphate Sal comum 0,29 0,29 0,29 0,29 0,29 Salt Total 100 100 100 100 100 Matéria seca (%) 87,71 88,30 88,93 89,42 90,00 Dry matter (%) Proteína bruta (%) 14,60 14,60 14,60 14,60 14,60 Crude protein (%) Proteína degrad. rúmen1 (%) 7,78 8,48 9,17 9,77 10,10 Ruminal degradable protein1 (%) Proteína não degrad. rúmen1 (%) 6,82 6,12 5,43 4,83 4,50 Ruminal undegradable protein1 (%) NDT (%) 76,60 76,60 76,60 76,60 76,60 TDN (%) ELm (kcal/kg) 1.825 1.828 1.831 1.833 1.833 NEm (kcal/kg) ELg (kcal/kg) 1.191 1.193 1.196 1.198 1.198 NEg (kcal/kg) FDN (%) 16,27 15,06 13,85 12,83 12,37 NDF (%) Cálcio2 ( % ) 0,92 0,92 0,92 0,92 0,92 Calcium2 (%) Fósforo2 ( % ) 0,58 0,58 0,58 0,58 0,58 Phosphorus2 (%) 1 Segundo dados do NRC (1988). 2 Teores da suplementação mineral and chemical composition of the experimental concentrates for the period from weaning to 120 days old 1 Segundo dados do NRC (1988). 2 1 According to data from NRC (1988). duas vezes ao dia, às 8 e 16 h; duas vezes ao dia, às 8 e 16 h; do balde, por ocasião do final de cada aleitamento, para estimular o animal a ingeri-lo, evitando maiores discrepâncias no consumo (Noller et al. 1975, citados por Prado, 1981), ocasionadas por diferentes com- portamentos dos animais. 4. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Do 7o dia até ao desaleitamento, concentrado comercial, com 20,7% de proteína bruta, 1,7% de cálcio e 0,57% de fósforo, ad libitum, 5. Do 7o dia até ao desaleitamento, feno inteiro ad libitum. Os animais provenientes de outras granjas leitei- ras (20 animais) foram selecionados e transportados Um pouco do concentrado foi colocada no fundo Um pouco do concentrado foi colocada no fundo R. Bras. Zootec., v.31, n.1, p.192-204, 2002 R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de B 196 Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses... 196 Tabela 3 - Composição percentual e química dos concentrados experimentais para o período de 121 a 180 dias de idade Table 3 - Percentual and chemical composition of the experimental concentrates for the period from 121 to 180 days old Variáveis Nível de substituição (%) Variables Replacement level (%) 0 25 50 75 100 Milho 78,41 58,80 39,20 19,65 - Corn Farinha de varredura - 19,60 39,20 57,85 74,77 Cassava meal Farelo de soja 9,15 10,42 11,69 13,68 17,03 Soybean meal Feno de tifton 85 7,45 5,81 4,17 2,87 2,21 Tifton 85 bermudagrass hay Farinha de peixe 1,50 1,50 1,50 1,50 1,50 Fish meal Suplemento mineral e vit. 2,00 2,00 2,00 2,00 2,00 Mineral and vit. suppl. Uréia - 0,38 0,76 1,00 1,00 Urea Calcário 0,75 0,75 0,75 0,75 0,75 Limestone Fosfato bicálcico 0,45 0,45 0,45 0,45 0,45 Dicalcium phosphate Sal comum 0,29 0,29 0,29 0,29 0,29 Salt Total 100 100 100 100 100 Total Matéria seca (%) 87,72 88,33 88,91 89,56 90,01 Dry matter (%) Proteína bruta (%) 13,50 13,50 13,50 13,50 13,50 Crude protein (%) Proteína degrad. rúmen1 (%) 7,01 7,73 8,45 9,04 9,38 Ruminal degradable protein1 (%) Proteína não-degradavel rúmen1 (%) 6,49 5,77 5,05 4,46 4,12 Ruminal undegradable protein1 (%) NDT (%) 76,70 76,70 76,70 76,70 76,70 TDN (%) ELm (kcal/kg) 1.828 1.831 1.834 1.836 1.837 NEm (kcal/kg) ELg (kcal/kg) 1.194 1.196 1.199 1.201 1.201 NEg (kcal/kg) FDN (%) 16,00 14,74 13,49 12,48 12,31 NDF (%) Cálcio2 ( % ) 0,92 0,92 0,92 0,92 0,92 Calcium2 (%) Fósforo2 ( % ) 0,58 0,58 0,58 0,58 0,58 Phosphorus2 (%) 1 Segundo dados do NRC (1988). 2 Teores da suplementação mineral. 1 5 litros de água. A partir daí, o manejo foi idêntico ao dos animais nascidos na propriedade. 1 Segundo dados do NRC (1988). 2 g ( ) 2 Teores da suplementação mineral. p ç 1 According to data from NRC (1988). JORGE et al. ç ( ) EM = 0,82 x ED ELm = -1,12 + 1,37 x EM -0,138 x ME2 + 0,0105 x EM3 ELg = -1,65 + 1,42 x EM - 0,174 x EM2 + 0,0122 x ME3 EM = 0,82 x ED , ELm = -1,12 + 1,37 x EM -0,138 x ME2 + 0,0105 x EM3 ELg = -1,65 + 1,42 x EM - 0,174 x EM2 + 0,0122 x ME3 Os animais foram pesados, em balança eletrôni- ca com 0,5 kg de precisão, logo ao nascimento ou após a chegada na propriedade e, posteriormente, a cada sete dias, até ao desaleitamento, quando tam- bém ocorreu nova pesagem. Posteriormente, foram feitas pesagens a cada 28 dias, para obtenção do ganho médio diário, totalizando três períodos em 84 dias de experimento. As pesagens foram após 15 horas de jejum, pela manhã, às 8 h. Foram realizadas anotações diárias da quantidade de concentrado fornecido e das sobras, para cada animal, para determinar o consumo total de matéria seca e conversão alimentar. O delineamento experimental foi inteiramente casualizado, com cinco tratamentos (0, 25, 50, 75 e 100% de substituição do milho pela farinha de varredu- ra de mandioca) e sete repetições. As análises estatís- ticas das variáveis estudadas (consumo, ganho médio diário, conversão alimentar e níveis de hematócrito, glicose e uréia sangüíneos) foram interpretadas por análises de variância e regressão, utilizando o Sistema de Análises Estatísticas e Genéticas - SAEG (UFV, 1983), de acordo com o seguinte modelo: g Yij = µ + Ni + b1 (PI-PM) + eij ij µ i 1 ( ) ij em que: Yijk = valor observado da variável estudada no indivíduo j, recebendo a ração com nível i de substituição do milho pela farinha de varredura; µ = constante geral da variável; Ni = efeito do nível i de substituição do milho pela farinha de varredura (i = 0, 25, 50, 75 e 100); b1 = coeficiente linear de regressão de Y, em função do peso inicial (PI); PI = peso inicial ao desaleitamento; PM = peso inicial médio; eij = erro aleatório associado a cada observação Yij. Foram realizadas amostragens semanais dos con- centrados, do feno fornecido e das sobras. Após a amostragem, o material foi acondicionado em sacos plásticos, devidamente identificados e guardados em freezer (-10oC), para posterior análise. JORGE et al. 197 As coletas de sangue foram no período da tarde, sendo acondicionadas em caixa térmica contendo gelo e processadas, logo a seguir. consumo de concentrado, sendo que, quando esta- vam consumindo ao redor de 800 g por dia, estes tiveram o fornecimento de leite cessado de uma vez, passando a consumir apenas o concentrado experi- mental. Uma semana antes do desaleitamento, foi ocorrendo a substituição gradativa do concentrado comercial pelos concentrados experimentais. Foram realizadas análises dos concentrados for- necidos, das sobras e do feno, para determinação de matéria seca (MS), proteína bruta (PB), energia bruta (EB), fibra em detergente neutro (FDN), extra- to etéreo (EE) e fibra bruta (FB) segundo Silva (1990). Para determinação do amido, foi utilizado o método enzimático descrito por Poore et al. (1989), adaptado por Pereira & Rossi Jr. (1995). O desaleitamento foi realizado em três datas diferentes, sendo a primeira em 18/06/1999 (18 animais), seguido por outro em 10/07 (12 animais) e, por fim, em 22/07 (5 animais). Os animais foram sorteados aleatoriamente entre os cinco tratamentos A determinação dos teores de nutrientes digestíveis totais (NDT) para farinha de varredura, milho, farelo de soja e feno de tifton baseou-se na composição química dos alimentos, utilizando a equação para alimentos energéticos, segundo Kearl (1982). Os animais foram vacinados contra aftosa, pneumoenterite e salmonelose e receberam dosa- gens de vitamina A e D, conforme NRC (1988). Foram submetidos a um controle sistemático de ecto e endo parasitas. Os bezerreiros foram limpos diariamente e mudados de local a cada vinte dias. Os animais foram presos por correntes, para impe- dir o contato direto entre os mesmos. Os recipientes utilizados para fornecimento de leite foram higienizados, após cada aleitamento e os baldes para o fornecimento de água, diariamente. g g ( ) %NDT= 40,2625 + 0,1969%PB + 0,4228%ENN + 1,1903EE% - 0,1379%FB %NDT= 40,2625 + 0,1969%PB + 0,4228%ENN + 1,1903EE% - 0,1379%FB Os valores de energia metabolizável (EM), ener- gia líquida de mantença (ELm) e energia líquida de ganho (ELg) foram calculados conforme recomenda- ções de Sniffen et al. (1992). R. Bras. Zootec., v.31, n.1, p.192-204, 2002 do seu nascimento até sete dias de idade. Ao chega- rem à propriedade, ficaram privados de leite durante as primeiras 12 horas, recebendo apenas soro por via oral, preparado segundo recomendações da Embrapa - CNPGL (Campos & Lizieire, 2000), consistindo da mistura de 45 g de sal e 250 g de açúcar, para cada Todos os animais foram avaliados qualitativamente quanto à concentração de imunoglobulinas no sangue, segundo as recomendações de Charles et al. (1994). O critério para desaleitamento dos animais foi o R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Resultados e Discussão A elevação dos níveis de substituição do milho pela farinha de varredura na dieta proporcionou com- portamento linear decrescente para o consumo médio total de matéria seca, com valores estimados, variando de 2,93 a 2,58 kg/dia. O mesmo comportamento ocorreu no primeiro e segundo períodos de 28 dias (2,24 a 1,70 e 3,04 a 2,42 kg MS/dia, respectivamente). No terceiro e último períodos de 28 dias, o consumo foi limitado a 3,56 kg MS/dia, sendo que todos os animais atingiram este consumo no início do período, mantendo o mesmo até o final. Os dados do consumo de matéria seca e de nutrien- tes serão expressos somente em relação ao consumo de concentrado dos animais (Tabela 4). Houve forneci- mento diário de feno de Tifton 85 para os animais, porém, as dificuldades de manejo e mensuração do fornecido e das sobras deste volumoso, nas condições em que o experimento foi realizado, tornaram difíceis as estimativas de consumo de feno, sendo estas feitas apenas por observações visuais, sem mensurações. O consumo deste, no período pré e pós-desaleitamento, foi baixo, podendo o fornecimento de concentrado à vonta- de, durante parte do experimento, ter contribuído para o baixo consumo de feno, como visualizado durante a condução do experimento. O consumo de matéria seca do concentrado expresso em porcentagem do peso vivo indica o alto consumo de concentrado, em relação à dieta total (Tabela 4). O consumo de matéria seca, em relação ao peso vivo dos animais, comportou-se da mesma forma, com resposta linear decrescente para o aumento dos níveis de substituição (2,40 a 2,18% do peso vivo). Houve redução linear para o consumo de proteína bruta, devido à redução no consumo de MS, com valores estimados, pela equação de regressão, vari- ando de 0,42 a 0,36 kg/dia. Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses... 198 o Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses Resultados e Discussão Da mesma forma, houve R Bras Zootec v 31 n 1 p 192-204 2002 Tabela 4 - Médias, equações de regressão ajustadas (ER) e coeficientes de variação (CV) para o consumo de nutrientes para as dietas experimentais Table 4 - Means, fitted regression equations (RE) and coefficients of variation (CV) for the nutrients intake of the experimental diets Item Nível de substituição (%) CV (%) ER Replacement level (%) RE 0 25 50 75 100 CTMS (kg/dia) 2,90 2,91 2,73 2,64 2,59 8,6 1 TDMI (kg/day) CTMS (%PV/dia) 2,43 2,35 2,26 2,18 2,23 7,1 2 TDMI (%LW/day) CMSP1 (kg/dia) 2,26 2,13 1,90 1,82 1,74 14,5 3 P1DMI (kg/day) CMSP2 (kg/dia) 2,93 3,07 2,72 2,51 2,43 18,1 4 P2DMI (kg/day) CMSP3 (kg/dia) 3,56 3,56 3,56 3,56 3,56 0,0 Y = 3,56 P3DMI (kg/day) CPB (kg/dia) 0,41 0,41 0,39 0,37 0,36 8,7 5 CPI (kg/day) CFDN (kg/dia) 0,46 0,43 0,37 0,33 0,31 8,6 6 NDFI (kg/day) CAMIDO (kg/dia) 1,69 1,76 1,72 1,70 1,68 8,6 Y = 1,71 STRCHI (kg/day) 1. Y = 2,927 - 0,00351**N r2 = 0,92 2. Y = 2,402 - 0,0022**N r2 = 0,81 3. Y = 2,236 - 0,00534**N r2 = 0,96 4. Y = 3,035 - 0,00611*N r2 = 0,83 5. Y = 0,416 - 0,00056**N r2 = 0,93 6. Y = 0,460 - 0,00159**N r2 = 0,97 * P<0,05) e **(P<0,01). N = Nível de substituição (%) . CTMS = consumo total médio de matéria seca; CMSP1, CMSP2 e CMSP3 = consumo de matéria seca no primeiro, segundo e terceiro períodos de 28 dias, respectivamente; CPB = consumo de proteína bruta; CFDN = consumo de fibra em detergente neutro e Camido = consumo de amido N = Level of substitution (%). TDMI = total dry matter intake; P1DMI, P2DMI and P3DMI = dry matter intake for the first, second and third periods; CPI = crude protein intake; NDFI = neutral detergent fiber intake (NDFI) and SARTCHI = starch intake. s de regressão ajustadas (ER) e coeficientes de variação (CV) para o consumo de nutrientes para mentais Tabela 4 - Médias, equações de regressão ajustadas (ER) e coeficientes de variação (CV) para o consumo de nutrientes para as dietas experimentais ariação (CV) para o consumo de nutrientes para R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. Foram colhidas amostras de sangue dos animais em tubos com vácuo, contendo heparina sódica, por ocasião do desaleitamento e do final ao experimento. Foi determinada a porcentagem de hematócritos das amostras, que posteriormente foram centrifugadas, sendo o plasma congelado (-20oC), para posteriores análises de glicose e uréia. j Os graus de liberdade para níveis de substituição do milho pela farinha de varredura de mandioca foram desdobrados em polinômios ortogonais. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. 199 substituição não diferiu (P>0,05) no consumo de ração (105,67 e 103,90 kg da 6a a 16a semanas, respectivamente). Concluíram, ainda, que bezerros desaleitados precocemente e submetidos a elevados fornecimentos de farinha de mandioca (50% na com- posição da ração - 100% de substituição ao milho) têm o consumo de ração diminuído, o que, da mesma forma, foi observado no presente experimento. Marques et al. (2000) também verificaram redução de consumo de MS (P<0,05), de 11,5 para 8,3 kg/dia, ao fornecerem dietas com silagem de milho, farelo de soja e farinha de varredura em substituição total ao milho, no desempenho de novilhas com 24 meses de idade e peso médio de 365 kg. redução linear no consumo de FDN (0,46 a 0,30 kg/dia). O consumo de amido não foi influenciado pelas rações (1,71 kg/dia). O apetite é regulado por mecanismos quimiostáticos e limitações físicas do aparelho digestivo (Van Soest, 1994). Fatores físicos são importantes na limitação do consumo voluntário. Estes fatores agem por limites impostos na capacidade do retículo e rúmen e pelo tempo de permanência do alimento no órgão. Em dietas de altas digestibilidades, como no caso de dietas com altos níveis de concentrado, que não levariam a um completo enchimento do rúmen, seria pouco prová- vel que os fatores físicos limitassem o consumo volun- tário das rações experimentais, à medida em que se elevassem os níveis de farinha de varredura. Consumos médios de matéria seca (MS) superi- ores aos observados no presente experimento foram encontrados por Araújo et al. (1998) e Signoretti et al. (1998), ao fornecerem a bezerros holandeses desaleitados, dos 60 até 180 dias de idade, níveis de volumoso (feno de tifton), variando de 10 a 55% na dieta. Esses autores observaram que o consumo de MS variou de 3,45 a 4,01 kg/dia. Todavia, vale ressaltar que, para a determinação do consumo médio de MS observado no presente trabalho, que variou de 2,93 a 2,58 kg/dia, foram considerados somente da- dos do concentrado, sendo que, no último período de 28 dias, este foi limitado para favorecer a ingestão de feno. Por outro lado, os consumos obtidos por Salles & Lucci (1998), ao testar níveis de monensina sódica no crescimento de 20 bezerros holandeses, com 83 dias de idade e alimentados com 20% de feno de coastcross e concentrado, também foram maiores, variando de 4,19 a 5,03 kg/dia. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Segundo Hill (1970), citado por Peixoto & Warner (1993) e Van Soest (1994), o alimento ao entrar na boca, estimula a secreção de saliva, que é atribuída ao estímulo reflexo das glândulas salivares, por intermé- dio dos receptores bucais. Em geral, alimentos tradici- onais utilizados na alimentação animal causam secre- ção de saliva rica em mucina e enzimas, o que facilita a deglutição, enquanto alimentos muito secos provo- cam considerável fluxo de saliva aquosa, com muito pouca mucina. Mastigação prolongada conduz à maior secreção salivar por meio de estímulo prolongado dos terminais na boca. A saliva é importante, porque sua função lubrificante facilita a mastigação e a deglutição do alimento ingerido. O conteúdo de mucina é particu- larmente útil por ajudar a formar adequadamente o bolo alimentar apropriado ao ato de deglutir. A farinha de varredura é seca (90% de matéria seca ou mais) e com textura de pó, de maneira que os bezerros não mastigam como fazem com o milho moído grosseiramente. Como consequência, menos saliva com alto teor de mucina é secretada, acarre- tando prejuízo na deglutição, provavelmente, levando a desencorajamento para elevar o consumo. Outro fato que pode prejudicar o consumo da farinha de varredura é o baixo teor de extrato etéreo na sua composição (menos de 1%), pois a gordura do ali- mento confere gosto e sabor. Em um sistema de produção testado pela Embrapa Gado de Leite, para o abate precoce de bezerros holandeses ao redor de 200 kg, citado em Campos & Lizieire (2000), foram utilizados concentrados cuja composição bromatológica foi semelhante à adotada neste experimento e oferecidos desde a segunda semana de idade, sempre à vontade, aos animais. Os bezerros holandeses consumiram, em média, 3,3 kg/ animal/dia, dos 71 a 120 dias de idade e 5,4 kg/animal/ dia, dos 121 aos 182 dias. Vale ressaltar que, neste sistema, não foi utilizado volumoso e, no período total (do nascimento ao abate), foram gastos 550 kg de concentrado, por bezerro. O consumo para o nível de 0% de substituição (100% milho) foi próximo ao observado, cujo concentrado foi semelhante ao utili- zado por Campos & Lizieire (2000). R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Peixoto & Warner (1993), em um experimento utilizando 64 bezerros holandeses, desmamados na sexta semana de vida, utilizando rações com 50% de milho e substituições de 50 e 100% por farinha de mandioca, também observaram diminuição no consu- mo de ração (P<0,05) para o tratamento com 100% de substituição (79,43 kg de ração da 6a a 16a semana de vida), sendo que o tratamento com milho e 50% de O menor consumo de MS registrado neste expe- R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Beze o Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses 200 recomendado um ganho de 0,45 a 0,50 kg/dia do nascimento aos 60 dias de idade. Houve redução linear para o ganho médio diário, à medida em que se aumentaram os níveis de substituição (1,03 a 0,90 kg/dia). O mesmo comportamento foi observado para o GDP1 (0,93 a 0,68 kg/dia) e para o GDP2 (1,10 a 0,89 kg/dia). No último período (GDP3), não houve efeito dos níveis de substituição do milho pela FV (P>0,05) sobre o ganho de peso vivo (1,09 kg/dia). rimento está correlacionado, principalmente, à limita- ção do fornecimento de concentrado no período final, para estimular o consumo de volumoso, e, também, à falta de dados sobre o consumo de feno. As médias e coeficientes de variação para idade e peso ao desaleitamento e no final do experimento, valores observados, equações de regressão ajustadas (ER) e coeficientes de variação (CV), para ganho médio diário total (GMD), ganho médio diário para os períodos 1 (GDP1), 2 (GDP2) e 3 (GDP3) e conversão alimentar da matéria seca (CAMS), em função dos níveis de substituição de milho pela farinha de varredura, são mostrados na Tabela 5. A redução do GMD dos animais, à medida em que se aumentou a inclusão de farinha de varredura nas dietas, foi causada pela diminuição ocorrida no CTMS, acarretando menores disponibilidades de nutrientes para os animais com maiores níveis de substituição. Isto pode ser visto na ingestão de proteína bruta (IPB), que decaiu linearmente com o aumento dos níveis. Essa menor quantidade de nutrientes ingerida deve ter limitado o ganho de peso, pela menor dispo- nibilidade destes para a utilização no crescimento. Tabela 5 - Médias e coeficientes de variação (CV) para idade e pesos ao desaleitamento e ao final do experimento, valores observados e equações de regressão ajustadas (ER) para o ganho de peso e conversão alimentar Table 5 - Means and coefficient of variation (CV) for age and weight at weaning and the end of experiment, observed values and fitted regression equations (RE) for weight gain and feed:gain ratio N = nível de substituição (%). GMD = ganho médio diário total; GDP1, GDP2 e GDP3 = ganho médio diário para o primeiro, segundo e terceiro períodos de 28 dias, respectivamente; CAMS = conversão alimentar da matéria seca. N = replacement level (%) R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Outro fator pode ter sido a diminuição gradativa da Os animais foram desaleitados com idades médias variando de 77 a 81 dias de idade. Apresentaram peso ao desaleitamento, variando de 78,0 a 81,6 kg de peso vivo. No período que precedeu ao desaleitamento, o ganho médio diário dos animais foi de 0,67 kg/dia. Este ganho foi satisfatório, pois, segundo Campos & Lizieire (2000), para a produção do bezerrão, tem sido Tabela 5 - Médias e coeficientes de variação (CV) para idade e pesos ao desaleitamento e ao final do experimento, valores observados e equações de regressão ajustadas (ER) para o ganho de peso e conversão alimentar Table 5 - Means and coefficient of variation (CV) for age and weight at weaning and the end of experiment, observed values and fitted regression equations (RE) for weight gain and feed:gain ratio Item Nível de substituição (%) CV (%) ER Replacement level (%) RE 0 25 50 75 100 Idade ao desaleitamento (dias) 79 81 77 80 81 15,3 - Weaning age (days) Peso ao desaleitamento (kg) 78,0 81,0 81,6 79,3 80,1 11,2 - Weaning weight (kg) Peso final (kg) 163,4 167,2 160,3 162,2 151,9 5,1 - Final weight (kg) Idade final (dias) 163 165 161 164 165 15,3 - Final age (days) GMD (kg/dia) 1,00 1,03 0,94 0,99 0,86 10,2 1 ADG (kg/dia) GDP1(kg/dia) 0,94 0,86 0,80 0,76 0,67 21,7 2 P1ADG (kg/day) GDP2 (kg/dia) 1,07 1,08 0,94 1,04 0,83 15,4 3 P2ADG (kg/day) GDP3(kg/dia) 0,99 1,14 1,07 1,17 1,06 18,8 Y = 1,09 P3AGD (kg/day) CAMS (kg/kg ganho) 2,9 2,9 2,9 2,7 3,0 9,7 Y = 2,9 FGRDM (kg/kg gain) 1. Y = 1,027 - 0,00131**N r2 = 0,60 2. Y = 0,934 - 0,00254**N r2 = 0,99 3. Y = 1,095 - 0,00204**N r2 = 0,61 ** (P<0,01) N = nível de substituição (%). GMD = ganho médio diário total; GDP1, GDP2 e GDP3 = ganho médio diário para o primeiro, segundo e terceiro períodos de 28 dias, respectivamente; CAMS = conversão alimentar da matéria seca. N = replacement level (%). ADG = average daily gain; P1ADG, P2ADG and P3ADG = average daily gain for the first, second and third periods, respectively; FGRDM = feed:gain ratio of dry matter. N = replacement level (%). R. Bras. Zootec., v.31, n.1, p.192-204, 2002 ADG = average daily gain; P1ADG, P2ADG and P3ADG = average daily gain for the first, second and third periods, respectively; FGRDM = feed:gain ratio of dry matter. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. 201 qualidade da proteína fornecida, com a utilização em níveis crescente de nitrogênio não protéico (uréia), com o fornecimento da farinha de varredura na formulação das dietas. Todavia, os níveis de uréia fornecidos esta- vam dentro dos limites recomendados pelo NRC (1988), para bezerros holandeses. dietas fornecidas. Com consumos semelhantes de ma- téria seca, não foi encontrada diferença entre os níveis de substituição, para o ganho de peso, estimado em 1,09 kg/dia para todos os animais. Esta mudança de compor- tamento pode ser observada na Figura 2. A CAMS não diferiu para nenhum dos níveis, apresentando valor estimado de 2,9 kg/kg e o mesmo foi observado nos três períodos. Vale ressaltar que, para o consumo de MS, foi computado apenas o concentrado. Isto confirma o CMS como fator determinante no GMD. Estes resultados estão em acordo com aqueles obtidos por Peixoto & Warner (1993), que encontraram diferenças para o consumo das rações e para ganho de peso, mas não para a conversão alimentar e concluíram que a diminuição no desenvolvimento dos animais foi devido ao menor consumo e não à menor eficiência na utilização das dietas, à base de farinha de mandioca. Pode-se confirmar o menor consumo como causa- dor da diminuição no ganho de peso, ao se avaliar este nos três períodos, separadamente, observando-se a mudança de tendência no comportamento das retas obtidas por intermédio das equações de regressão (Figura 1). No período 1, logo após o desaleitamento, o estresse da mudança de alimentação líquida para a sólida, além da substituição do milho no concentrado comercial, utilizado no período pré-desaleitamento, pela farinha de varredura nas rações experimentais, um alimento mais seco e pulverulento que o milho, reduzi- ram o consumo do concentrado. No segundo período, quando os animais estavam mais adaptados à consistên- cia da farinha de varredura, pode-se observar diminui- ção na diferença entre os ganhos de peso e entre os níveis de substituição. No período 1, a diferença entre o ganho médio diário do nível zero para o de 100% de substituição, foi 27% menor para o nível 100%. No período 2, essa diferença foi de 18,6%. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Isto pode indicar adaptação dos animais ao alimento e, também, possivel- mente, ganho compensatório. Destaca-se a avaliação do período 3, no qual os consumos de matéria seca foram semelhantes entre os níveis, pela limitação das Apesar da diminuição do ganho de peso com o incremento dos níveis de substituição da FV, o GMD obtido para o nível 100% pode ser considerado satisfatório (0,90 kg /dia). Este valor é intermediário ao encontrado em vários trabalhos na literatura. Lucci et al. (1974), citados por Lucci (1989), num experimento com 24 bezerros holandeses, conduzi- dos dos 91 aos 180 dias de idade, recebendo mistura concentrada com teor protéico de 20% mais feno de capim-gordura de baixa qualidade, à vontade, verifi- caram ganho diário de 0,70 kg/dia, atingindo-se 143 0,0 1,0 2,0 3,0 4,0 0 25 50 75 100 CTMS CMSP1 CMSP2 CMSP3 Níveis de substituição (%) Replacement levels (%) Consumo de MS (kg/dia) DM intake (kg/day) 0,60 0,70 0,80 0,90 1,00 1,10 1,20 0 25 50 75 100 GMD GDP1 GDP2 GDP3 Níveis de substituição (%) Replacement levels (%) Ganho médio diário (kg/dia) Average daily gain (kg/day) R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Figura 1 - Consumo médio total de matéria seca (CTMS) e consumos médios de matéria seca no primeiro, segundo e terceiro períodos de 28 dias (CMSP1, CMSP2 e CMSP3, respectivamente), em função dos níveis de substituição. Figure 1 - Total dry matter intake and dry matter intakes for the first, second, and third periods of 28 days, in function of the replacement levels. 0,0 1,0 2,0 3,0 4,0 0 25 50 75 100 CTMS CMSP1 CMSP2 CMSP3 Níveis de substituição (%) Replacement levels (%) Consumo de MS (kg/dia) DM intake (kg/day) Figura 2 - Ganho médio diário total (GMD) e ganhos médios diários para o primeiro, segundo e terceiro períodos de 28 dias (GDP1, GDP2 e GDP3, respectivamente), em função dos níveis de substituição. Figure 2 - Mean weight gain and average daily weight gain for the first, second and third periods of 28 days, in function of the replacement levels. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Salles & Lucci (1998) encontraram valores de ganho de peso superior aos do presente experimento, variando de 1,06 a 1,37 kg/dia, nos diferentes níveis de inclusão de monensina sódica em bezerros holandeses desaleitados precocemente. lizar, pode-se aumentar gradativamente a inclusão, até ao nível de substituição total, quando não houver mais o problema ocasionado pelo consumo. Os valores observados de hematócrito, glicose e uréia sangüínea para o período do desaleitamento (80 dias de idade) e final do experimento (164 dias de idade), coeficientes de variação e equações de re- gressão estão descritos na Tabela 6. Os valores de hematócrito, glicose e uréia sangüíneos não diferiram no período do desaleitamento e no período relativo ao final do experimento. A inclusão de farinha de varredura não influenciou os parâmetros sangüíneos estudados. Verifica-se que os valores obtidos para os parâmetros sangüíneos, com relação à época da colheita, foram maiores (P<0,01) na desmama que no final do experimento, tanto para hematócrito (30,7 e 26,0%), para glicose (93,2 e 82,6 mg/100 mL) e para uréia (22,4 e 11,2 mg/100 mL). As recomendações da Embrapa (Campos & Lizieire, 2000) para a produção do bezerrão são que os animais atinjam ganho entre 0,90 e 1,00 kg/dia, na fase entre 60 a 120 dias de idade e de 1,20 a 1,30 kg/ dia, de 121 a 180 dias de idade. Ainda relataram resultados em que os animais apresentaram ganhos médios diários de 0,45 kg, do nascimento aos 70 dias de idade, 0,99 kg dos 71 aos 120 dias de idade, e 1,20 kg dos 121 aos 182 dias de idade, quando foram abatidos, em média, com 196 kg de peso vivo. Os ganhos de 0,67 kg/dia na fase pré experimental, e de 0,93 e 1,10 kg/dia para o nível 0% para os períodos 1 e 2, estão em acordo com os dados e recomenda- ções da Embrapa. O ganho de peso dos animais do período 3 foi baixo, o que foi devido à limitação no fornecimento de concentrado para os animais, não permitindo, assim, a expressão de maiores ganhos. Barnes et al. (1985) encontraram valores de 10,3 mg/100 mL para uréia e 60,6 mg/100 mL para glicose, em bezerras Holandesas, com seis meses de idade e alimentadas com rações com 15,5% de proteína bruta. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 0,60 0,70 0,80 0,90 1,00 1,10 1,20 0 25 50 75 100 GMD GDP1 GDP2 GDP3 Níveis de substituição (%) Replacement levels (%) Ganho médio diário (kg/dia) Average daily gain (kg/day) 0,60 0,70 0,80 0,90 1,00 1,10 1,20 0 25 50 75 100 GMD GDP1 GDP2 GDP3 Níveis de substituição (%) Replacement levels (%) Ganho médio diário (kg/dia) Average daily gain (kg/day) 0,60 0,70 0,80 0,90 1,00 1,10 1,20 0 25 50 75 100 GMD GDP1 GDP2 GDP3 Níveis de substituição (%) Replacement levels (%) Ganho médio diário (kg/dia) Average daily gain (kg/day) 0,0 1,0 2,0 3,0 4,0 0 25 50 75 100 CTMS CMSP1 CMSP2 CMSP3 Níveis de substituição (%) Replacement levels (%) Consumo de MS (kg/dia) DM intake (kg/day) Níveis de substituição (%) Replacement levels (%) R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Figura 1 - Consumo médio total de matéria seca (CTMS) e consumos médios de matéria seca no primeiro, segundo e terceiro períodos de 28 dias (CMSP1, CMSP2 e CMSP3, respectivamente), em função dos níveis de substituição. Figure 1 - Total dry matter intake and dry matter intakes for the first, second, and third periods of 28 days, in function of the replacement levels. Figura 2 - Ganho médio diário total (GMD) e ganhos médios diários para o primeiro, segundo e terceiro períodos de 28 dias (GDP1, GDP2 e GDP3, respectivamente), em função dos níveis de substituição. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 o Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Beze 202 kg aos 180 dias. Araújo et al. (1998), estudando níveis de volumoso na dieta de 10 a 55%, para animais de 60 a 180 dias de idade, obtiveram valores médios, vari- ando de 1,01 a 0,71 kg/dia. Neste mesmo trabalho, o GMD para os primeiros 28 dias pós desaleitamento, variou de 0,94 a 0,66 kg/dia, de 0,82 kg/dia para o segundo período e 0,81 kg/dia para o terceiro. Estes autores ainda concluíram que o ganho médio geral de peso vivo, obtido no experimento (0,87 kg/dia), foi bom. Signoretti et al. (1998) obtiveram valores de ganho de peso variando de 1,23 a 0,92 kg/dia. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Todavia, os valores observados no presente experimento foram superiores aos de Park (1985), que utilizou bezerros holandeses com 100 kg de peso vivo submetidos a uma dieta com 12% de proteína bruta e encontrou 8,0 mg de uréia/100 mL e 75,8 mg de glicose/ 100 mL. Diaz-Castaneda & Brisson (1989) observa- ram, em bezerros holandeses com aproximadamente 60 dias de idade, alimentados com rações contendo 28,7% de proteína bruta, níveis de uréia variando de 11 a 18 mg/100 mL e de glicose de 65 a 85 mg/100 mL. Neathery et al. (1991) encontraram valores de 32,4% de hematócrito e 102 mg de glicose/100 mL e 11,2 mg uréia/100 mL em bezerros holandeses alimen- tados com rações com 21,0% de proteína bruta, com 124 kg de peso vivo e 1,18 kg/dia de ganho de peso. Com base na literatura citada, os ganhos de peso obtidos para todos os níveis de substituição do milho pela farinha de varredura podem ser considerados satisfatórios. O que determinará o nível a ser utilizado dependerá dos preços de mercado do milho e da disponibilidade da farinha de varredura, que é um resíduo industrial e não está disponível em todas as regiões, muitas vezes, não sendo encontrada em quantidades significativas. Outro fator a ser conside- rado é o planejamento do ganho de peso dos animais e, a partir de ganhos alvo, escolher o melhor nível ou a melhor época de utilização, pois é necessário uma adaptação por parte dos animais ao consumo da farinha. O aumento gradativo dos níveis de inclusão de farinha de varredura, logo após ao desaleitamento, também é uma alternativa viável, começando com níveis menores. À medida que o consumo se estabi- Os valores observados estão de acordo com Matsuzaki et al. (1997), que determinaram valores de 80 mg glicose/100 mL e 9 mg uréia/100 mL, em bezerros holandeses com 180 dias de idade e peso vivo de 200 kg, alimentados com concentrado à base de 1% do peso vivo, com 16,6% de PB e 73,0% de NDT e feno de gramínea. Os níveis de glicose e uréia sangüíneas são con- siderados por refletir a quantidade de amido e prote- ína ingeridos ou a relação entre estes nutrientes consumidos (Blowey et al., 1973; citados por Matsuzaki et al., 1997). Esta afirmação, possivelmen- R. Bras. Zootec., v.31, n.1, p.192-204, 2002 JORGE et al. JORGE et al. Literatura Citada te, explica a não influência dos níveis de substituição da FV, nos teores de glicose e uréia sanguíneos, pois o consumo de amido não diferiu entre os tratamentos. ARAÚJO, G.G.L.; COELHO DA SILVA, J.F.; VALADARES FILHO et al. Ganho de peso, conversão alimentar e caracte- rísticas da carcaça de bezerros alimentados com dietas con- tendo diferentes níveis de volumoso. Revista Brasileira de A diminuição nos níveis de uréia do desaleitamento para o período do final do experimento pode ter sido relacionada com os níveis protéicos a que os animais estavam submetidos, pois, até o desaleitamento, es- tes receberam concentrado comercial com 20,7% de proteína bruta, enquanto, no período final do experi- mento, o concentrado continha 13,5% (Tabela 3). Zootecnia, v.27, n.5, p.1006-1012, 1998. BARNES, M.A.; KAZMER. G.W.; AKERS, R.M. et al. Influence of selection for milk yield on endogenous hormones and metabolites in Holstein heifers and cows. Journal of Animal Science, v.60, n.1, p.271-284. Science, v.60, n.1, p.271-284. CALDAS NETO, S.F.; ZEOULA, L.M.; PRADO, I.N. et al. Degradabilidade ruminal de concentrados compostos com milho, raspa de mandioca e resíduos das farinheiras. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 37, 2000. Viçosa. Anais... Viçosa: Sociedade Brasileira de Zootecnia, 2000b, p.378. CALDAS NETO, S.F.; ZEOULA, L.M.; PRADO, I.N. et al. Degradabilidade ruminal de concentrados compostos com milho, raspa de mandioca e resíduos das farinheiras. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE R. Bras. Zootec., v.31, n.1, p.192-204, 2002 203 Tabela 6 - Valores observados de hematócrito, glicose e uréia sanguíneo para o período do desaleitamento e final do experimento, coeficientes de variação e equações de regressão Table 6 - Values of blood hematocrit, glucose and urea at weaning and in the end of experiment, coefficient of variation and fitted regression equations Período (dias de idade) Nível de substituição (%) CV(%) ER Period (days of age) Replacement level (%) RE 0 25 50 75 100 Hematócrito (%) Hematocrit Desaleitamento (80 dias) 31,9 29,6 29,9 31,7 30,5 10,5 Y=30,7a Weaning (80 days) Final do experimento (164 dias) 25,9 27,7 27,0 25,1 24,5 12,8 Y=26,0b End of the experiment (164 days) Glicose (mg/100 mL plasma) Glucose Desaleitamento (80 dias) 97,9 95,9 92,3 88,0 91,9 11,6 Y=93,2a Weaning (80 days) Final do experimento (164 dias) 89,1 86,3 76,0 82,9 78,9 16,4 Y=82,6b End of the experiment (164 days) Uréia (mg/100 mL plasma) Urea Desaleitamento (80 dias) 21,0 17,4 29,7 20,7 23,0 58,7 Y=22,4a Weaning (80 days) Final do experimento (164 dias) 11,1 9,7 11,4 8,4 15,1 66,5 Y=11,2b End of the experiment (164 days) Médias nas mesmas colunas para cada parâmetro, seguidas de letras diferentes diferem (P<0,01) pelo teste Tukey. 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Logan: International Feedstuffs Institute, Utah Agricultural Experimental Station, Utah State University, 1982. 271p. PRADO, I.N. Substituição gradativa do leite integral de vaca pelo leite de soja com adição de 3% de gordura de porco no aleitamento artificial de bezerros holandesados. Lavras: Universidade Federal de Lavras, 1981. Dissertação (Mestrado em Zootecnia) - Universidade Federal de Lavras, 1981. LUCCI, C.S. Bovinos leiteiros jovens. São Paulo: Nobel, 1989. 371p. SALLES, M.S.V.; LUCCI, C.S. Conclusões Monensina para bezerros rumi- nantes em crescimento acelerado. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 33., 1998, Botucatu. Anais... Botucatu: Sociedade Brasileira de Zootecnia, 1998. p.446-448. MARQUES, J.A.; PRADO, I.N.; ZEOULA, L.M. et al. Avali- ação da mandioca e seus resíduos industriais em substituição ao milho no desempenho de novilhas confinadas. Revista Brasileira de Zootecnia, v.29, n.5, p.1528-1536, 2000. MARQUES, J.A.; PRADO, I.N.; ZEOULA, L.M. et al. Avali- ação da mandioca e seus resíduos industriais em substituição ao milho no desempenho de novilhas confinadas. Revista SIGNORETTI, R.D.; COELHO DA SILVA, J.F.; VALADARES FILHO, S.C. et al. Consumo e digestibilidade aparente, em bezerros holandeses alimentados com dietas contendo dife- rentes níveis de volumoso. In: REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE ZOOTECNIA, 33., 1998. Botucatu. Anais... Botucatu: Sociedade Brasileira de Zootecnia, 1998. p.422-424. MARTINS, A.S.; ZEOULA, L.M.; PRADO, I.N. et al. Degradabilidade ruminal in situ da matéria seca e proteína bruta das silagens de milho e sorgo e de alguns alimentos concentrados. Revista Brasileira de Zootecnia, v.28, n.5, p.898-905, 1999. MATSUZAKI, M.; TAKIZAWA, S.; OGAWA, M. Plasma insulin, metabolite concentrations, and carcass characteristics of Japanese Black, Japanese Brown, and Holstein steers. Journal of Animal Science, v.75, p.3287-3293, 1997. SILVA, D.J. Análise de alimentos: métodos químicos e bioló- gicos. Viçosa: Universidade Federal de Viçosa, 1990. 165p. MELLOTI, S.D. Contribuição para o estudo da composição química e valor nutritivo dos resíduos da industrialização da mandioca, Manihot utilissima, POHL., no Estado de São Paulo. Boletim de Indústria Animal, v.29, p.339-349, 1972. SNIFFEN, C.J.; O’CONNOR, J.D.; VAN SOEST, P.J. et al. A net carbohydrate and protein system for evaluating cattle diets II. Carbohydrate and protein availability. Journal of Animal Science, v.70, n.11, p.3562-3577. UNIVERSIDADE FEDERAL DE VIÇOSA - UFV. SAEG - Sistema de análises estatísticas e genéticas. Viçosa, MG: 1983. (Manual do usuário). NATIONAL RESEARCH COUNCIL - NRC. Nutrient requirements of dairy cattle. Washington, D.C., 1988. 158p. NEATHERY, M.W.; CROWE, C.T.; HARTNELL, G.F. et al. Effects of sometribove on performance, carcass composition, and chemical blood characteristics of dairy calves. Journal of Dairy Science, v.74, p.3933-3939, 1991. Van SOEST, P.J. Nutritional ecology of ruminant. New York: Cornell University Press, 1994. 476p. PEIXOTO, R.R.; WARNER, R.G. Avaliação da farinha de mandioca como componente de rações para terneiros leiteiros e desaleitamento precoce. Revista Brasileira de Mandioca, v.12, n.1/2, p.39-47, 1997. PARK, C.S. Influence of dietary protein on blood cholesterol and related metabolites of growing calves. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 Conclusões Journal of Animal Science, v.61, n.4, p.924-930, 1985. Recebido em: 31/10/00 Aceito em: 06/08/01
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Topological frustration of artificial spin ice
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ARTICLE Received 3 May 2016 | Accepted 21 Nov 2016 | Published 13 Jan 2017 Received 3 May 2016 | Accepted 21 Nov 2016 | Published 13 Jan 2017 NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 1 Department of Physics, University of Maryland, College Park, Maryland 20742, USA. 2 Department of Materials Science and Engineering, University of Maryland, College Park, Maryland 20742, USA. Correspondence and requests for materials should be addressed to J.C. (email: cumings@umd.edu). Topological frustration of artificial spin ice Jasper Drisko1, Thomas Marsh2 & John Cumings2 Frustrated systems, typically characterized by competing interactions that cannot all be simultaneously satisfied, display rich behaviours not found elsewhere in nature. Artificial spin ice takes a materials-by-design approach to studying frustration, where lithographically patterned bar magnets mimic the frustrated interactions in real materials but are also amenable to direct characterization. Here, we introduce controlled topological defects into square artificial spin ice lattices in the form of lattice edge dislocations and directly observe the resulting spin configurations. We find the presence of a topological defect produces extended frustration within the system caused by a domain wall with indeterminate configuration. Away from the dislocation, the magnets are locally unfrustrated, but frustration of the lattice persists due to its topology. Our results demonstrate the non-trivial nature of topological defects in a new context, with implications for many real systems in which a typical density of dislocations could fully frustrate a canonically unfrustrated system. 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 level of control and lack of direct characterization in these studies leave numerous unanswered questions. D islocations are topological defects1 ubiquitous in crystalline materials that can cause a diverse range of phenomena across vastly different systems. Examples of these phenomena include: theoretically predicted ferromagnetic dislocations in an antiferromagnetic lattice2,3, which have recently been experimentally observed in antiferromagnetic NiO4; one-dimensional fermionic excitations in topological insulators5; plasticity in metallic alloys6; and a recent report on confined structural states at dislocations in an FeMn alloy7. The topological nature of dislocations means their presence can be measured far away from the actual defect site by following a closed loop around the dislocation core, resulting in displacement by a lattice constant and requiring an extra vector to complete the loop, known at the Burgers vector. While non-topological defects such as vacancies or substitutions can impart useful and favourable properties for crystals in, for example, semi- conductor engineering, the presence of such a defect is generally unknown in parts of the material far removed from it. However, the non-trivial presence of even a single dislocation could produce long-range topological effects that permeate the crystal. Typical dislocation densities in metals are on the order of 105–1012 cm2, while in ceramics they can be much lower, as low as 104–106 cm2 (ref. 8). For this reason, dislocations generally receive less attention in ceramic materials. Topological frustration of artificial spin ice The topological nature of dislocations means their presence can be measured far away from the actual defect site by following a closed loop around the dislocation core, resulting in displacement by a lattice constant and requiring an extra vector to complete the loop, known at the Burgers vector. While non-topological defects such as vacancies or substitutions can impart useful and favourable properties for crystals in, for example, semi- conductor engineering, the presence of such a defect is generally unknown in parts of the material far removed from it. However, the non-trivial presence of even a single dislocation could produce long-range topological effects that permeate the crystal. Typical dislocation densities in metals are on the order of 105–1012 cm2, while in ceramics they can be much lower, as low as 104–106 cm2 (ref. 8). For this reason, dislocations generally receive less attention in ceramic materials. D q Here, we present a controlled and direct study of the topological frustration, specifically due to topological defects, in otherwise ideal crystals, where our use of the term topological frustration follows that of Araki et al.29. We introduce controlled topological defects in the form of edge dislocations into thermally active square ASI systems and directly observe the resulting spin configurations on annealing. We observe that the system creates large regions of ground-state antiferromagnetic order, as expected for the square ice geometry. However, the dislocations create domain walls that break this order, and their locations and configurations are not constrained, giving a source of entropy among the observed results. The individual plaquettes of the system are locally unfrustrated, but nevertheless the whole structure remains frustrated due to its topology, leading to a high number of low lying energetic states. Schematics of topological frustration as well as other, more commonly studied types of frustration are shown in Fig. 1a–c. Results One-dislocation crystals. Square ASI nanomagnet arrays were originally created to model the six-fold vertex degeneracy in pyrochlore spin ice9,16. The system has proven an excellent way to study frustration and its effects, but falls slightly short of a completely analogous model to the pyrochlores due to the inequivalent interaction strengths between the four magnets at each vertex. The variation in the distance between adjacent and opposite spins at each vertex lifts the six-fold degeneracy of the ice-rule state and leads to a ground state given by a perfect tiling of type I vertices (Fig. 1g), which is unique up to a global reversal of spins. We have previously demonstrated perfect ground-state ordering in square ASI samples fabricated from thin films of FePd3 (ref. 21). The samples show ground-state order after an annealing procedure where they are heated above the Curie temperature (TC) of the FePd3 (B150 C) and cooled slowly to room temperature at a rate of 1 C min  1. Our observation of large domains of ground-state ordering in the square geometry is robust and highly reproducible, demonstrating the benefits of FePd3 as a material system for ASI studies. In this work, we have modified the perfect square geometry by introducing edge dislocations into square ASI (see ‘Methods’ section). Figure 1e presents a transmission electron microscopy (TEM) image of a dislocation in our samples. A Burgers circuit (Fig. 1d) is drawn schematically around the defect site to illustrate its topological nature. Samples are heated to 165 C in an Ar atmosphere and cooled at a rate of 1 C min  1, similar to our previous studies21. We image our samples using Lorentz TEM19 (Fig. 1f) and utilize digital image processing to analyse the resulting images (Fig. 1h,i). We fabricate samples with 36 distinct dislocation geometries, 6 geometries with one dislocation and 30 with two dislocations, and pattern up to 16 crystals of each geometry onto each substrate (for full fabrication details, see ‘Methods’ section). Our main results are summarized in Fig. 1i. We find that the Our main results are summarized in Fig. 1i. We find that the dislocation samples show large domains of ground-state ordering, but always have a chain of type II and type III higher energy vertices originating from the dislocation point. These chains are required because of the topological nature of the dislocation. NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications Topological frustration of artificial spin ice A class of ceramics that has attracted considerable recent scientific interest are the spin ices, which have been shown to exhibit geometric frustration9,10 in the magnetic moments of rare earth atoms on the pyrochlore lattice of corner sharing tetrahedra, analogous to Pauling’s description of hydrogen disorder in hexagonal water ice at low tempe- ratures11. A finite residual entropy at low temperature, resulting from a macroscopically degenerate ground state, is characteristic of these fully frustrated systems. Despite measurements confirming a residual entropy in spin ice9 and implying a disordered and degenerate low-temperature state, a unique ground state has nevertheless been predicted, based on long- range dipolar interactions in the system12. However, this ordered state has proven difficult to observe experimentally9,13 and may be complicated by a competing ground state, based on exchange interactions14. Notably, one recent study did observe a partial recovery of entropy, below the Pauling value, for single crystal samples of Dy2Ti2O7 thermally equilibrated for very long-time scales15. These considerations are of course influenced by the crystalline perfection of the materials, the nature and density of defects, and the precise configuration of spins around these defects—details that are challenging to experimentally determine or control. Artificial spin ices (ASIs) present a possible pathway to resolving these problems through lithographically patterned two dimensional (2D) arrays of nanoscale bar magnets16–23, in which the precise structure of the lattice may be controlled by design and the interactions are well-described by a dipolar model23–26. The ordering principles of ASI have been studied through demagnetizing protocols or, more recently, through thermal activation and annealing17,18,20–22. Thermal activation has been successful in observing theoretically predicted long-range ordered states in both the square and kagome ASI geometries17,21,23, and it is a promising direction for future studies. An important consideration in the relationship between structure and frustration that has received relatively little attention is the presence of topological str ct ral defects D islocations are topological defects1 ubiquitous in crystalline materials that can cause a diverse range of phenomena across vastly different systems. Examples of these phenomena include: theoretically predicted ferromagnetic dislocations in an antiferromagnetic lattice2,3, which have recently been experimentally observed in antiferromagnetic NiO4; one-dimensional fermionic excitations in topological insulators5; plasticity in metallic alloys6; and a recent report on confined structural states at dislocations in an FeMn alloy7. Results A closed path around the defect is topologically altered, making it impossible to support continuous ground-state order, and thus a frustrated chain must be present. The chains in the single- dislocation geometries always continue to an edge of the finite crystal, and a representative example is shown in Fig. 2, displaying the extended frustration of the system. Other domains and domain walls may be present in the sample as are seen in An important consideration in the relationship between structure and frustration that has received relatively little attention is the presence of topological structural defects. Topological influences on frustration have been studied in protein folding27, in the carbon bonding of graphene nano- flakes28, in nematic liquid crystals29, and in spin configurations at the (001) surface of antiferromagnetic Cr30,31. The work in refs 30,31 is the most relevant to our results as it is related to topological effects in ordered magnetic systems. However, the NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 a c f g h b d AF FM FM FM Type I = Type II = Type III = Type IV = (None observed) i AF AF AF e b b Figure 1 | Topological frustration. (a–c) Schematics of different origins of frustration. (a) Disorder frustration: disordered bonds between Ising spins on a square lattice cannot be satisfied with any spin configuration, leading to frustration. (b) Geometric frustration: antiferromagnetically (AF) coupled Ising spins on a triangular lattice are frustrated due to the geometry of the system. (c) Topological frustration. Ferromagnetic (FM) spins perpendicular to a Mobius strip are locally unfrustrated, but the system is still frustrated due to its topology. (d) Burgers circuit and vector around an edge dislocation. A loop around the dislocation (red arrows) with an equal number of lattice constants in each direction cannot be completed without the addition of the Burgers vector (green arrow). Also shown is the conventional ‘T’ notation (green) representing the dislocation location and direction. (e) In-focus TEM image of a dislocation point defect in square ASI. The Burgers circuit and vector are overlaid in the image. (f) Lorentz contrast TEM image of the same defect site as in e. The asymmetry in contrast across each magnet indicates the direction of its magnetization. For more details see ref. 9. Scale bar, 500 nm. Results There are 16 possible configurations for 4 spins meeting at each vertex in the square lattice that are divided into four vertex types I–IV with increasing energy. A characteristic spin configuration is given for each type, with the remaining configurations enumerated by four-fold rotation and global spin flip symmetry operations. Two and three spin vertices are simply mapped onto the corresponding four-spin type, excluding the highest energy, type IV vertex because we do not observe any in our samples. (h) Lorentz contrast TEM image of a sample that has been annealed with arrows overlaid indicating the direction of each macro spin. (i) Vertex map of the same image in h representing the same information, but with the domain wall clearly defined by the type II and type III vertices in a single domain of ground-state order. Scale bar, 2 mm. studies of canonical square ASI17, but one of these chains must always begin at the dislocation point. Other than this, the frustrated chains and conventional domain walls are locally indistinguishable, exhibiting the same basic phenomena. The lowest energy configuration for either would be the shortest possible, straight chain of only type II vertices, extending in a o114 direction, but this is not generally observed. Type III vertices are almost always present in both the frustrated chains and the conventional domain walls likely because the type III vertices play an essential role in domain wall motion in the square ice lattice22. In addition, since a frustrated wall’s presence is required by topology, there is only a fractionally small change in its total energy to include type III vertices and take a longer, meandering path through the lattice. It appears that both such walls are at least partly entropically driven, as opposed to purely energetically driven, as the lowest energy configurations do not dominate. We note that a vacancy, a single missing magnet without lattice distortion, is not a topological defect and does not induce frustration or nucleate a domain wall; a square ASI lattice can still display perfect order with a single spin removed (Supple- mentary Fig. 1). In addition, we observe that repeated annealing of the same crystal results in different domain configurations (Supplementary Fig. 2). Finally, topological frustration is distinct from vertex frustration, which is found, for example, in the shakti lattice20 because vertex frustration is only due to the geometry of the system, not its topology. Two-dislocation crystals. Results (g) Vertex types in square ASI and the legend for vertex map images in i and throughout the manuscript. There are 16 possible configurations for 4 spins meeting at each vertex in the square lattice that are divided into four vertex types I–IV with increasing energy. A characteristic spin configuration is given for each type, with the remaining configurations enumerated by four-fold rotation and global spin flip symmetry operations. Two and three spin vertices are simply mapped onto the corresponding four-spin type, excluding the highest energy, type IV vertex because we do not observe any in our samples. (h) Lorentz contrast TEM image of a sample that has been annealed with arrows overlaid indicating the direction of each macro spin. (i) Vertex map of the same image in h representing the same information, but with the domain wall clearly defined by the type II and type III vertices in a single domain of ground-state order. Scale bar, 2 mm. a d AF FM FM FM c c a d b f d h h i Type III Type IV (None observed) Figure 1 | Topological frustration. (a–c) Schematics of different origins of frustration. (a) Disorder frustration: disordered bonds between Ising spins on a square lattice cannot be satisfied with any spin configuration, leading to frustration. (b) Geometric frustration: antiferromagnetically (AF) coupled Ising spins on a triangular lattice are frustrated due to the geometry of the system. (c) Topological frustration. Ferromagnetic (FM) spins perpendicular to a Mobius strip are locally unfrustrated, but the system is still frustrated due to its topology. (d) Burgers circuit and vector around an edge dislocation. A loop around the dislocation (red arrows) with an equal number of lattice constants in each direction cannot be completed without the addition of the Burgers vector (green arrow). Also shown is the conventional ‘T’ notation (green) representing the dislocation location and direction. (e) In-focus TEM image of a dislocation point defect in square ASI. The Burgers circuit and vector are overlaid in the image. (f) Lorentz contrast TEM image of the same defect site as in e. The asymmetry in contrast across each magnet indicates the direction of its magnetization. For more details see ref. 9. Scale bar, 500 nm. (g) Vertex types in square ASI and the legend for vertex map images in i and throughout the manuscript. NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications Results The second is the transition from 1 to 5 lattice spacings shows the emergence of defect independence and at around a separation of 5 lattice constants, the system enters a regime of nearly inde- pendent defects. Overall, we find the presence of a domain wall at a dislocation to be extremely robust. We studied five samples fabricated from different depositions of FePd3 and collected over 1,600 images of annealed crystals, and in every image, there is a domain wall originating from every dislocation and connecting to either the edge of the crystal or to another dislocation. inclusion of two topological defects actually allows for the pos- sibility of a larger continuous ground-state domain (Fig. 3b). From our observations, we compute the probability of domain walls not connecting dislocations in our samples for 30 different two-dislocation geometries (Supplementary Fig. 3) and plot the probability versus the distance between the two dislocations in Fig. 4. An annotated version of Fig. 4 indicating the geometries that contribute to each data point is found in Supplementary Fig. 3h. We find that for a spacing of 410 lattice constants, it is extremely rare for a domain wall to connect the two dislocations. At spacings of 5–10 lattice constants, there is still a low prob- ability for a domain wall to span the defects. At 5 lattice constants apart, there is a sharp decrease down to 1 lattice constant spacing. Two points should be noted about this behaviour. The first is that even when the defects are 1 lattice constant apart, there is still a reasonable rate for the two defects to behave independently and nucleate completely separate domain walls. The second is the transition from 1 to 5 lattice spacings shows the emergence of defect independence and at around a separation of 5 lattice constants, the system enters a regime of nearly inde- pendent defects. Overall, we find the presence of a domain wall at a dislocation to be extremely robust. We studied five samples fabricated from different depositions of FePd3 and collected over 1,600 images of annealed crystals, and in every image, there is a domain wall originating from every dislocation and connecting to either the edge of the crystal or to another dislocation. Figure 3 | Vertex map of crystals with two topological defects. Lorentz contrast TEM images with vertex types indicated. Results For crystals with two dislocations, the two defects generally have separate domain walls that each meander to the edges of the crystal (Fig. 3a). Occasionally, a single domain wall will connect the two dislocations, as the 3 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 a b gure 3 | Vertex map of crystals with two topological defects. Lorentz ontrast TEM images with vertex types indicated. (a) Domain walls begin at ch dislocation and end at the edges of the crystal. (b) A single domain all connects the two dislocations. Inset shows a schematic representation the dislocation locations and direction. Scale bar, 2 mm. Figure 2 | Vertex map of a crystal with one topological defect. Lorentz contrast TEM image with vertex types indicated as in Fig. 1g. A meandering vertex chain originates from the dislocation among a few ground-state domains separated by traditional domain walls. Inset: sample schematic showing location and direction of dislocation. Scale bar, 2 mm. a a Figure 2 | Vertex map of a crystal with one topological defect. Lorentz contrast TEM image with vertex types indicated as in Fig. 1g. A meandering vertex chain originates from the dislocation among a few ground-state domains separated by traditional domain walls. Inset: sample schematic showing location and direction of dislocation. Scale bar, 2 mm. b b inclusion of two topological defects actually allows for the pos- sibility of a larger continuous ground-state domain (Fig. 3b). From our observations, we compute the probability of domain walls not connecting dislocations in our samples for 30 different two-dislocation geometries (Supplementary Fig. 3) and plot the probability versus the distance between the two dislocations in Fig. 4. An annotated version of Fig. 4 indicating the geometries that contribute to each data point is found in Supplementary Fig. 3h. We find that for a spacing of 410 lattice constants, it is extremely rare for a domain wall to connect the two dislocations. At spacings of 5–10 lattice constants, there is still a low prob- ability for a domain wall to span the defects. At 5 lattice constants apart, there is a sharp decrease down to 1 lattice constant spacing. Two points should be noted about this behaviour. The first is that even when the defects are 1 lattice constant apart, there is still a reasonable rate for the two defects to behave independently and nucleate completely separate domain walls. NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications Results (a) Domain walls begin at each dislocation and end at the edges of the crystal. (b) A single domain wall connects the two dislocations. Inset shows a schematic representation of the dislocation locations and direction. Scale bar, 2 mm. P 1.0 0.8 0.6 0.4 0.2 0.0 Distance between dislocations (a) P of domain walls not connecting dislocations 0 5 10 15 20 25 30 Figure 4 | Probability of domain walls not connecting dislocations. Probability of domain walls not connecting dislocations versus distance between dislocations in crystals with two topological defects. Some data points include two or three geometries of equal distance apart. Distances are expressed in units of the lattice constant aE500 nm. Error bars are one s.d. and are calculated from counting statistics. Each geometry includes B40 crystals. P 1.0 0.8 0.6 0.4 0.2 0.0 Distance between dislocations (a) P of domain walls not connecting dislocations 0 5 10 15 20 25 30 Numerical modelling. We also perform simulations of our system to gain greater understanding of the behaviour. We use a kinetic Monte Carlo technique, which is known to be an accurate method for modelling thermal ASI systems18,21, and we include a small amount of disorder in the widths of individual magnets in the model (see ‘Methods’ section). These variations in the samples are due to artifacts from lithography in fabrication and have shown to be important in matching with previous experimental results21. We find remarkably similar behaviour between our simulations and TEM data, where the dislocation points always terminate a domain wall, but they are surrounded by large domains of ground-state order. Monte Carlo results also show domain walls separating spontaneous ordinary domains and Figure 4 | Probability of domain walls not connecting dislocations. g | y g Probability of domain walls not connecting dislocations versus distance between dislocations in crystals with two topological defects. Some data points include two or three geometries of equal distance apart. Distances are expressed in units of the lattice constant aE500 nm. Error bars are one s.d. and are calculated from counting statistics. Each geometry includes B40 crystals. occasionally connecting two dislocations, consistent with our experimental observations. Results We also note that some simulations (not shown) with less disorder tend to produce lattices with larger regions of perfect ground-state ordering and fewer canonical NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 would result in an extended 2D surface of frustration of the system. domain walls. The simulations shown here include a representative amount of disorder for our experimental crystals, though naturally some experimental crystals will have higher or lower degrees of disorder and will display correspondingly larger or smaller regions of ground-state order surrounding the topologically required domain walls. We show the steady state behaviour of some of our simulations in Fig. 5 and movies of the evolution of the simulations in the Supplementary Movies 1 and 2. In the movies, the lattices start in a randomized state and achieve most of their ordering relatively quickly, first eliminating the highest energy type IV vertices and progressing to mainly type I ground-state order. Once the sample has reached a ground-state domain-dominated phase, the domain walls slowly fluctuate and move around, often shrinking some of the smaller domains. In the domain wall motion, we note fluctuations of type III vertices very similar to previous Monte Carlo simulations of perfect square geometry ASI22. We note that topological frustration, while it derives from crystalline disorder, is fundamentally different from standard disorder-induced frustration. Here, the system shows extended frustration as the topology of the defect causes long-range effects in the lattice. In 2D ASI, the dislocation is a 0D point defect, but it promotes itself into a 1D line of frustration. In a 3D material, dislocations are 1D defects, and this domain walls. The simulations shown here include a representative amount of disorder for our experimental crystals, though naturally some experimental crystals will have higher or lower degrees of disorder and will display correspondingly larger or smaller regions of ground-state order surrounding the topologically required domain walls. We show the steady state behaviour of some of our simulations in Fig. 5 and movies of the evolution of the simulations in the Supplementary Movies 1 and 2. In the movies, the lattices start in a randomized state and achieve most of their ordering relatively quickly, first eliminating the highest energy type IV vertices and progressing to mainly type I ground-state order. Results Once the sample has reached a ground-state domain-dominated phase, the domain walls slowly fluctuate and move around, often shrinking some of the smaller domains. In the domain wall motion, we note fluctuations of type III vertices very similar to previous Monte Carlo simulations of perfect square geometry ASI22. We note that topological frustration, while it derives from crystalline disorder, is fundamentally different from standard disorder-induced frustration. Here, the system shows extended frustration as the topology of the defect causes long-range effects in the lattice. In 2D ASI, the dislocation is a 0D point defect, but it promotes itself into a 1D line of frustration. In a 3D material, dislocations are 1D defects, and this ARTICLE [ ] a q 2π = –— 0 0 1 q · b = –π q · b = –π Figure 6 | Dislocations in pyrochlore spin ice. (a) Reduced unit cell showing the locations of the rare earth atoms and their magnetic moment directions in the possible q ¼ (2p/a)[001] dipolar ground state. This state has alternating planes of ordered tetrahedra, here coloured red and blue, corresponding to the planes of ordering in (b). (b) Schematic showing how a dislocation in pyrochlore spin ice could disrupt ground-state ordering. Each cube is 1/8 the pyrochlore unit cell shown in a. This example has a dislocation with a Burgers vector (shown as a white arrow) in the (a/2)[101] direction, which is a primitive basis vector for the FCC parent structure of the pyrochlore lattice so that this does not cause any structural mismatch away from the dislocation. This dislocation does create a domain wall in the q ¼ (2p/a)[001] ground-state order parameter, highlighted in yellow. The location of this domain wall is arbitrary, but it must be present. Note that q . ba2pn and thus the system is topologically frustrated. Supplementary Movies 1 and 2, and related freezing phenomena are also observed in pyrochlore spin ices34,35. Despite this, domain walls separating ground-state regions should still carry an additional energy per unit area, producing a surface tension that would tend to minimize the significance of the domain walls, in the limiting case producing a single configuration with only minimal entropy. If the system remains sufficiently dynamic, the mere presence of dislocations may thus not prevent the formation of extended ground-state regions, separated by discrete domain walls. Since the domain walls carry a p phase shift of the order parameter (an antiphase boundary), the domain walls may simply find a minimum-energy configuration connecting two nearby dislocations. On the other hand, if the system is otherwise frozen, even a dislocation-free crystal may still show residual entropy without long-range order. Thus, the role of dislocations is not necessarily simple. However, we point out that crystalline dislocations—since they are topological defects—behave differe- ntly in three dimensions than in the 2D systems we study here. A recent and thorough analysis of Dy2Ti2O7 results speculates that random disorder, in the form of stuffed spins or oxygen vacancies, could be the cause of discrepancies between various specific heat measurements on the material14. NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications ARTICLE a a a [ [ ] ] a a b b q q · b = –π 2 2π = = – –— 1 0 0 1 0 –1 a a the single crystal in ref. 15 could be showing a slight emergence of the ground state because it contains far fewer topological defects. However, it is important to note that larger single crystals maintain lattice coherence over greater distances and are thus actually more susceptible to topological frustration. As an example, a cubic crystal of side length N unit cells and volume N3 unit cells could need only on the order of Nd ¼ N dislocations to be fully frustrated, if the emanating frustrated surfaces are non-interacting, similar to the domain walls we observe in our specimens—we discuss this in more detail below. Under this assumption, the dislocation density would be rd ¼ Nd/N2, and the density required to frustrate a system scales as 1/N, implying that larger single crystals are actually more likely to be impacted by the presence of dislocations than smaller crystals. This is counter to the conventional expectation that larger single crystals show more intrinsic ground-state behaviours. On the contrary, smaller single crystals, or single crystals with lower densities of dislocations, should be better able to reach a fully ordered ground state when topological frustration is present. the single crystal in ref. 15 could be showing a slight emergence of the ground state because it contains far fewer topological defects. However, it is important to note that larger single crystals maintain lattice coherence over greater distances and are thus actually more susceptible to topological frustration. As an example, a cubic crystal of side length N unit cells and volume N3 unit cells could need only on the order of Nd ¼ N dislocations to be fully frustrated, if the emanating frustrated surfaces are non-interacting, similar to the domain walls we observe in our specimens—we discuss this in more detail below. Under this assumption, the dislocation density would be rd ¼ Nd/N2, and the density required to frustrate a system scales as 1/N, implying that larger single crystals are actually more likely to be impacted by the presence of dislocations than smaller crystals. This is counter to the conventional expectation that larger single crystals show more intrinsic ground-state behaviours. Discussion An important question remains about how our observations relate to dislocations in 3D crystalline materials and the outstanding issue of whether a unique ground state could be realized in spin ice materials. We consider previous measure- ments of residual entropy in spin ice in the context of sample preparation and dislocation density. The material studied in the seminal report by Ramirez et al.9 was a pressed powder sample, which should result in a high density of dislocations both due to damage during compaction and also due to grain boundaries in the resulting structure. This material, with a presumably high density of dislocations, showed a level of entropy equal to the Pauling ice value, but the 2  1  1 mm3 single crystal studied by Pomaranski et al.15 showed less entropy, a small amount below the Pauling value. Pomaranski et al. also studied powder samples, which showed similar spin-freezing and frustrated behaviour as the samples in ref. 9. We postulate that the topological frustration due to a high density of dislocations in both powder samples could be complicating and slowing ground-state ordering, while a b c d Figure 5 | Monte Carlo simulations. Final frames of kinetic Monte Carlo simulations with one (a) and two (b–d) dislocation lattices. The domain w patterns observed are remarkably similar to our experimental data with dislocations always nucleating a domain wall that extends to the edge of the crystal or to another dislocation among canonical ground-state ordering and domains. The spread in widths used in these runs are 5 Å (a), 6 Å (b), 6 Å and 6 Å (d) (see ‘Methods’ section). b b a d d c c Figure 5 | Monte Carlo simulations. Final frames of kinetic Monte Carlo simulations with one (a) and two (b–d) dislocation lattices. The domain wall patterns observed are remarkably similar to our experimental data with dislocations always nucleating a domain wall that extends to the edge of the finite crystal or to another dislocation among canonical ground-state ordering and domains. The spread in widths used in these runs are 5 Å (a), 6 Å (b), 6 Å (c) and 6 Å (d) (see ‘Methods’ section). NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 5 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 ARTICLE On the contrary, smaller single crystals, or single crystals with lower densities of dislocations, should be better able to reach a fully ordered ground state when topological frustration is present. a a [ [ ] ] a a b b q q · b = –π 2 2π = = – –— 1 0 0 1 0 –1 b g p g p Implications of topological frustration could extend far beyond the systems discussed above, and we first lay out a framework for such considerations as follows. Many materials, simple and exotic alike, display ordered states that can generally be characterized by an order parameter and associated ordering vector, qa0. If the topological Burgers vector of a defect is perpendicular to a given q, or contains a complete wave of the order parameter, with q . b ¼ 2pn, where n ¼ 0 or an integer, the system will not be frustrated. However, if these conditions do not hold, the system will exhibit topological frustration. In our square ASI, there are two q vectors corresponding to the doubly degenerate ground state, q ¼ ±(p/a)[11], which tile type I vertices onto the lattice. Our choices of Burgers vectors are in the [10] or [01] directions, thus q . ba2pn, and the system will always be topologically frustrated. In the 3D pyrochlore spin ices, there are 6 q vectors for 6 realizations of the predicted long-range ordered state, q ¼ (2p/a) o100412, as shown in Fig. 6a. To our knowledge, no prior studies have identified the density or nature of dislocations in pyrochlore samples, but it would be straightforward and instructive to analyse their expected influence. The pyrochlore structure is derived from the fluorite structure, which readily forms dislocations with Burgers vectors along one of the primitive FCC lattice vectors, (a/2) o110432, as shown in Fig. 6b. For each possible Burgers direction, 4 out of the 6 dipolar ground- state q vectors will not be perpendicular to the Burgers vector and will thus be necessarily frustrated, producing a domain wall surface emanating from each such dislocation line, even in the lowest energy state. If a crystal contains dislocations with all FCC primitives, then all 6 ground states will exhibit such effects, and the specimen could be prevented from finding a unique ground state, a possibility we discuss further below. ARTICLE In kinetic Monte Carlo simulations, each element is assigned a flip rate given by t  1 ¼ v0 exp E0 þ DE kBT   ð1Þ ð1Þ where n0 is a prefactor, DE is the change in energy given by nearest neighbour dipolar coupling, T is the temperature and E0 is the intrinsic energy barrier. We are less concerned with the parameters n0 and T as they affect every rate in the same way and are generally used to evolve time in the model, where we are mainly interested in the long-time steady state configuration of the system. After assigning rates to each spin, the rates are summed and used to create a probability distribution so that elements in energetically unfavourable arrangements have a higher probability of flipping. We then randomly pick one element weighted by the probabilities, flip it, and recalculate all the flip rates. As in our previous studies21, we include a small amount of disorder in the form of width variations in individual elements. We use Gaussian distributions with average 120 nm and characteristic spreads on the order of 1–10 Å. This width disorder factors into the simulation in two ways: first, the intrinsic energy barrier, E0, is approximated by the shape anisotropy energy of the element, which is the product of the demagnetizing factor, volume, and the magnetization of the material squared18,41. Both the demagnetizing factor and the volume are affected by the range of element widths. Second, the disorder enters through the magnetic dipolar coupling, given by E ¼  m0 4p rj j3 3ðm1  ^rÞðm2  ^rÞ  m1  m2 ð Þ ð2Þ ð2Þ Similar frustration could be present in other systems with qa0 order parameters. Prime examples may include spin-density-wave materials36, such as antiferromagnets and ferrimagnets, anti- ferroelectrics, such as SrTiO3 (ref. 37), charge-density-wave materials38 and pair-density-wave superconductors39. In cases where the dislocation densities are low, or where the frustrated domain walls have stronger surface tension between dislocations, the frustration may not prevent the formation of the order parameter, but rather it will only degrade it or delay it some- what. In such cases, we expect topological frustration may still thermally broaden the phase transition, degrading the formation of the qa0 order parameter. Topological defects may even play a role in the frustration of ice-XI, the thermodynamic ground state of water not known to form under typical experimental conditions40. Methods S l f b 5. Ran, Y., Zhang, Y. & Vishwanath, A. One-dimensional topologically protected modes in topological insulators with lattice dislocations. Nat. Phys. 5, 298–303 (2009). Sample fabrication. To define our lithography patterns, square lattices are first modelled as a network of connecting springs. The springs are all of equal spring constant and attempt to keep the lengths between neighbouring nodes the same. We also include a term that aims to keep the angles between connections coming out of each node the same, for example, 90 for four connections or 120 for three connections. We remove a chain of nodes starting from a given point, doubling the spring lengths along the chain of removed points. We then let the system relax and after it has equilibrated, we record the x and y location of all nodes. We then use these relative coordinates to as a guide for our lithography patterns. A movie of the relaxation process is given in Supplementary Movie 3. A description of all geometries studied is given in Supplementary Fig. 3. 6. Aifantis, E. C. The physics of plastic deformation. Int. J. Plast. 3, 211–247 (1987). 7. Kuzmina, M., Herbig, M., Ponge, D., Sandlobes, S. & Raabe, D. Linear complexions: Confined chemical and structural states at dislocations. Science 349, 1080–1083 (2015). 8. Callister, W. & Rethwisch, D. Materials Science and Engineering: an Introduction (John Wiley & Sons, 2007). y 9. Ramirez, A. P., Hayashi, A., Cava, R. J., Siddharthan, R. & Shastry, B. S. Zero point entropy in ‘spin ice’ Nature 399 333 335 (1999) 9. Ramirez, A. P., Hayashi, A., Cava, R. J., Siddharthan, R. & Sha Zero-point entropy in ‘spin ice’. Nature 399, 333–335 (1999). Our ASI arrays are fabricated from 23 nm thick FePd3. We have reported film grown and fabrication details previously21. Elements are 120 nm wide and the nominal lattice constant is 500 nm, though this varies due to the relaxation in the connected spring model. Element lengths are given by subtracting a fixed amount from each node, with the three connected vertices treated by subtracting a slightly larger amount to prevent elements from touching. 10. Harris, M. J., Bramwell, S. T., McMorrow, D. F., Zeiske, T. & Godfrey, K. W. Geometrical frustration in the ferromagnetic pyrochlore Ho2Ti2O7. Phys. Rev. Lett. 79, 2554–2557 (1997). 11. Pauling, L. The structure and entropy of ice and of other crystals with some randomness of atomic arrangement. J. Am. ARTICLE Stuffed spins have indeed been shown to influence magnetic relaxation times in these systems33, but the actual disorder and the true source of the specific heat discrepancies remain unresolved. We propose that topological frustration due to dislocations may be an additional contributing source of random disorder, and we consider the implications as follows. As noted above, we have assumed in the prior discussion that the domain walls due to dislocations are non-interacting, as appears to be the case in our studies of 2D ASI. This appearance may be influenced by dynamical arrest, where the domain configurations become locked in our experiments due to local disorder, and the motion of the domain walls slows down significantly. This effect is apparent in NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 In 2D crystals, a dislocation is a point defect, but in three dimensions it is a topological line defect and must either close in a loop or extend to the edges of a crystal. If line dislocations in a 3D crystal occur on a simple parallel lattice, then a trivial arrangement of domain walls may connect them, producing a straightforward ground-state configuration. However, an arbitrary configuration of dislocations could produce compli- cated arrangements of domain walls, which may not even be unique. In this case, a material which may otherwise be able to find a ground state could see a robust frustration simply due to the presence of topological defects. Furthermore, it has been proposed that a second competing ground state with longer ordering period may play a role in spin ice behaviour14. For this order parameter, a dislocation would not produce a simple antiphase boundary but instead a more complicated phase shift. Thus, dislocations may play an even more significant role than described above. The possible influence of topological frustration on spin ordering in these systems is therefore not straightforward and warrants more attention in future experimental and theoretical investigations. spins, same size spins, and same boundary conditions as experimental samples. In kinetic Monte Carlo simulations, each element is assigned a flip rate given by spins, same size spins, and same boundary conditions as experimental samples. ARTICLE In general, the effects of topological frustration could be observed in a wide variety of materials systems, and ASI could serve as a valuable platform for future studies to develop these general considerations. where r is the vector connecting the midpoints of the dipolar spins pointing from 1 to 2 and ^r is the unit vector along r. The magnetic moments m1 and m2 are the magnetization of the material times the volume of the element and are thus also affected by the width disorder. The vector orientations of the individual magnets in the relaxed lattices are also taken into account with this dipolar coupling. The nanomagnets’ small deviations from regular square lattice orientations slightly broaden the four vertex energy levels into bands, but not enough so that they overlap or reorder. Movies of the simulations are given in the Supplementary Information. Data availability. The data that support the findings of this study are available from the corresponding author upon reasonable request. References 1. Mermin, N. D. The topological theory of defects in ordered media. Rev. Mod. Phys. 51, 591–648 (1979). y 2. Nakamura, T. & Kawamura, K. The frustration of antiferromagnetic bonds in the screw dislocation. Phys. Status Solidi B 122, 141–150 (1984). 3. Dzyaloshkinskii, I. E. Domains and dislocations in antiferromagnets. JETP Lett. 25, 98–100 (1977). 4. Sugiyama, I. et al. Ferromagnetic dislocations in antiferromagnetic NiO. Nat. Nanotechnol. 8, 266–270 (2013). NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14009 19. Qi, Y., Brintlinger, T. & Cumings, J. Direct observation of the ice rule in an artificial kagome spin ice. Phys. Rev. B 77, 094418 (2008). 38. Gru¨ner, G. The dynamics of charge-density waves. Rev. Mod. Phys. 60, 1129–1181 (1988). g p y 20. Gilbert, I. et al. Emergent ice rule and magnetic charge screening from vertex frustration in artificial spin ice. Nat. Phys. 10, 670–675 (2014). 39. Berg, E., Fradkin, E., Kivelson, S. A. & Tranquada, J. M. Striped superconductors: how spin, charge and superconducting orders intertwine in the cuprates. New J. Phys. 11, 115004 (2009). Drisko, J., Daunheimer, S. & Cumings, J. FePd3 as a material fo 21. Drisko, J., Daunheimer, S. & Cumings, J. FePd3 as a material for studying thermally active artificial spin ice systems. Phys. Rev. B 91, 224406 (2015). 40. Petrenko, V. F. & Whitworth, R. W. Physics of Ice (Oxford University Press, 1999). 22. Budrikis, Z. et al. Domain dynamics and fluctuations in artificial square ice at finite temperatures. New J. Phys. 14, 035014 (2012). 41. Aharoni, A. Demagnetizing factors for rectangular ferromagnetic prisms. J. Appl. Phys. 83, 3432–3434 (1998). 23. Mo¨ller, G. & Moessner, R. Artificial square ice and related dipolar nanoarrays. Phys. Rev. Lett. 96, 237202 (2006). Acknowledgements y 24. Chern, G.-W., Mellado, P. & Tchernyshyov, O. Two-stage ordering of spins in dipolar spin ice on the kagome lattice. Phys. Rev. Lett. 106, 207202 (2011). y 24. Chern, G.-W., Mellado, P. & Tchernyshyov, O. Two-stage ordering of This work was supported by NSF CAREER Grant No. DMR-1056974. We also acknowledge the support of the Maryland NanoCenter and its AIMLab and FabLab. spins in dipolar spin ice on the kagome lattice. Phys. Rev. Lett. 106, 207202 (2011). 25. Mo¨ller, G. & Moessner, R. Magnetic multipole analysis of kagome and artificial spin-ice dipolar arrays. Phys. Rev. B 80, 140409 (2009). Author contributions 26. Farhan, A. et al. Direct observation of thermal relaxation in artificial spin ice. Phys. Rev. Lett. 111, 057204 (2013). J.C. and J.D. conceived of the experiment. J.C. supervised the experiments, simulations and data analysis. J.D. deposited thin films, fabricated ASI samples, performed the experiments and ran Monte Carlo simulations. T.M. processed Lorentz TEM images and analysed data with J.D. J.D. wrote the manuscript with input from all authors. y 27. von Heijne, G. Membrane-protein topology. Nat. Rev. Mol. Cell Biol. 7, 909–918 (2006). 28. Wang, W. L., Yazyev, O. V., Meng, S. & Kaxiras, E. Topological frustration in graphene nanoflakes: magnetic order and spin logic devices. Phys. Rev. Lett. 102, 157201 (2009). Methods S l f b Chem. Soc. 57, 2680–2684 (1935). 12. Melko, R., den Hertog, B. & Gingras, M. Long-range order at low temperatures in dipolar spin ice. Phys. Rev. Lett. 87, 067203 (2001). Heating experiments and imaging. After fabrication, samples are heated to 165 C in an Ar atmosphere and cooled at a rate of 1 C min  1. The resulting magnetic configurations are imaged in a JEOL JEM 2100 LaB6 microscope. A degaussing procedure is run on the microscope’s objective lens before inserting the sample to remove any remnant field that could bias the magnets. The field at the sample is measured to be o1 G after this procedure. Lorentz contrast images are then taken and analysed with the aid of computerized automatic image processing. 13. Morris, D. J. P. et al. Dirac strings and magnetic monopoles in the spin ice Dy2Ti2O7. Science 326, 411–414 (2009). 14. Henelius, P. et al. Refrustration and competing orders in the prototypical Dy2Ti2O7 spin ice material. Phys. Rev. B 93, 024402 (2016). 15. Pomaranski, D. et al. Absence of Pauling’s residual entropy in thermally equilibrated Dy2Ti2O7. Nat. Phys. 9, 353–356 (2013). 16. Wang, R. F. et al. Artificial ‘spin ice’ in a geometrically frustrated lattice of nanoscale ferromagnetic islands. Nature 439, 303–306 (2006). 17. Zhang, S. et al. Crystallites of magnetic charges in artificial spin ice. Nature 500, 553–557 (2013). Numerical methods. Monte Carlo simulations are performed using a kinetic Monte Carlo technique. We use the same procedure to define our simulation lattices as we do our lithography patterns. Simulations have the same number of 18. Farhan, A. et al. Exploring hyper-cubic energy landscapes in thermally active finite artificial spin-ice systems. Nat. Phys. 9, 375–382 (2013). 18. Farhan, A. et al. Exploring hyper-cubic energy landscapes in thermally active finite artificial spin-ice systems. Nat. Phys. 9, 375–382 (2013). 7 NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications r The Author(s) 2017 NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications Additional information 29. Araki, T., Buscaglia, M., Bellini, T. & Tanaka, H. Memory and topological frustration in nematic liquid crystals confined in porous materials. Nat. Mater. 10, 303–309 (2011). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing financial interests: The authors declare no competing financial interests. 30. Kleiber, M., Bode, M., Ravlic´, R. & Wiesendanger, R. Topology-induced spin frustrations at the Cr(001) surface studied by spin-polarized scanning tunneling spectroscopy. Phys. Rev. Lett. 85, 4606–4609 (2000). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ y y 31. Ravlic´, R., Bode, M., Kubetzka, A. & Wiesendanger, R. Correlation of dislocation and domain structure of Cr(001) investigated by spin-polarized scanning tunneling microscopy. Phys. Rev. B 67, 174411 (2003). How to cite this article: Drisko, J. et al. Topological frustration of artificial spin ice. Nat. Commun. 8, 14009 doi: 10.1038/ncomms14009 (2017). g g py y 32. Chin, G. Y. in Deformation of Ceramic Materials (Springer, 1975). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 33. Revell, H. M. et al. Evidence of impurity and boundary effects on magnetic monopole dynamics in spin ice. Nat. Phys. 9, 34–37 (2012). 34. Snyder, J., Slusky, J. S., Cava, R. J. & Schiffer, P. How ‘spin ice’ freezes. Nature 413, 48–51 (2001). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 35. Matsuhira, K., Hinatsu, Y. & Sakakibara, T. Novel dynamical magnetic properties in the spin ice compound Dy2Ti2O7. J. Phys. Condens. Matter 13, 737–746 (2001). 36. Fawcett, E., Alberts, H. L., Galkin, V. Y., Noakes, D. R. & Yakhmi, J. V. Spin- density-wave antiferromagnetism in chromium alloys. Rev. Mod. Phys. 66, 25–127 (1994). 37. Osterman, D. P., Mohanty, K. & Axe, J. D. Observation of the antiferroelectric order parameter in surface layers of SrTiO3. J. Phys. C Solid State Phys. 21, 2635–2640 (1988). NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 8:14009 | DOI: 10.1038/ncomms14009 | www.nature.com/naturecommunications 8
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Author Correction: Split intein-mediated selection of cells containing two plasmids using a single antibiotic
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© The Author(s) 2020 Author Correction: Split intein-mediated selection of cells containing two plasmids using a single antibiotic Author Correction: Split intein-mediated selection of cells containing two plasmids using a single antibiotic Navaneethan Palanisamy , Anna Degen , Anna Morath, Jara Ballestin Ballestin , Claudia Juraske, Mehmet Ali Öztürk, Georg A. Sprenger , Jung-Won Youn , Wolfgang W. Schamel & Barbara Di Ventura Correction to: Nature Communications https://doi.org/10.1038/s41467-019-12911-1, published online 31 October 2019. The original version of this Article contained an error in Fig. 7. In panels c and d, the y-axes for L-PAPA concentration in mM were incorrectly labelled as nM. This has been corrected in both the PDF and HTML versions of the Article. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 NATURE COMMUNICATIONS | (2020) 11:276 | https://doi.org/10.1038/s41467-019-13716-y | www.nature.com/naturecommunication
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Fluorine-Doped M-N-C Catalysts for Efficient Oxygen Reduction Reaction
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Fluorine-Doped M-N-C Catalysts for Efficient Oxygen Reduction Reaction Zhichuan Zheng  Beijing University of Posts and Telecommunications Xuekun Hong  Changshu Institute of Technology Dajun Wu  Changshu Institute of Technology Ning Sun  Beijing University of Posts and Telecommunications Yawei Kuang  Changshu Institute of Technology Debao Zhang  Changshu Institute of Technology Xiaxi Yao  Changshu Institute of Technology Peng Du  (  pengdu@bupt.edu.cn ) Beijing University of Posts and Telecommunications Kai huang  Beijing University of Posts and Telecommunications Ming Lei  Beijing University of Posts and Telecommunications Research Article Keywords: Oxygen reduction reaction, Manganese phthalocyanine, M-N-C catalysts, M-N4 site, pre- fluorinated carbon nanotubes, charge distribution Zhichuan Zheng  Beijing University of Posts and Telecommunications Xuekun Hong  Changshu Institute of Technology Dajun Wu  Changshu Institute of Technology Ning Sun  Beijing University of Posts and Telecommunications Yawei Kuang  Changshu Institute of Technology Debao Zhang  Changshu Institute of Technology Xiaxi Yao  Changshu Institute of Technology Peng Du  (  pengdu@bupt.edu.cn ) Beijing University of Posts and Telecommunications Kai huang  Beijing University of Posts and Telecommunications Ming Lei  Beijing University of Posts and Telecommunications Research Article Keywords: Oxygen reduction reaction, Manganese phthalocyanine, M-N-C catalysts, M-N4 site, pre- fluorinated carbon nanotubes, charge distribution Posted Date: February 15th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2558022/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/20 Page 1/20 Page 1/20 Version of Record: A version of this preprint was published at Advanced Composites and Hybrid Materials on April 21st, 2023. See the published version at https://doi.org/10.1007/s42114-023-00663-y. Page 2/20 Abstract In recent years, transition metal-nitrogen-carbon (M-N-C) composites are expected to be an alternative to platinum group metal (PGM) among various nonprecious metal catalysts investigated. However, the major challenge comes from insufficient electrocatalytic performance and durability for oxygen reduction reaction (ORR). In addition to the selection of suitable central metal active sites, the electrocatalytic activity and stability of the M-N-C catalysts can be enhanced by adjusting the electronic structure of the catalysts. In this work, M-N-C/F composites were synthesized by loading transition metal phthalocyanine complexes onto pre-fluorinated carbon nanotubes through a simple pyrolysis method. Pyrroline-N(PN) and graphite-N(GN) formed after thermal treatment can act as electron acceptors to modulate their charge distribution on the M-N4 sites, and the use of pre-fluorinated nanotubes also allows for a more controlled introduction of fluoride ions that are well coordinated to transition metals, both of which can modulate and modify the electronic structure of M-N-C catalysts. The obtained manganese phthalocyanine/fluorinated carbon nanotubes at 800°C (MnPc/FCNT800) exhibits a competitive electrocatalytic ORR performance with the half-wave potential (E1/2) of 0.9 V and only 12.1% decay after 20 h long-term chronoamperometry (CA) test in 1.0 M KOH electrolyte, outperforming the commercial Pt/C. Overall, this work paves the way of the electronic structure modification and design of such M-N-C composites for sustainable energy applications. Introduction These nanoparticles do not act directly as actual active sites, but as guest groups to enhance the activity of nearby M-N-C sites [25]. Therefore, the materials reconstructed by decomposition and recombination after pyrolysis modify the electronic structure of M-N-C [8]. The enhancement of other active site N-C is generally accomplished by tailoring the electronic structure of the catalyst. For example, catalysts with atomically dispersed M-N4 coordination usually suffer from sluggish oxygen activation kinetics resulted from the symmetrical charge distribution. And the integration of heteroatoms is a promising way to regulate the charge distribution of MN4 structures [12, 13, 20, 21]. Typically, pyrrole-N(PN) and graphitized-N(GN) can be introduced as electron acceptors near MN4 to improve its ORR activity [22, 23]. In addition, pyrolysis is also an effective method to modulate the electronic structure of electrocatalysts as metal-based nanoparticles (usually metals, nitrides or carbides) are readily formed during synthesis [24]. These nanoparticles do not act directly as actual active sites, but as guest groups to enhance the activity of nearby M-N-C sites [25]. Therefore, the materials reconstructed by decomposition and recombination after pyrolysis modify the electronic structure of M-N-C [8]. Moreover, the metal phthalocyanine is found to show significantly optimized electronic structure when further combined with carbon materials, which can be attributed to the high specific surface area, excellent electrical conductivity, chemical stability, and easily adjustable surface chemistry of carbon materials [9, 26–33]. Furthermore, the decorated heteroatoms with lower (B, P, S, and transition metals) or higher (N) electronegativities distinct from that of carbon can polarize adjacent carbon atoms and change the electronic properties of the carbon skeletons [34, 35], which will modify the related electrocatalytic performance [36]. Normally, the d orbitals of the central metal atom perpendicular to the M-N-C plane are not fully occupied and may interact with small molecule ligands. Thus, external ligands can act as guest groups to coordinate with the central metal atom and modulate the electronic structure of the M-N-C sites. For example, halogen and pseudo-halogen ions, such as chloride and cyanide ions, have strong coordination with transition metal atoms, which can substitute the nitrogen coordination of the central metal [37]. Therefore, fluoride species well coordinated with transition metals can be introduced by pyrolysis of M-N-C supported on the pre-fluorinated carbon nanotubes to obtain fluorine- doped M-N-C/F catalysts. Introduction To achieve the Dual Carbon Goals, the development of renewable energy devices such as fuel cells and zinc-air batteries is of crucial importance, while the practical application is significantly restricted by the sluggish kinetics of the oxygen reduction reaction (ORR) at their cathodes [1–4]. Commercial platinum carbon (Pt/C) is regarded as an ideal ORR catalyst [5, 6]. Nevertheless, its high cost and low-durability significantly limit the practical application [7]. Therefore, the exploitation of platinum group metal-free (PGM) for kinetically sluggish oxygen reduction reactions (ORR) is crucial to the further application of clean energy [8–10]. A primary concern for applications of non-precious metal catalysts is their insufficient performance and durability in the membrane electrode assemblies (MEAs) under practical hydrogen-air conditions [11]. Over recent years, metal-nitrogen-carbon (M-N-C) catalysts with M-N4 active centers such as transition metal phthalocyanine complexes are widely accepted as one of the promising candidates to replace Pt/C due to their superior intrinsic activity [7, 12–14]. In general, M and N-C centers act synergistically as the active center for ORR in M-N-C catalysts [1]. While the central metal atoms are usually recognized as the actual ORR activity sites, thus the most direct and effective strategy to regulate the catalytic activity of the catalyst is to select the appropriate central metal atom [5, 15]. Theoretical and experimental studies have demostrated that the fourth-cycle Fe [16, 17], Co, Mn, Ni, and their neighboring transition metal elements exhibit the highest ORR activity [18]. Among them, the acceptable ORR activity and weak interaction with H2O2 make manganese-based materials one of the most durable electrocatalysts. Thus, Page 3/20 Page 3/20 such composites are perceived as the most promising noble metal-free electrocatalysts in proton exchange membrane fuel cells [11, 19]. The enhancement of other active site N-C is generally accomplished by tailoring the electronic structure of the catalyst. For example, catalysts with atomically dispersed M-N4 coordination usually suffer from sluggish oxygen activation kinetics resulted from the symmetrical charge distribution. And the integration of heteroatoms is a promising way to regulate the charge distribution of MN4 structures [12, 13, 20, 21]. Typically, pyrrole-N(PN) and graphitized-N(GN) can be introduced as electron acceptors near MN4 to improve its ORR activity [22, 23]. In addition, pyrolysis is also an effective method to modulate the electronic structure of electrocatalysts as metal-based nanoparticles (usually metals, nitrides or carbides) are readily formed during synthesis [24]. Materials Fluorinated carbon nanotubes, fluorine content 48–58 wt% was purchased by Nanjing XFNANO Materials Tech. Co., Ltd. Manganese phthalocyanine (C32H16N8Mn), cobalt phthalocyanine (C32H16N8Co), nickel phthalocyanine (C32H16N8Co) and phthalocyanine (C32H16N8) were purchased from Alfa Aesar, absolute ethanol (C2H5OH, 99.8%) and methanol (99.8%) was purchased by Aladdin. All chemicals were used as received without further purification except for special declaration in this work. Preparation of catalyst samples First, 45 mg of manganese phthalocyanine (MnPc), 41 mg of cobalt phthalocyanine (CoPc), 41 mg of Ni phthalocyanine (NiPc), 45 mg of phthalocyanine (Pc) and 100 mg of each fluorinated carbon nanotubes (FCNT) were weighed into different beakers using a weighing balance to ensure that the wt% of Mn, Co, and Ni was 3%. 50 mL of anhydrous ethanol was poured along the wall of the beaker and dispersed well in the ultrasonic device, and then stirred at a uniform speed for 12 h using a magnetic stirring device. After uniform dispersion, it was extracted and filtered, dried for 24 h and then placed in a tube heating furnace for high temperature thermal decomposition under argon gas protection. The temperature was increased to 800 ℃ at 1 ℃/min, held for one hour, cooled down and then the target sample was removed and stored under vacuum. The samples MnPc/FCNT800, CoPc/FCNT800, NiPc/FCNT800, Pc/FCNT800 were obtained after pyrolysis at 800 ℃ in the tube furnace. Introduction Benefiting from the efficient and controllable fluorination process, the physicochemical properties of such carbon-based materials are expected to well regulated, such as the significantly increase of solubility and conductivity, which facilitates the synthesis process and conduction of electrons [38, 39]. The large surface area of nanotubes covered by fluorination is also considered as an advantage to facilitate the introduction of functional groups in the subsequent reactions [40]. Moreover, the selection of fluorinated carbon nanotubes as substrates enables better thermal treatment and structural enhancement, and pre-fluorination also provides the potential to adjust the interfacial bonding between nanotubes and matrix by modifying the side groups, which is more controllable compared to the direct introduction of halogen atoms for doping [37, 41]. M-N-C catalysts are synthesized by a sample pyrolysis method of cleaving transition metal M-N-C catalysts are synthesized by a sample pyrolysis method of cleaving transition metal phthalocyanine complexes and fluorinated carbon nanotubes at 800°C for enhanced electrocatalytic oxygen reduction. The doping of electronegative F element polarizes the adjacent carbon atoms and regulates the electronic structure of M-N-C. And the generated pyrrole-N (PN) and graphitized-N (GN) can Page 4/20 Page 4/20 also modulate the charge distribution at pyrolysis up to 800°C. The as-prepared MnPc/FCNT800 exhibits superior electrocatalytic ORR activity with a half-wave potential (E1/2) of 0.90 V, outperforming those of comparative samples and commercial Pt/C. Moreover, the remarkable long-term stability of MnPc/FCNT800 was demonstrated by CA test for 20 h with only 12.1% decay. The enhanced ORR performace can be ascribed to the better doping of heteroatoms to regulate the charge distribution and the stronger electron giving ability of manganese. This work provides a new avenue for the development of non-precious metal catalysts towards the cathodic oxygen precipitation reaction of M-N-C catalysts in fuel cells. Material characterizations Transmission electron microscopy (TEM, JEM-2100F, Japan) and scanning transmission electron microscopy (FE-SEM, S-4800, Japan) were applied to observe the sample morphologies. A D/MAX 2500 diffractometer (Rigaku, Japan) fitted with Cu Kα radiation was used to acquire the X-ray diffraction (XRD) patterns. To study the surface chemistry of these samples, a Thermo Scientific ESCALAB 250Xi with Al Kα (1487.6 eV) was used as the excitation source and C 1s peak (284.8 eV) was applied as the calibration to acquire the X-ray photoelectron spectroscopy (XPS). Electrochemical measurements Electrochemical measurements Page 5/20 Page 5/20 The electrochemical measurements were performed with an Autolab potentiostat (PGSTAT-204N) equipped with a typical three-electrode alkaline electrochemical cell system. A graphite rod and Hg/HgO (1M KOH) were used as the counter and reference electrodes. And a glassy carbon (GC) electrode with a surface area of 0.196 cm2 served as the substrate for the working electrode. 850 µL of ethanol, 100 µL of 5wt% Nafion solution and 50 µL of water were used to make up 3mg of the catalyst. Then 30 µL catalyst ink was deposited on a GC to give a 0.45 mg cm− 2 catalyst loading with a 30 min ultrasonic treatment. All polarization curves were collected at a scan rate of 50 mV s− 1and calibrated to the reversible hydrogen electrode according to the following equation: ERHE = EHg/HgO + 0.0592 × pH + 0.098 V - iR (1) The linear sweep voltammograms (LSVs) and Cyclic voltammograms (CVs) were performed in N2- or O2- saturated 1 M KOH solution with a scan rate of 50 mV s-1. And the rotating rate of rotating-disk electrode was 1600 rpm. In addition, the EIS potential for ORR was 0.85V (vs RHE), and the electrochemical impedance spectroscopy (EIS) analysis was applied with an iR compensation level of 90%. Accelerated durability tests (ADTs) for 5000 cyclic voltammetry cycles of various catalysts were conducted at the range of 0.6-1.0 V vs RHE. To further verify the stability, CA test was taken at a constant voltage of 0.85 V vs. RHE in 1M O2-saturated KOH for 20 h. The K-L equation is used to calculate the kinetic current density (JK) and the electron transfer number (n): The linear sweep voltammograms (LSVs) and Cyclic voltammograms (CVs) were performed in N2- or O2- saturated 1 M KOH solution with a scan rate of 50 mV s-1. And the rotating rate of rotating-disk electrode was 1600 rpm. In addition, the EIS potential for ORR was 0.85V (vs RHE), and the electrochemical impedance spectroscopy (EIS) analysis was applied with an iR compensation level of 90%. Accelerated durability tests (ADTs) for 5000 cyclic voltammetry cycles of various catalysts were conducted at the range of 0.6-1.0 V vs RHE. To further verify the stability, CA test was taken at a constant voltage of 0.85 V vs. RHE in 1M O2-saturated KOH for 20 h. Synthesis and morphology As shown in Fig. 1, M-N-C/F catalysts were synthesized by a simple pyrolysis method. Homogeneous mixtures of pre-fluorinated carbon nanotubes and transition metal phthalocyanine compounds are pyrolyzed at 800 ℃ with the formation of transition metals, carbides or nitrides. And the fluorination as well as the generation of new species are beneficial to enhance the electrocatalytic activity of M-N-C composites. Figure 2a presents the SEM images of MnPc/FCNT800, and the morphology of catalysts after pyrolysis still maintains the mesoporous structure of fluorinated carbon nanotubes, while the porous structure of carbon nanotubes is beneficial to increase the active sites and improve the ORR catalytic activity and stability of the materials [26]. The TEM images of MnPc/FCNT800, CoPc/FCNT800 and NiPc/FCNT800 are displayed in Fig. 2b, Fig. 2c and Fig. 2d, respectively. Compared with Fig. 2e, the similar morphological structures indicate that the nanoparticles obtained after pyrolysis can be well immobilized on fluorinated carbon nanotubes. As shown in Fig. 2f, a MWCNT-like, onion and graphitic structures can be observed in the HRTEM images of MnPc/FCNT800, which can be explained as the phase transformation induced by the electron-beam [42]. And it can be illustrated that the use of pre- fluorinated carbon nanotubes improves the physical and chemical properties of carbon nanotubes and has a catalytic effect on the oxygen reduction reaction [39, 40]. As displayed in Fig. S1, the EDS elemental mapping images of the MnPc/FCNT800 exhibite that F, Mn, C, and N elements were uniformly distributed, which confirmed that the required materials had been successfully synthesized. And the EDS elemental mapping images of the CoPc/FCNT800 and NiPc/FCNT800 are shown in Fig. S2 and Fig. S3 respectively. Electrochemical measurements The K-L equation is used to calculate the kinetic current density (JK) and the electron transfer number (n): 1/J = 1/JK + 1/JL = 1/JK + 1/0.2nFCD2/3v-1/6w1/2 (2) where J is the measured current density, JK is the kinetic current density, JL is the limiting current densities, ω is the rotation rate in angular velocity, n is the electron transfer number, F is the Faraday constant (96485 C mol-1), C is the bulk concentration of O2 (1.2×10− 6 mol cm-3), D is the diffusion coefficient of O2 (1.9×10-5cm2 s-1), and ν is the kinematic viscosity of the electrolyte (0.01 cm2 s-1). where J is the measured current density, JK is the kinetic current density, JL is the limiting current densities, ω is the rotation rate in angular velocity, n is the electron transfer number, F is the Faraday constant (96485 C mol-1), C is the bulk concentration of O2 (1.2×10− 6 mol cm-3), D is the diffusion coefficient of O2 (1.9×10-5cm2 s-1), and ν is the kinematic viscosity of the electrolyte (0.01 cm2 s-1). he following K-L equation can determine the specific kinetic current density: JK = JL * J/(JL - J) (2) JK = JL * J/(JL - J) (2) Furthermore, A CHI760E electrochemical workstation was used to perform RRDE measurements. The H2O2 yield (H2O2%) and the electron transfer number (n) were monitored by the following equations: Page 6/20 Page 6/20 H2O2(%) = 200IR/N/(IR/N + ID) (4) n = 4ID/(IR/N + ID) (5) where ID is the disk current, IR is the ring current, and N = 0.4 is the ring collection efficiency. where ID is the disk current, IR is the ring current, and N = 0.4 is the ring collection efficiency. where ID is the disk current, IR is the ring current, and N = 0.4 is the ring collection efficiency. Structural characterizations 4b-d, the electron binding energy of the metal in the 2p3/2 orbital is shifted in the negative direction after pyrolysis, which can be explained by the fact that the metal gives electrons to the nitrogen and the metal valence decreases. The XPS spectra of F1s before pyrolysis are shown in Fig. 4e, with two peaks indicating physical adsorbed and trapped fluorine (686.6 eV) and covalent Cfx (687.7 eV) [53]. And the XPS spectra of F1s after pyrolysis at 800°C are shown in Fig. 4g, where the physical adsorbs and trapped fluorine disappears and with the appearance of C-F (685.6 eV), the proportion of CFx decreases [54]. Compared with the covalent C-F bond, the ionic C-F bond is more active [55]. And the doping of the F atom can polarize the adjacent carbon atom and change the electronic properties of the carbon skeleton as the pyrolysis proceeds [37]. As displayed in Fig. 4f and 4h, the XPS spectra of N1s before pyrolysis could be deconvoluted into four peaks at 398.5, 399.6, 400.3 and 401.2 eV, referring to the pyridinic- N, metal-N, pyrrolic-N and graphitic-N, respectively [7]. Among them, metal-N usually refers to the complexes formed by metals and nitrogen, such as Mn-Nx, Co-Nx and Ni-Nx [17]. Moreover, pyridinic-N and metal-N are usually the reason for higher ORR activity in alkaline electrolytes, as central atom could coordinate with them to form M-Nx configuration [20]. And the increased pyrrole-N(PN) and graphitized-N(GN) after pyrolysis can be introduced as electron acceptors near MN4, which will be helpful to regulate the charge distribution of M-N-C/F catalysts and improve their ORR activity [22, 23]. Correspondingly, the electron binding energy of the catalyst after pyrolysis in the XPS spectra of N1s is positively shifted and the Structural characterizations The XRD results before and after pyrolysis are displayed in Fig. 3a-d, where a strong peak located at 26.60° can be interpreted as a amorphous bun peak of carbon. A diffraction peak at 42° can be indexed for the (100) plane of carbon, and this peak is replaced by a graphite peak when the temperature increases, which can be explained by the higher graphitization of carbon nanotubes during pyrolysis [43, 44]. The peaks between 30° and 50° can be interpreted as diffraction peaks of transition metals and their compounds [11, 45], such as metals, nitrides or carbides [8, 24]. And the metal-based nanoparticles can act as guest groups to enhance the activity of nearby M-N-C sites [25], which can be beneficial to modulate the electronic structure of M-N-C/F catalysts [10, 46]. Page 7/20 The chemical states and coordination environment of the prepared catalysts were characterized by XPS. As shown in Fig. 4b, Mn Pc/FCNT shows a peak at 642.3 eV (Mn 2p3/2), which is negatively shifted by 1.0 eV in Mn Pc/FCNT800. For Mn Pc/FCNT and Mn Pc/FCNT800, Mn3+ and Mn4+ are located at 641.5/642.8 eV and 641.0/642.5 eV [47]. In addition, there is a peak located at 638.6 eV in Mn Pc/FCNT800, which can be explain as the metallic manganese [48], indicating the partial reduction of manganese through carbothermal reduction. Furthermore, the generation of metal monomers and the metal carbides could generate new active sites and increase the reaction area of the catalyst in conjunction with the M-Nx ligand structure [17]. For Co Pc/FCNT800 and Pc/FCNT800, the peaks at 778.1 eV (Fig. 4c) and 852.6eV (Fig. 4d) can also be interpreted as the central metal obtained by reduction during pyrolysis [24]. In Fig. 4c, Co Pc/FCNT shows a peak at 781.3 eV (Co 2p3/2), which is negatively shifted by 0.9 eV in Co Pc/FCNT800. For Co Pc/FCNT and Co Pc/FCNT800, Co2+ and Co3+ are located at 781.2/786.6 eV and 780.8/785.8 eV [49, 50]. As shown in Fig. 4d, Ni Pc/FCNT shows a peak at 855.4 eV (Ni 2p3/2), which is negatively shifted by 0.5 eV in Ni Pc/FCNT800. For Ni Pc/FCNT and Ni Pc/FCNT800, Ni2+ and Ni3+ are located at 854.4/856.4 eV and 854.9/856.2 eV [51, 52]. As presented in Fig. Electrochemical characterization The higher electrochemical active surface area (ECSA) of MnPc/FCNT800 compared to Pt/C can be attributed to the porous structure on the carbon nanotubes loaded with MnPc/FCNT800, which is able to provide more active sites and has advantages in accessing the active centers. Electrochemical characterization The ORR performances of the synthesized composites and Pt/C (20 wt%) were tested in 1 M KOH solution using a rotating disc electrode (RDE) with O2-saturated. LSV curves show that MnPc/FCNT800 exhibits the best ORR catalytic activity among all the M-N-C catalysts, with a E1/2 of 0.9 V, while the E1/2 for Pt/C is 0.87 V (Fig. 5a). Figure 5b exhibited the Nyquist plots of the synthesized catalysts, which were Page 8/20 Page 8/20 obtained from electrochemical impedance spectroscopy (EIS) measurements. And MnPc/FCNT800 shows the smallest charge-transfer resistance, which could facilitate the charge transfer process of the oxygen electrode. In addition, MnPc/FCNT800 exhibits the smallest Tafel slopes of 60.4 mV dec− 1, which indicates that the reaction kinetics have been significantly enhanced in MnPc/FCNT800 (Fig. 5c). And fluorination has been shown to be an effective way to enhance catalyst activity as the excellent electrocatalytic ORR performance exceeds most previously M-N-C catalysts in both overpotential and tafel slope (Table S1). Moreover, after 5000 continuous potential cycles during ADT, MnPc/FCNT800 is demonstrated a negligible decay in the onset potential and the half-wave potential (Fig. 5d). As shown in Fig. 5e, MnPc/FCNT800 exhibits an ORR activity decay of only 12.1% in a 20 h long-term CA test, which is much lower than the 38.3% of Pt/C (Fig. 5e). These results confirm that the MnPc/FCNT800 shows the excellent electrocatalytic activity and stability for oxygen reduction reactions under alkaline conditions. Figure 6a presents the LSV curves recorded for the MnPc/FCNT800 catalyst in an O2-saturated 1 M KOH electrolyte at different rotation rates, with a scan rate of 50 mV s− 1, which will help determine the number of transferred electrons per O2 molecule and yield of hydrogen peroxide production during ORR. As displayed in Fig. 6b, the high electron transfer rate and low H2O2 yield (< 5%) indicate the presence of a direct four-electron ORR electrocatalytic pathway on MnPc/FCNT800. Furthermore, the calculation result of Koutecky-Levich (K-L) equation (Fig. 6c) reveals that the kinetic current density (JK) of MnPc/FCNT800 at 0.85 V (62.157 mA cm− 2) is much higher than that of commercial Pt/C (7.966 mA cm− 2), which confirms the prominent ORR activity of MnPc/FCNT800. Figure 6d shows the cyclic voltammogram (CV) curves of MnPc/FCNT800 and Pt/C recorded in O2-saturated 1 M KOH electrolyte at a scan rate of 50 mV s− 1. Compliance with ethical standards Conflict of interest: The authors declare no conflict of interest. Conflict of interest: The authors declare no conflict of interest. Author contributions Zhichuan Zheng, Xuekun Hong, Xiaxi Yao, Peng Du and Ming Lei put forward the idea and validated the paper. Zhichuan Zheng, Dajun Wu and Kai Huang investigated the literature and synthetized the electrocatalysts. Zhichuan Zheng and Ning Sun participated in the micromorphology characterizations. Zhichuan Zheng, Yawei Kuang and Debao Zhang performed the structural characterizations and conducted the electrocatalytic performance testing. All of the authors participated in the writing of original draft and contributed to the paper. Conclusion In summary, a simple pyrolysis method was applied to synthesis M-N-C catalysts loaded on fluorinated carbon nanotubes for oxygen reduction reactions. The morphology of CNTs after pyrolysis still maintain the mesoporous structure and metal-based nanoparticles formed during synthesis can be well immobilized on fluorinated carbon nanotubes, which contribute to the increase of active sites and improvement of electrocatalytic performance. And the fluorine atoms on CNTs can coordinate with the transition metals and polarize the carbon atoms, thus adjusting the M-N4 structure of the transition metal phthalocyanine complexes. In this work, the MnPc/FCNT800 catalyst shows outstanding electrocatalytic activity and stability for ORR, such as a half-wave potential of 0.9 V and only 12.1% decay after 20 h long-term CA test, which exceeds the commercial catalysts as well as most of the reported M-N-C catalysts. The excellent performance of M-N-C/F catalysts in ORR can be be attributed to fluorine atom doping and optimization of electronic structure of M-N-C composites. And this work also provides a simple and effective way to design and develop non-precious metal-based catalysts for efficient ORR. Page 9/20 Page 9/20 Funding This study is supported by the National Natural Science Foundations of China (Grant Nos. 62274017 and 61974011),  the Fundamental Research Funds for the Central Universities (2021XD-A04-2), the Fund of State Key Laboratory of Information Photonics and Optical Communications (Beijing University of Posts and Telecommunications, P.R. China) and BUPT Excellent Ph.D. Students Foundation (CX2022240). References 1. Tavakkoli M, Flahaut E, Peljo P, Sainio J, Davodi F, Lobiak E V, Mustonen K, Kauppinen E I (2020) Mesoporous single-atom-doped graphene–carbon nanotube hybrid: Synthesis and tunable electrocatalytic activity for oxygen evolution and reduction reactions. ACS Catal. 10 (8): 4647-4658. http://dx.doi.org/10.1021/acscatal.0c00352 1. 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Geng H Z, Rosen R, Zheng B, Shimoda H, Fleming L, Liu J, Zhou O (2002) Fabrication and properties of composites of poly (ethylene oxide) and functionalized carbon nanotubes. Adv. Mater. 14 (19): 1387-1390. http://dx.doi.org/10.1002/1521-4095(20021002)14:19<1387::AID-ADMA1387>3.0.CO;2- 40. Geng H Z, Rosen R, Zheng B, Shimoda H, Fleming L, Liu J, Zhou O (2002) Fabrication and properties of composites of poly (ethylene oxide) and functionalized carbon nanotubes. Adv. Mater. 14 (19): 1387-1390. http://dx.doi.org/10.1002/1521-4095(20021002)14:19<1387::AID-ADMA1387>3.0.CO;2- Q Page 13/20 41. Li S, Tang X, Zhang Y, Lan Q, Hu Z, Li L, Zhang N, Ma P, Dong W, Tjiu W, Wang Z, Liu T (2022) Corrosion-resistant graphene-based magnetic composite foams for efficient electromagnetic absorption. ACS Appl. Mater. Interfaces 14 (6): 8297-8310. http://dx.doi.org/10.1021/acsami.1c23439 42. 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Chem. 59 (23): 17134-17142. http://dx.doi.org/10.1021/acs.inorgchem.0c02420 54. Nakajima T, Koh M, Gupta V, Zemva B, Lutar K (2000) Electrochemical behavior of graphite highly fluorinated by high oxidation state complex fluorides and elemental fluorine. Electrochim. Acta 45 (10): 1655-1661. http://dx.doi.org/10.1016/s0013-4686(99)00389-8 54. Figures Figures Figure 1 Schematic diagram of the synthesis of MnPc/FCNT800 by pyrolysis. Figure 1 Schematic diagram of the synthesis of MnPc/FCNT800 by pyrolysis. Schematic diagram of the synthesis of MnPc/FCNT800 by pyrolysis. Page 15/20 Page 15/20 Figure 2 (a) SEM image of MnPc/FCNT800, (b) TEM image of MnPc/FCNT800, (c) TEM image of CoPc/FCNT800, (d) TEM image of NiPc/FCNT800, (e) TEM image of Pc/FCNT800, (f) HRTEM images of MnPc/FCNT800. Figure 2 (a) SEM image of MnPc/FCNT800, (b) TEM image of MnPc/FCNT800, (c) TEM image of CoPc/FCNT800, (d) TEM i f NiP /FCNT800 ( ) TEM i f P /FCNT800 (f) HRTEM i f M P /FCNT800 (a) SEM image of MnPc/FCNT800, (b) TEM image of MnPc/FCNT800, (c) TEM image of CoPc/FCNT800, (d) TEM image of NiPc/FCNT800, (e) TEM image of Pc/FCNT800, (f) HRTEM images of MnPc/FCNT800. (a) SEM image of MnPc/FCNT800, (b) TEM image of MnPc/FCNT800, (c) TEM image of CoPc/FCNT800, (d) TEM image of NiPc/FCNT800, (e) TEM image of Pc/FCNT800, (f) HRTEM images of MnPc/FCNT800. ( ) g / , ( ) g / , ( ) g / , (d) TEM image of NiPc/FCNT800, (e) TEM image of Pc/FCNT800, (f) HRTEM images of MnPc/FCNT800. Page 16/20 Figure 3 XRD patterns of (a) MnPc/FCNT800 and MnPc/FCNT, (b) CoPc/FCNT800 and CoPc/FCNT, (c) NiPc/FCNT800 and NiPc/FCNT, and (f) Pc/FCNT800and Pc/FCNT. Figure 3 XRD patterns of (a) MnPc/FCNT800 and MnPc/FCNT, (b) CoPc/FCNT800 and CoPc/FCNT, (c) NiPc/FCNT800 and NiPc/FCNT and (f) Pc/FCNT800and Pc/FCNT Figure 3 XRD patterns of (a) MnPc/FCNT800 and MnPc/FCNT, (b) CoPc/FCNT800 and CoPc/FCNT, (c) NiPc/FCNT800 and NiPc/FCNT, and (f) Pc/FCNT800and Pc/FCNT. Page 17/20 Figure 4 XPS spectra of(a) survey, (b) Mn 2p, (c) Co 2p, (d) Ni 2p, (e) F 1s bef 800°C pyrolysis, (g) F 1s after 800°C pyrolysis, (h) N 1s after 800°C XPS spectra of(a) survey, (b) Mn 2p, (c) Co 2p, (d) Ni 2p, (e) F 1s before 800°C pyrolysis, (f) N 1s before 800°C pyrolysis, (g) F 1s after 800°C pyrolysis, (h) N 1s after 800°C pyrolysis. Page 18/20 Page 18/20 Page 18/20 Figure 5 ORR electro-catalytic performance in O2-saturated 1 M KOH solution. (a) LSV curves, (b) Nyqu obtained from EIS measurements, (c) Tafel plots, (d) ADT test, (e) Chronoamperometry (CA) c Figure 5 ORR electro-catalytic performance in O2-saturated 1 M KOH solution. (a) LSV curves, (b) Nyquist plot obtained from EIS measurements, (c) Tafel plots, (d) ADT test, (e) Chronoamperometry (CA) curves. Figures Figure 5 Figure 5 Figure 6 (a) ORR polarization curves of MnPc/FCNT800 at various rotation rates, (b) ORR electron transfer rate and H2O2 yield of MnPc/FCNT800, Pt/C, (c) Comparison of JK values for different catalysts, (d) Electrochemical active area in O2-saturated 1 M KOH solution of MnPc/FCNT800, Pt/C. (a) ORR polarization curves of MnPc/FCNT800 at various rotation rates, (b) ORR electron transfer rate and H2O2 yield of MnPc/FCNT800, Pt/C, (c) Comparison of JK values for different catalysts, (d) Electrochemical active area in O2-saturated 1 M KOH solution of MnPc/FCNT800, Pt/C. Figure 5 ORR electro-catalytic performance in O2-saturated 1 M KOH solution. (a) LSV curves, (b) Nyquist plots obtained from EIS measurements, (c) Tafel plots, (d) ADT test, (e) Chronoamperometry (CA) curves. 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English
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Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis
Frontiers in plant science
2,022
cc-by
9,558
TYPE Original Research PUBLISHED 07 November 2022 DOI 10.3389/fpls.2022.1005711 TYPE Original Research PUBLISHED 07 November 2022 DOI 10.3389/fpls.2022.1005711 TYPE Original Research PUBLISHED 07 November 2022 DOI 10.3389/fpls.2022.1005711 Angelica sinensis, rhizosphere microbiota, metabolomics, plant development stage, correlation, quality COPYRIGHT © 2022 Chen, Feng, Yan, Liu, Zhou, Guo, Yu and Duan. 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. Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis OPEN ACCESS EDITED BY Linkun Wu, Fujian Agriculture and Forestry University, China REVIEWED BY Wei Zhang, Nanjing Normal University, China Youn-Sig Kwak, Gyeongsang National University, South Korea *CORRESPONDENCE Hui Yan yanhui@njucm.edu.cn Pei Liu liupei@njucm.edu.cn Jin-Ao Duan dja@njucm.edu.cn SPECIALTY SECTION This article was submitted to Plant Symbiotic Interactions, a section of the journal Frontiers in Plant Science RECEIVED 28 July 2022 ACCEPTED 17 October 2022 PUBLISHED 07 November 2022 EDITED BY Linkun Wu, Fujian Agriculture and Forestry University, China REVIEWED BY Wei Zhang, Nanjing Normal University, China Youn-Sig Kwak, Gyeongsang National University, South Korea Jing-Mei Chen, Wei-Meng Feng, Hui Yan*, Pei Liu*, Gui-Sheng Zhou, Sheng Guo, Guang Yu and Jin-Ao Duan* Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, China Angelica sinensis is a medicinal plant widely used to treat multiple diseases in Asia and Europe, which contains numerous active components with therapeutic value. The interaction between root and rhizosphere microorganisms is crucial for the growth and quality formation of medicinal plants. But the micro-plant-metabolite regulation patterns for A. sinensis remain largely undetermined. Here, we collected roots and rhizosphere soils from A. sinensis in seedling stage (M) and picking stage (G), respectively cultivated for one year and two years, generated metabolite for roots, microbiota data for rhizospheres, and conducted a comprehensive analysis. Changes in metabolic and microbial communities of A.sinensis over growth were distinct. The composition of rhizosphere microbes in G was dominated by proteobacteria, which had a strong correlation with the synthesis of organic acids, while in M was dominated by Actinobacteria, which had a strong correlation with the synthesis of phthalide and other organoheterocyclic compounds, flavonoids, amines, and fatty acid. Additionally, co-occurrence network analysis identified that Arthrobacter was found to be strongly correlated with the accumulation of senkyunolide A and n- butylidenephthalide. JGI 0001001.H03 was found to be strongly correlated with the accumulation of chlorogenic acid. Based on rhizosphere microorganisms, this study investigated the correlation between root metabolism and rhizosphere microbiota of A. sinensis at different growth stages in traditional geoherb region, which could provide references for exploring the quality formation mechanism of A. sinensis in the future. CITATION Chen J-M, Feng W-M , Yan H, Liu P, Zhou G-S, Guo S, Yu G and Duan J-A (2022) Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis. Front. Plant Sci. 13:1005711. doi: 10.3389/fpls.2022.1005711 Introduction NIAHS because of its unique cultivation conditions, traditional cultivation techniques, and processing techniques, as well as the indigenous microbial flora of Min County, which plays a vital role in shaping the quality of A. sinensis. Angelica sinensis (Oliv.) Diels (Umbelliferae family), known as Dang Gui (in Chinese), is a traditional medicinal and edible plant which was recommended as a first-line treatment for gynecological diseases and is widely used in Asia and Europe. A. sinensis is mainly used in treating female menstrual disorders and amenorrhea, as well as invigorating and replenishing blood, and lubricating the intestines (Hook, 2014; Wei et al., 2016). Numerous pharmacological effects of A. sinensis include enhanced immune function, anti-arrhythmic, heart protection, anti-atherosclerotic, and inhibited platelet aggregation and smooth muscle inflammation to prevent myocardial infraction events (Hu et al., 1991; Liu et al., 2001; Zhou et al., 2015; Yao et al., 2015). Diverse habitats and climates have shaped the qualities and efficacy of medicinal plants. In China, it is generally believed that Geo-authentic Chinese medicinal materials are selected by long-term clinical application which considered with satisfactory clinical efficacy and stable quality and produced in specific ecological environment. A Chinese group standard, Daodi Chinese Medicinal Material – MinDanggui, defines the geoherb region of A. sinensis explicitly. Standard states that the geoherb region of A. sinensis are Min County and its surrounding areas, such as Tanchang, Zhang, Weiyuan, Zhuoni, Lintan Counties in Gansu province, which are located in the eastern branch of Minshan Mountains in southern Gansu province and the transitional zone where loess Plateau in Longzhong and the Qinghai – Tibet Plateau meet (Huang, Guo, & Zhan, 2020). A. sinensis is a typical ecological dominant medicinal plant, and ecological environment is an essential factor affecting the quality of traditional Chinese medicine (TCM). Min County and its surrounding areas are located between 33°46′~35°07′ N, 103°14′~104°59′ E, the altitude ranges from 2040 m to 3747 m, with cool humid climate, moderate rainfall, rich brown soil and black soil, which are favorable to the growth of A. sinensis and are the main areas for the production of Angelica sinensis (Zhang et al., 2016). In 2014, the Radix Angelica sinensis planting system in Min County of China was selected as the second batch of China- Secondary compounds in plants (PSCs) are crucial messengers in plant ecology through chemical interactions with plant hosts and the outside world. Introduction For example, they can attract herbivorous predators and pollinators to help spread seeds by releasing secondary metabolites. Plant chemical barriers are also formed to prevent invasion by pathogens and herbivorous predators. In turn, pathogen and predator pressures, microbes, and microclimates all influence the levels and types of secondary metabolites that plants synthesize and release (Ehlers et al., 2020). Studies have shown that phthalides, organic acids, and polysaccharides are the primary secondary metabolites in determining the biological activities and pharmacological properties of A. sinensis (Wei et al., 2016). The root microbial community could affect the growth and yield of A. sinensis. Studies have shown that Bacillus isolated from the rhizosphere can promote biomass accumulation and plant growth, and can increase the contents of butylidenephthalide (36 ~ 415%) of A. sinensis (Feng et al., 2020). Nonetheless, little is known about the mechanisms of the accumulation of A. sinensis metabolites and the microbial communities. Soil microbial diversity is closely related to ecosystem functions such as nutrient decomposition and recycling, and positively related to plant productivity in the earth’s microbiome (Delgado-Baquerizo et al., 2020). Plant rhizosphere is the most abundant part of microbial activity. Plants selectively assemble specialized functional rhizosphere microbiota from bulk soil for plant fitness. On the other hand, many rhizosphere microorganisms benefit plants by assisting with acquiring supplements from soil and suppressing pathogen invasion (Bulgarelli et al., 2013; Leach et al., 2017; Ling et al., 2022). The variety and composition of rhizosphere bacterial communities result from a combination of plant species and soil properties (Ling et al., 2022). However, these plant microbes are not constant; they vary with environmental stimuli, including abiotic stress and biotic factors. The microbiota structure is impacted by the physical and chemical aspects of the environment (Pang et al., 2021). It has been determined that the rhizosphere microbiota is a highly modular but unstable network system by the effects of plant hosts and other microbes. This characteristic reflects the interaction between microorganisms and their adaption to dynamic conditions (Ling et al., 2022). Studies have shown that root metabolism and microbiota are sensitive to and driven by changes in plant growth stages. Results showed a correlation between stable and dynamic root microbial taxa and root lipids during plant growth (Bourceret et al., 2022). Plants can influence their microbiome by secreting a variety of metabolites, which in turn can influence the metabolome of host plants (Pang et al., 2021). Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis CITATION Chen J-M, Feng W-M , Yan H, Liu P, Zhou G-S, Guo S, Yu G and Duan J-A (2022) Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis. Front. Plant Sci. 13:1005711. doi: 10.3389/fpls.2022.1005711 CITATION Chen J-M, Feng W-M , Yan H, Liu P, Zhou G-S, Guo S, Yu G and Duan J-A (2022) Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis. Front. Plant Sci. 13:1005711. doi: 10.3389/fpls.2022.1005711 COPYRIGHT © 2022 Chen, Feng, Yan, Liu, Zhou, Guo, Yu and Duan. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science 01 frontiersin.org 10.3389/fpls.2022.1005711 Chen et al. Soil DNA extraction and sequencing According to manufacturer’s approach, total DNA was extracted from per sample using the HiPure Soil DNA Kits (Magen, Guangzhou, China). The bacterial sequence was amplified with the primers 806R (5’-GGACTACHVGG GTATCTAAT-3’) and 341F (5’-CCTACGGGNGGCWGCAG- 3’). PCR reactions were performed in triplicate in 50 mL mixture containing 3 mL of 25 mM MgSO4, 5 mL of 2 mM dNTPs, 5 mL of 10 × KOD Buffer, 1.5 mL of each primer (10 mM), 100 ng of template DNA, and 1mL of KOD Polymerase. The bacterial V3-V4 hypervariable region of 16S rRNA was amplified by PCR (94°C for 2 min, followed by 30 cycles at 98°C for 10 s, 62-66°C for 30 s, and 68°C for 30 s, and a final extension at 68°C for 5 min) using primers. Related PCR reagents were from TOYOBO, Japan. According to the manufacturer, the PCR amplicons were extracted from 2% agarose gels and purified using the AMPure XP Beads (Beckman Agencourt, USA), and quantified using ABI StepOnePlus Real- Time System (Life Technologies, Foster City, USA). Purified amplicons were pooled in equimolar and paried-end sequenced (PE250) on an Illumina platform in accordance with the standard protocols. Research on rhizosphere microbiome for sustainable ecosystem development has focused on identifying the core of plant microbiota and clarifying the functional mechanisms that regulating plant-microbiome interactions. (Busby et al., 2017; de Vries et al., 2020). Here, we collected 14 batches of annual seedlings from the traditional geoherb region of A. sinensis and planted them in the same experimental field in Min county, Gansu province. The same measures of cultivation and management were carried out during the planting period, and it was allowed to grow for 180 days before harvest. Seedlings and mature roots of A. sinensis were measured the root metabolites and medicinal components using UPLC-QTOF-MS/MS and performed 16S rRNA sequencing on rhizosphere samples. We generated metabolites profiles, medicinal components and microbial community composition for seedlings (M) and mature roots (G) of A. sinensis, profiled the differential metabolites and medicinal components of A. sinensis over growth, deciphered the compositional characteristics of microbes colonizing A. sinensis roots in different growth stage, and characterized the dynamic regulations between the accumulation of secondary metabolites and rhizosphere microbial community. We focused the following questions during the A. sinensis transplanting cultivation model: (1) Does the synthesis of secondary metabolites and the contents of principal bioactive constituents of A. Sample collection Collection of 14 batches of seedlings of annual A. sinensis in Min County, Tanchang County, Zhang County, Lintan County and Zhuoni County in Gansu Province, China. The seedlings were planted in Min County Medicinal Plants Growing Technology Extension Centre (34°22′30” N, 104°53′20” E; black soil, pH ≈8.0) with same measures of cultivation and management during the planting period, and it was cultivated to grow for 180 days then harvest. Three samples for each batch were randomly selected, each sample was comprised of 5 healthy plants, and 42 samples were taken for each growth stage. The seedlings and mature roots were both authenticated by Dr. Hui Yan. Carefully, uproot fresh plants and shake loose bulk soil that clings to the roots. Remove the rhizosphere soil which tightly attached to roots with a sterile brush. Fresh plants and soil samples were stored at -80 °C refrigerator for analyses. Soil DNA extraction and sequencing sinensis change at seedling stage and picking stage? (2) The assembly of rhizosphere microbial community changes with the different growth stage. How does the rhizosphere microbial community of A. sinensis assemble over growth? (3) Are there any correlations between the accumulation of secondary metabolites and the change of rhizosphere microbial community? Materials and methods microbial communities and the composition of root exudates vary in the stages of plant growth. The underlying mechanism may be related to the substrates preference of microorganisms (Zhalnina et al., 2018). On the other hand, the same plant growing in different habitats may affect the type and content of plant secondary metabolites, and these differences may be closely related to indigenous microbes (Köberl et al., 2013). A. sinensis is an herbaceous perennial plant. In cultivation, it has a three-year growth cycle. Traditionally, seedlings are fostered in uncultivated alpine meadows in the first year, transplanting the seedlings, harvesting the fleshy roots in the second year, and collecting the seeds in the third year (An et al., 2020). The cultivation pattern of transplanting determined that A. sinensis had to adapt to different habitats and corresponding microbial environments during its growth. Studies of specific ecosystems observed that alpha- diversity in rhizosphere of cultivated land continued to decline (-0.8% ~ -9.3%), while in grassland and forest ecosystems, there were no significant differences in Faith’s phylogenetic diversity and species richness between the rhizosphere and bulk soil (Ling et al., 2022). Therefore, the microbial environment in different habitats has different shaping effects on the quality of medicinal plants. Root length, soil pH, climate temperature, rainfall, root diameter, and plant weight are also considerable factors affecting the composition of A. sinensis rhizosphere microbial community (An et al., 2020). Introduction The chemical assemblage of Abbreviations: PSCs, Plant secondary compounds; UPLC-QTOF-MS/MS, Ultra Performance Liquid Chromatography-Quadrupole Times of Flight- Mass/Mass; LC-MS/MS, liquid chromatography coupled with high- resolution tandem MS; PCA, Principal components analysis; OPLS-DA, Orthogonal partial least-squares discrimination analysis; NMDS, Nonmetric multidimensional scaling; VIP, Variable importance for projection; ANOVA, Analysis of varia; OUT, Operational taxonomic units; PCR, Polymerase chain reaction; DBP, Dibutyl phthalate; DMP, Dimethyl phthalate; BXs, Benzoxazinoids. 02 Frontiers in Plant Science frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 Statistical analyses Using IBM SPSS Statistics 19 and R (4.2.0) to perform all statistical analyses. The mean and standard deviation were calculated by multiple comparison analysis and analysis of variance (ANOVA) for statistical tests. The univariate approach depends on t-tests (or their nonparametric alternatives). Spearman correlation coefficients were calculated using R v 4.2.0. RStudio v 2022.02.1 and SIMICA v14.1 were employed to perform PCA and OPLS-DA. Differences between groups were considered significant when p < 0.05. TBtools v 1.09876 (Chen et al., 2020) was employed to perform heatmaps and hierarchical clustering. Statistical analysis of rhizosphere microorganisms taxonomic differences between groups using STAMP v 2.1.3. Cytoscape v3.9.1 and MetScape v3.1.3 were employed to visualize the co-occurrence networks of microbiota and metabolome data. Results were built and optimized using OriginPro 2021. Bioinformation analysis FASTP (Chen et al., 2018) (version 0.18.0) mainly used for the quality control of the raw reads which containing less than 50% of bases with quality (Q-value)>20 and more than 10% of unknown nucleotides (N). The splicing was done by FLASH (Magoc and Salzberg, 2011) (version 1.2.11). The clean tags were Frontiers in Plant Science 03 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 desolvation gas flow 600 L/h; sample cone voltage 30 V; cone gas flow 50 L/h; extraction cone voltage 2.0 V; secondary collision energy 25 ~ 45 V. Quantitative mass spectrometric analysis was performed using Xevo Triple Quadrupole MS (Waters Corp., Milford, MA USA) equipped with electrospray. ESI-MS spectra were obtained by using MRM mode. grouped into operational taxonomic units (ASVs) by UPARSE (Edgar, 2013) (version 9.2.64) according to the similarity ≥97%. The representative OUT sequences was analyzed by RDP classifier (Wang et al., 2007) (version 2.2) based on SILVA (Pruesse et al., 2007) database (version 132), were classified into organisms according to the confidence threshold of 0.8. Diversity indices of data were performed in QIIME (version 1.9.1) and R packages (version 2.5.3). Statistic analysis of Anosim test, Tukey’s HSD test, Welch’s t-test, Kruskal-Wallis H test, Wilcoxon rank test and Adonis was calculated in R project Vegan package (version 2.5.3). Medicinal components and metabolite analysis The ACQUITY™UPLC system was used to perform the medicinal components of A. sinensis with an A Thermo Syncronis C18 (2.1 mm × 100 mm, 1.7 mm) column. The 0.1% formic acid in chromatographic (A) and acetonitrile (B) were used as mobile phase. The gradient elution were as follow: 0 ~ 2 min, 5% ~ 10% B; 2 ~ 10 min, 10% ~ 40% B; 10 ~ 13 min, 40% ~ 40% B; 13 ~ 19 min, 40% ~ 50.8% B; 19 ~ 23 min, 50.8% ~ 90% B; 23 ~ 24 min, 90% ~ 90% B; 24 ~ 24.5 min, 90% ~ 5% B; 24.5 ~ 26 min, 5% ~ 5% B The detection wavelength are 260 nm, 280 nm and 320 nm. The medicinal components (ferulic acid, chlorogenic acid, Z- ligustilide, senkyunolide A, senkyunolide H, senkyunolide I, n- butylphthalide, coniferyl ferulate) were measured by comparing with the calibration curves. The 8 reference compounds bought from National Institutes for Food and Drug Control (China), Shanghai Macklin Biochemical Co., Ltd (China) and Nanjing Jin Yibai Biological Technology Co., Ltd (China) respectively. The Waters ACQUITY™Synapt Q-TOF mass spectrometer equipped with an electrospray ionization (ESI) source. ESI-MS spectra was generated in both positive (ESI+) and negative (ESI-) ion modes through scanning from 100-1000 Da. The flow rate was 0.4 mL·min-1, column temperature was kept at 35°C, with 2 mL injection volume. The optimized conditions include: capillary voltage 3 kV for both positive ion mode and negative ion mode; source temperature 120°C; desolvation gas temperature 350°C; Bacterial alpha-diversity and beta-diversity The depth of sequencing in this experiment has basically covered all the species in the sample, as shown in the Figure S1. As shown in Table 1, the indices representing alpha-diversity were estimated as community richness and evenness. The diversity indices were lower in G compared with M, however, no significant differences in bacterial richness or diversity between M and G, as indicated by Sobs, Shannon, Simpson, TABLE 1 Richness and alpha-diversity indices of the different ASVs between group M and G. Group Sobs Shannon Simpson Chao1 Mean SD Mean SD Mean SD Mean SD M 4082.881a 379.9242 9.30265a 0.778844 0.98772a 0.020017 4951.628a 391.3516 G 3837.619a 357.408 9.00981a 0.748578 0.98145a 0.019449 4667.415a 368.1226 Group Ace Goods coverage Pielou Pd Mean SD Mean SD Mean SD Mean SD M 5125.335a 394.3544 0.98369a 0.001361 0.7754a 0.05825 359.3468a 26.01045 G 4777.339b 366.216 0.98609b 0.001127 0.7562a 0.05588 340.168a 24.19418 Different letters in the same column indicate a significant difference (ANOVA followed by Tukey s-b(k), n=14, p<0.05, average value, SD standard deviation). TABLE 1 Richness and alpha-diversity indices of the different ASVs between group M and G. 04 Frontiers in Plant Science Chen et al. 10.3389/fpls.2022.1005711 10.3389/fpls.2022.1005711 Actinobacteria declined, showed a negative response to plant growth. The relative abundance of Proteobacteria increased, showed a positive response to plant growth. The relative abundance of Acidobacteria showed no significant difference during the plant growth. This observation is consistent with many other successional studies: including Arabidopsis thaliana (Roller et al., 2016), wheat (Bulgarelli et al., 2015), rice (Lu et al., 2006), switchgrass (Mao et al., 2014), maize (Peiffer et al., 2013) and Avena barbata (Zhalnina et al., 2018). It is indicating that the reconstruction of rhizosphere microbial communities had a general rule in different plant species and soil types. Research showed (Zhalnina et al., 2018), rhizosphere microbes that respond positively to plant growth are predicted to have longer generation times based on codon-usage bias, which means that their genomes are characterized by slower growth rates. Since slower-growing organisms can have higher substrate utilization efficiency (Chaparro et al., 2013), growth efficiency may be preferred over growth rate in the rhizosphere. Top 10 phyla with significant differences between group M and G are shown in Figure 2B. Chao1, Pielou, and Pd. There are significant differences in coverage of ASV with low abundance between M and G, as indicated by Goods coverage index. Bacterial alpha-diversity and beta-diversity The ACE index was significantly lower in G compared with M, showing that the richness and evenness of community in M were significantly higher than in G. NMDS was utillzed to analyze the comparability of the assemblage of bacterial communities between group M and G at the ASV level (Figure 1). Group M was located in the left part of the graph, and the distribution of different samples was discrete. The group G was gathered at the right, and the distribution of different samples was centralized. The results showed a distinctive difference in bacterial community structures between group M and G. Taxonomic composition analysis The species composition of rhizosphere microorganisms was analyzed at phylum and genus level. Figure 2A shows Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Verrucomicrobia, Gemmatimonadetes, Chloroflexi, Firmicutes, Patescibacteria are most abundant at the phylum level, representing about 96.6% of the microorganisms detected in the 84 soil samples. Compared with group M, the abundance of Proteobacteria, Gemmatimonadetes, Patescibacteria, and Firmicutes have significantly (p <0.05) increased while the abundance of Actinobacteria, Bacteroidetes, Verrucomicrobia has significantly (p <0.05) decreased in group G. Proteobacteria, Acidobacteria, Actinobacteria were the most three abundant in the rhizosphere of A. sinensis. The relative abundance of At the genus level, the dominant genera were Pseudomonas, Sphingomonas, RB41, Flavobacterium, Pedobacter, Candidatus Udaeobacter, Stenotrophomonas, Acinetobacter, Gemmatimonas, Chthoniobacter. The hierarchical clustering of TOP 35 genera showed that there were significant differences in microbial community structure between group M and G as shown in Figure 3A. The dominant genera also showed significant differences. Compared with group M, the abundance of Acinetobacter, Gemmatimonas, and Sphingomonas has significantly (p <0.05) increased while the abundance of RB41, Pedobacter, Candidatus Udaeobacter, Chthoniobacter has FIGURE 1 Unweighted unifrac nonmetric multidimensional scaling of the bacterial community compositions in the soil between group M and G. FIGURE 1 Unweighted unifrac nonmetric multidimensional scaling of the bacterial community compositions in the soil between group M and G 05 Frontiers in Plant Science frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 A B FIGURE 2 Taxonomic composition and abundance distribution of bacteria at the phylum level between group M and G (A). The top 10 phyla with significant differences between group M and G (B). A A A B FIGURE 2 Taxonomic composition and abundance distribution of bacteria at the phylum level between group M and G (A). The top 10 phyla with significant differences between group M and G (B). B B B FIGURE 2 Taxonomic composition and abundance distribution of bacteria at the phylum level between group M and G (A). The top 10 phyla w significant differences between group M and G (B). significantly (p <0.05) decreased in group G as shown in Figure 3B. In addition, the contents of comuside, phenylethyl primeveroside and QUIZALOFOP-ETHYL, 2-dicyclohexylphosphino-2’,6’- dimethoxybiphenyl and [12]-gingerdione in group G were higher than M, while the levels of phenylpropanoids, phthalide and other organoheterocyclic compounds, flavonoids, amines, and fatty acid were lower compared to group M. These results suggest that different growth stages of A. Variations in metabolites of Angelica sinensis between M and G Untargeted metabolomics facilitates botanical metabolomics studies through efficient high-throughput screening techniques. At present, LC-MS/MS has been widely used for metabolomics studying (Tsugawa et al., 2021). In this study, UPLC-QTOF-MS/ MS method was used to characterize metabolites in A. sinensis samples between M and G. Principal components analysis (PCA) (Figure 4A) and the OPLS-DA model (Figure S2) showed that the metabolic profiles varied significantly (p < 0.05) between groups, and the samples were clustered into two groups significantly. The hierarchical clustering heatmap (Figure 4B) showed the differences between group M and G, suggesting that the synthesis of secondary metabolites pattern at seedling stage and collection period of medicinal material are distinct. Further analysis (Table S1) combined with a hierarchical clustering heatmap (Figure 4B) revealed that group G had a higher level of organic acids, such as dicaffeoylquinic acid, chlorogenic acid, genipic acid, and ferulic acid. Taxonomic composition analysis sinensis have different secondary metabolite synthesis patterns, which indicate that changes in the composition of the secondary metabolites over time may contribute to the observed successional patterns in the rhizosphere microbiota (Figure 1). Metabolites with the Variable Importance for Projection (VIP) value greater than 1 and the p-value less than 0.05, combined with s-plot (covariance greater than 0.05) were screened as markers that contributing to grouping. A total of 29 markers were identified. (Table 2) (Zhang et al., 2019). Frontiers in Plant Science Quality evaluation in medicinal components of Angelica sinensis between M and G To further explain the cause of differences in metabolites of A. sinensis between group M and G, phthalides and organic acids Frontiers in Plant Science 06 frontiersin.org frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 A B FIGURE 3 Heatmap of TOP 35 bacteria at the genus level between group M and G (A). The top 10 genus with significant differences between group M and G (B). A B FIGURE 3 Heatmap of TOP 35 bacteria at the genus level between group M and G (A). The top 10 genus with significant differences between group M and G (B). means of shaping the microbial composition of the rhizosphere. The hierarchical clustering heatmap showed that the differential metabolites were clustered into two groups (Figure 4C), and the distribution and type of differential metabolites are highly consistent with Figure 4B. The results showed that the rhizosphere microbial community have different assembly patterns between group M and G, and the rhizosphere microbial community had a key contribution to grouping and quality-related factors of A. sinensis. Combined Figures 4B, C, we found that Actinobacteria, Proteobacteria and Bacteroidetes were predominant in synthesizing secondary metabolites such as phenylpropanoids, phthalide, and other organoheterocyclic compounds, flavonoids, amines, and fatty acid in the seedling stage or early growth stage of A. sinensis. Proteobacteria, Acidobacteria and Firmicutes were predominant in the synthesis of organic acid in the drug- producing stage or later growth stage of A. sinensis. Aromatic organic acids, such as cinnamic acid, have been found to shape plant rhizosphere microbes and influence plant-microbe interactions (Sasse et al., 2018; Cotton et al., 2019). Therefore, the were comprehensively analyzed. The result showed that the contents of medicinal components differed remarkably (p < 0.05) between groups. Specially, the level of coniferyl ferulate, senkyunolide A, and ligustilide of A. sinensis in group M were significantly (p < 0.01) higher than in group G (Figure 5). The level of chlorogenic acid in group G was significantly higher than in group M (p < 0.001). Correlation between differential microbial community and differential secondary metabolites synthesis of medicinal components and differential secondary metabolites is closely related to the growth stage of A. sinensis. The synthesis and secretion of secondary metabolites also affect the assembly of the rhizosphere microbial community. There showed a complex correlation between differential microbial community and differential secondary metabolites (Figure 6). For differential secondary metabolites, the flavonoids mulberroside F showed the greatest correlation with the differential bacterial members (Figure 6), and the phenol 4-Tert- Amylphenol, the amines dehydrophytosphingosine, the organic acid genipic acid and pantothenic acid, the organoheterocyclic loliolide are also important nodes that correlate with the differential bacterial members. 4-Tert-Amylphenol, loliolide and mulberroside F were negatively correlated with JGI 0001001.H03, Candidatus Solibacter, Aquicella and Bryobacter, while genipic acid and comuside was positively correlated with Candidatus Solibacter and Bryobacter, genipic acid was positively correlated with Candidatus Solibacter (r > 0.7). Various rhizodeposits may differentially influence the composition of the rhizosphere microbiome composition (Pascale et al., 2020). Recently studies showed that selected secondary including flavonoid, coumarin, benzoxazinoid, phytohormones, and triterpenes affect the succession of rhizosphere microorganisms of host plants. (Pang The characteristics of metabolites and rhizosphere microbial communities succession of Angelica sinensis over growth The utilization of specific chemical components of exudates by microorganisms has potential metabolic differences, which may be crucial for bacterial success in the rhizosphere (Zhalnina et al., 2018). Thus, the chemical composition of plant secondary metabolites synthesis and plant exudates may represent key Frontiers in Plant Science 07 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 A B C FIGURE 4 PCA scores for the comparison of metabolomic profiles between M and G (A). Hierarchical clustering heatmap of the differential metabolites of A. sinensis between group M and G (B). Covariation between differential abundant microbes and metabolites match against standards between group M and G (Spearman’s rank correlation) (n=3) (C). A C B B C FIGURE 4 PCA scores for the comparison of metabolomic profiles between M and G (A). Hierarchical clustering heatmap of the differential metabolites of A. sinensis between group M and G (B). Covariation between differential abundant microbes and metabolites match against standards between group M and G (Spearman’s rank correlation) (n=3) (C). Frontiers in Plant Science Correlation between the differential microbial community, differential secondary metabolites and medicinal components In the case pf medicinal plants, specific microorganisms may be directly related to the biosynthesis of medicinal components of the host plant. For example, Lysobacter was identified strongly associated with gene CYP72A154, which was required glycyrrhizic acid biosynthesis of Glycyrrhiza uralensis Fish (Zhong et al., 2022). Therefore, to further elucidate the relation between differential microbial community, differential secondary metabolites and medicinal components, two interactive networks were constructed (Figures 6, 7). The significance test and correlation of differential microbial community, differential secondary metabolites and medicinal components were showed in Figures S3, S4. Frontiers in Plant Science frontiersin.org 08 Chen et al. 10.3389/fpls.2022.1005711 TABLE 2 Differential metabolites for discriminating A. sinensis samples between M and G. Identification tR (min) Theoretical accurate mass (m/z) Q-TOF-MS (m/z) (ESI+/ESI-) Mass accuracy (ppm) MS/MS fragment ion (m/z) Glucose-1,3-mannose oligosaccharide 1.05 341.1089[M-H]- 341.1072[M-H]- -4.98 Phenylethanolaminium 1.53 120.0814[M+H-H2O]+ 120.0811[M+H-H2O]+ -2.50 Pantothenic acid 1.72 218.1034[M-H]- 218.1016[M-H]- -8.25 Vanillic acid 3.66 167.0344[M-H]- 167.0334[M-H]- -5.99 123.0447[M-H]- Mulberroside F 3.85 401.1453[M-H]- 401.1439[M-H]- -3.49 565.1557[M-H]-、 387.1641[M-H]- Chamaechromone 3.99 541.1140[M-H]- 541.1182[M-H]- 7.76 Phenylethyl primeveroside 4.79 415.1610[M-H]- 415.1592[M-H]- -4.34 cis-Ferulic acid 5.19 177.0552[M+H-H2O]+ 177.0553[M+H-H2O]+ 0.56 Chlorogenic acid 5.99 353.0873[M-H]- 353.0863[M-H]- -2.83 Dicaffeoylquinic acid 6.32 515.119[M-H]- 515.1181[M-H]- -1.75 QUIZALOFOP-ETHYL 6.4 353.0693[M-H20-H]- 353.0701[M-H20-H]- 2.27 (Z)-Resveratrol 4’-glucoside 6.7 389.1242[M-H]- 389.1223[M-H]- -4.88 373.1855[M-H]- Tangeritin 8.22 371.1136[M-H]- 371.1119[M-H]- -4.58 Loliolide 8.7 177.0916[M-H2O-H]- 177.0903[M-H2O-H]- -7.34 4-Tert-Amylphenol 10.15 163.1128[M-H]- 163.1113[M-H]- -9.20 1-(2,4,5-Trimethoxyphenyl)-1,2-propanedione 10.48 219.0657[M-H20-H]- 219.0645[M-H20-H]- -5.48 p-Hydroxyphenethyl trans-ferulate 10.64 313.1076[M-H]- 313.1064[M-H]- -3.83 134.0361[M-H]-、 193.0495[M-H]- 1-(3,4-Dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6- heptadiene-3,5-dione 11.34 353.1031[M-H]- 353.1016[M-H]- -4.25 203.0697[M-H]- Coniferyl ferulate 11.34 353.1031[M-H]- 353.1016[M-H]- -4.25 193.0488[M-H]-、 134.0362[M-H]- E-Ligustilide 14.55 191.1072[M+H]+ 191.1069[M+H]+ -1.57 Comuside 15.46 313.1156[M+2ACN+2H]+ 313.1154[M+2ACN +2H]+ -0.64 Senkyunolide D 15.48 221.0814[M-H]- 221.0804[M-H]- -4.52 Dehydrophytosphingosine 15.87 316.2846[M+H]+ 316.2853[M+H]+ 2.21 (10E,12Z)-(9S)-9-Hydroperoxyoctadeca-10,12-dienoic acid 15.88 311.2228[M-H]- 311.221[M-H]- -5.78 9(S)-HPODE 20 293.2117[M-H20-H]- 293.2104[M-H20-H]- -4.43 Genipic acid 20.42 185.0808[M+H]+ 185.0816[M+H]+ 4.32 2-Dicyclohexylphosphino-2’,6’-dimethoxybiphenyl 22 409.2302[M-H]- 409.2344[M-H]- 10.26 1-Hydroxy-3,6,7-trimethoxy-2-(2-hydroxy-3-methyl-3- butenyl)-8-(3-methyl-2-butenyl)-xanthone 23.15 453.1919[M-H]- 453.1938[M-H]- 4.19 [12]-Gingerdione 23.5 377.2686[M+H]+ 377.2673[M+H]+ -3.45 (r > 0.7), flavonoids and phenol were positively correlated with Thermomonas (r > 0.7). et al., 2021). In this paper, we classified the differential secondary metabolites of A. sinensis according to its chemical structure, and made a further Venn comparative analysis on the primary, and secondary metabolites of carbohydrates and glycosides, organic acid, flavonoids, phthalides, and other organoheterocyclic compounds, phenol (Figures 8A, B). Frontiers in Plant Science frontiersin.org Correlation between the differential microbial community, differential secondary metabolites and medicinal components The results showed that carbohydrates and glycosides, organic acid, and phenol were positively correlated with Candidatus Koribacter (r > 0.7), carbohydrates and glycosides, organic acid were positively correlated with Candidatus Solibacter (r > 0.7), flavonoids, phenol, Phthalide, and other Organoheterocyclic compounds were positively correlated with Pedobacter and Bosea (r > 0.7), flavonoids and organic acid were positively correlated with Iamia et al., 2021). In this paper, we classified the differential secondary metabolites of A. sinensis according to its chemical structure, and made a further Venn comparative analysis on the primary, and secondary metabolites of carbohydrates and glycosides, organic acid, flavonoids, phthalides, and other organoheterocyclic compounds, phenol (Figures 8A, B). The results showed that carbohydrates and glycosides, organic acid, and phenol were positively correlated with Candidatus Koribacter (r > 0.7), carbohydrates and glycosides, organic acid were positively correlated with Candidatus Solibacter (r > 0.7), flavonoids, phenol, Phthalide, and other Organoheterocyclic compounds were positively correlated with Pedobacter and Bosea (r > 0.7), flavonoids and organic acid were positively correlated with Iamia Flavonoids, phenol, phthalide, and other organoheterocyclic compounds were negatively correlated with JGI 0001001. H03, Candidatus Solibacter, Aquicella (r > 0.7), carbohydrates and glycosides, organic acids were negatively correlated with Clostridium sensu stricto 12, flavonoids and organic acid were negatively correlated with Burkholderia.Caballeronia. Paraburkholderia (r > 0.7), flavonoids and phenol were negatively correlated with Ralstonia (r > 0.7). From the above results, we can see that flavonoids, phthalides, and other organoheterocyclic compounds have consistent correlation with specific bacterial communities; carbohydrates and Glycosides, and Frontiers in Plant Science 09 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 FIGURE 5 Comparison of the medicinal components of A. sinensis samples between group M and G. **P ≤0.01; ***P ≤0.001. FIGURE 5 Comparison of the medicinal components of A. sinensis samples between group M and G. **P ≤0.01; ***P ≤0.001. the previous analysis, the level of chlorogenic acid in group G was significantly higher than in group M (p < 0.001), while the level of senkyunolide A was significantly lower than in group M (p < 0.001). And the content of n-butylidenephthalide in group M was also higher than in group G. Therefore, Arthrobacter and JGI 0001001.H03 may be the key microorganism related to the quality of A. sinensis. Arthrobacter is known for its nutritionally versatile nature and wade prevalence in stressful environments, with efficient survivability under high attitude stress conditions (Mukhia et al., 2021). Correlation between the differential microbial community, differential secondary metabolites and medicinal components And the strains of Arthrobacter also have the ability to degrade Dibutyl phthalate (DBP), dimethyl phthalate (DMP), 4-chlorophenol efficiently (Kim et al., 2008; Wang et al., 2019; Liu et al., 2020). Therefore, we speculate that Arthrobacter may be able to provide energy to A. sinensis decomposing harmful substances in high altitude and cold environment, and promote the synthesis of medicinal components such as n-butylidenephthalide and senkyunolide A in the early growth of A. sinensis. organic acids have consistent correlation with the other specific bacterial communities, which have opposite trend with the former. In short, flavonoids, organic acids, carbohydrates and glycosides, phthalide, and other organoheterocyclic compounds are strongly related to microbes, which may be the main driving factors of microbial community assembly in the rhizosphere of A. sinensis. Correlation between the medicinal compounds and differential bacterial community There showed a complex correlation between differential microbial community and medicinal components (Figure 7). Chlorogenic acid, senkyunolide A, and n-butylidenephthalide showed the greatest correlation with rhizosphere bacterial communities among 8 medicinal components. Chlorogenic acid was positively correlated with Rhodobacter, JGI 0001001.H03 (r > 0.7), while negatively correlated with Aridibacter, Arthrobacter, Cellulomonas, Luteolibacter (r > 0.7). Senkyunolide A was positively Kineosporia, Arthrobacter, Devosia, Sphingorhabdus, and Brevundimonas (r > 0.7). n-butylidenephthalide was positively with Arthrobacter, while negatively correlated with Alkanindiges, JGI 0001001.H03, Chthonomonas (r > 0.7). Interestingly, the Venn comparative analysis showed that Arthrobacter was positively correlated with senkyunolide A and n-butylidenephthalide, while negatively correlated with chlorogenic acid (r > 0.7) (Figure 8C). And JGI 0001001.H03 was positively correlated with chlorogenic acid, while negatively correlated with n-butylidenephthalide (r > 0.7) (Figure 8C). In frontiersin.org Discussion The primary active components of A. sinensis include phthalides, organic acids and polysaccharides (Wei et al., 2016). Research on the production of medicinal bioactive components from rhizosphere microbiome and metabolomic perspective for medicinal plants is still lacking. Since phthalides and organic acids are the marker components for assessing the quality of A. sinensis, studying how to stimulate the accumulation of these two main bioactive components has become a key technical point for improving the quality of A. sinensis. The accumulation of Frontiers in Plant Science 10 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 FIGURE 6 Co-occurrence network of the differential secondary metabolites and differential microbial taxa in rhizosphere of A. sinensis. FIGURE 6 Co-occurrence network of the differential secondary metabolites and differential microbial taxa in rhizosphere of A. sinensis. picking stage. We exploited quality-related mechanisms of authentic Angelica sinensis using a combination of metabolites content and microbial community to identify regulation of microbes and secondary metabolites synthesis mechanisms. secondary metabolite and the rhizosphere microbes are essential for improved active constituents of A. sinensis. Here, we measured root metabolites, contents of medicinal components, and sequenced the rhizosphere microbes for A. sinensis under seedling stage and FIGURE 7 Co-occurrence network of the medicinal components and differential microbial taxa in rhizosphere of A. sinensis. FIGURE 7 Co-occurrence network of the medicinal components and differential microbial taxa in rhizosphere of A. sinensis. Frontiers in Plant Science 11 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 A B C FIGURE 8 Venn comparative analysis of main differential secondary metabolites of A. sinensis and differential microbes correlated positively (A). Venn comparative analysis of main differential secondary metabolites of A. sinensis and differential microbes correlated negatively (B). Venn comparative analysis of the correlation between differential medicinal components of A. sinensis and differential microbes (C). A C B B C C FIGURE 8 Venn comparative analysis of main differential secondary metabolites of A. sinensis and differential microbes correlated positively (A). Venn comparative analysis of main differential secondary metabolites of A. sinensis and differential microbes correlated negatively (B). Venn comparative analysis of the correlation between differential medicinal components of A. sinensis and differential microbes (C). levels of phenylpropanoids, phthalide, and other organoheterocyclic compounds, flavonoids, amines, and fatty acids were lower compared to seedling stage, indicating that different growth stages of A. sinensis have different secondary metabolite synthesis patterns. Root exudates are a mix of a wide variety of compounds, including primary and secondary metabolites. Discussion Our study showed that the composition of rhizosphere microbes in picking stage were dominated by proteobacteria, which had a strong correlation with the synthesis of organic acids. The composition of rhizosphere microbes in seedling stage were dominated by Actinobacteria, which had a strong correlation with the synthesis of phthalide and other organoheterocyclic compounds, flavonoids, amines, and fatty acid. Secondly, in view of the co- occurrence network analysis, we have comprehension of the integrated microbe-medicinal associations. This finding was exemplified by rhizosphere microbes Arthrobacter and JGI 0001001.H03, which were thought to be the key microorganisms related to the quality of A. sinensis. Arthrobacter was found to be strongly associated with the accumulation of senkyunolide A and n-butylidenephthalide, JGI 0001001.H03 was found to be strongly associated with the accumulation of chlorogenic acid. Microbe-differential secondary metabolites indicated that flavonoids, organic acids, carbohydrates and glycosides, phthalide, and other organoheterocyclic compounds were closely and strongly related to differential rhizosphere microbes, which may be the main driving factors affecting the assemble of microbial community in rhizosphere of A. sinensis. Collectively, These findings provide such a basis for further exploration into the relationship between rhizosphere microorganisms and the medicinal bioactive markers over growth, are also helpful in guiding future cultivation of A. sinensis. specific microbial taxa (Hu et al., 2018; Kudjordjie et al., 2019). Released compounds have been shown to influence the assembly of rhizosphere microbiota, thus improving the ability of plants to adapt to their environments (Bulgarelli et al., 2013). Correlation between differential secondary metabolites and differential microbes were linked to the quality of A. sinensis, as well as environmental factors. Our study revealed a regulatory microbe-metabolite network for the correlation of microorganisms and differential metabolites at the molecular level, showed that the composition of rhizosphere microbes in picking stage were dominated by proteobacteria, which had a strong correlation with the synthesis of organic acids. The composition of rhizosphere microbes in seedling stage were dominated by Actinobacteria, which had a strong correlation with the synthesis of phthalide and other organoheterocyclic compounds, flavonoids, amines, and fatty acid. On the one hand, the succession of rhizosphere microbial communities based on different growth stages is related to bacteria generation time, as slower-growing microorganisms have higher rhizodeposits utilization efficiency (Roller et al., 2016). Discussion Primary metabolites, including carbohydrates, amino acids, and organic acids, are secreted in larger quantities than secondary metabolites, such as flavonoids, glucosinolates, auxins.etc. Among them, the differential metabolites of group M and G, such as ferulic acid, vanillic acid, pathothenic and glucosides can be secreted to the rhizosphere (Badri et al., 2009; Vives-Peris et al., 2020). The composition of root exudates is not constant, its composition varies with developmental stage, environmental conditions, plant species, soil type, nutrition, and root traits, and among other factors. (Jones, 1998; Aulakh et al., 2001; Badri and Vivanco, 2009; Iannucci et al., 2013). In maize, several studies have shown that benzoxazinoids (BXs) are synthesized and secreted in plant early stage that display allelopathic activities and insecticidal action, and affect root -related microbiota by inhibiting plant pathogens and colonization by Extensive studies have proved the soil microenvironment influenced the growth and level/type of active components in A. sinensis (Zhu et al., 2021). In this study, we found that the developmental stage is also an important factor affecting the accumulation of medicinal components of A. sinensis. Compared with seedling stage of A. sinensis, the level of chlorogenic acid in picking stage was significantly increased, while the level of coniferyl ferulate, senkyunolide A and ligustilide were significantly decreased in picking stage (Figure 5). It has been shown that the accumulation of secondary metabolites of A. sinensis is tightly connected to the growth periods. The four chemical markers of A. sinensis are Z- ligustilide, Z-butylidenephalide, butylphthalide, linoleic acid, which were remarkable differences in their contents during growth, and concentrations of these markers were relatively higher in September and October (Qian et al., 2013). Through further analysis of metabolomics of A. sinensis root, we found that secondary metabolites had significant differences between different growth stages (seedling stage and picking stage) of A. sinensis. Organic acid, such as dicaffeoylquinic acid, chlorogenic acid, genipic acid, and ferulic acid, were mostly synthesized at the picking stage. While the Frontiers in Plant Science 12 frontiersin.org frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 while phe nylpropanoids, phthalide , and other organoheterocyclic compounds, flavonoids, amines, fatty acids were mainly synthesized at the seedling stage. The differences in the biosynthesis of different types of secondary metabolites over growth are also related to the changes of composition of microorganisms in the rhizosphere of A. sinensis. Discussion On the other hand, the growth and succession of bacteria in the rhizosphere is not only determined by the root exudates, but also can be predicted by the substrate preferences of rhizosphere bacteria. This study provides direct evidence that specific rhizosphere exudates manipulate rhizosphere microbial community assembly. In the rhizosphere, substrate preferences may provide a selection advantage (Zhalnina et al., 2018). Proteobacteria contained significantly more organic acid transporter genes than Actinobacteria, suggesting that positive and negative bacteria respond to plant growth differed in their predicted metabolic potential to utilize organic acids (Zhalnina et al., 2018). These factors interact and affect the assembly patterns of the rhizosphere microbial community of A. sinensis at different growth stages. Therefore, we speculate that the stage-specific microbes may be associated with the particular root exudates that drive the microbes to respond quickly (Zhang et al., 2017). Conclusion In this study, we have explored quality-related mechanisms of authentic A. sinensis at the metabolite and microbiota levels based on samples, and first identified the differences of A. sinensis under different growth status at the medicinal components and metabolite levels: we have confirmed that A. sinensis at the picking stage accumulated significantly more chlorogenic acid than seedling stage, while the contents of coniferyl ferulate, senkyunolide A and ligustilide were significantly decreased. The synthesis of differential secondary metabolites at different growth stages also showed a similar trend: organic acids were mainly synthesized at the picking stage, Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm. nih.gov/, PRJNA893538. References Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver, L. V. T. 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Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ fpls.2022.1005711/full#supplementary-material SUPPLEMENTARY FIGURE 4 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. Correlation between the medicinal components and differential microbial taxa in rhizosphere of A. sinensis. (*, P <=0.05; **, P <= 0.01; ***, P <=0.001.). Publisher’s note This research was supported financially by National Natural Science Foundation of China (81773848), Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-D-202005), China Agriculture Research System of MOF and MARA (CARS-21), Ministry of Finance Central Level of the Special (2060302). This work was also partly sponsored by Jiangsu Province 333 Highlevel Talents Training Project, Qing Lan Project, Six talents peaks project in Jiangsu Province (JNHB-066). All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions The experiments were conceived and designed by HY, PL, and J-AD. The experiments were carried out by J-MC, who also drafted the manuscript. J-MC and W-MF analyzed the data. HY, W-MF, PL also contributed to manuscript editing, literature search, and figures generation. G-SZ, SG, GY, and J-AD contributed to the discussion of the manuscript. All authors contributed to the article and approved the submitted version. 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Factors associated with children and young people’s mental health in the English-speaking Caribbean region: Systematic review and narrative synthesis
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PLOS ONE PLOS ONE RESEARCH ARTICLE Methods Editor: A. K. M. Alamgir, Access Alliance Multicultural Health and Community Services: Access Alliance, CANADA Multiple academic databases and grey literature sources were searched until January 2022. We then identified primary research focusing on CYP’s mental health in the English-speak- ing Caribbean region. Data was extracted and summarized to form a narrative synthesis of the factors associated with CYP’s mental health. The synthesis was then organised accord- ing to the social-ecological model. The Joanna Briggs Institute’s critical appraisal tools were used to examine the quality of the reviewed evidence. The study protocol was registered with PROSPERO, CRD42021283161. Received: December 12, 2022 Accepted: February 20, 2023 Published: March 8, 2023 Copyright: © 2023 Liverpool 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. Background Citation: Liverpool S, Draoui Y, Tucker J, Pereira B, Prescod J, Owen M, et al. (2023) Factors associated with children and young people’s mental health in the English-speaking Caribbean region: Systematic review and narrative synthesis. PLoS ONE 18(3): e0282666. https://doi.org/ 10.1371/journal.pone.0282666 Studies conducted in regions consisting of low and middle income and developing countries often report high prevalence of mental health problems among children and young people (CYP). To identify some of the contributing factors we examined the available evidence from research in one such setting. Shaun LiverpoolID1,2*, Yasmin Draoui1, Judea Tucker1, Brent Pereira3, Jamal Prescod4, Michael Owen1, Catherine Trotman4 Shaun LiverpoolID1,2*, Yasmin Draoui1, Judea Tucker1, Brent Pereira3, Jamal Prescod4, Michael Owen1, Catherine Trotman4 Shaun LiverpoolID1,2*, Yasmin Draoui1, Judea Tucker1, Brent Pereira3, Jamal Prescod4, Michael Owen1, Catherine Trotman4 1 Faculty of Health, Social Care and Medicine, Edge Hill University, Ormskirk, United Kingdom, 2 Evidence Based Practice Unit, Anna Freud National Centre for Children and Families, London, United Kingdom, 3 Department of Counselor Education, The Chicago School of Professional Psychology, Chicago, IL, United States of America, 4 Faculty of Social Sciences, University of the West Indies, Cave Hill, St Michael, Barbados * Shaun.liverpool@edgehill.ac.uk * Shaun.liverpool@edgehill.ac.uk Results From 9684 records, 83 publications representing CYP ages 3 to 24 years from 13 countries met our inclusion criteria. The evidence was varied in quality, quantity and consistency for 21 factors associated with CYP’s mental health. Adverse events and negative peer to peer and sibling relationships were consistently associated with mental health problems, while helpful coping strategies were associated with better mental health. There were mixed find- ings for age, sex/gender, race/ethnicity, academic level, comorbidity, positive affect, health risks behaviours, religion/prayer, parent history, parent to parent and parent to child relation- ships, school/employment, geography and social status. There was also some limited Introduction The mental health and wellbeing of children and young people (CYP) continues to be a global public health concern [1–3]. International evidence suggests that at least 1 in 10 CYP experi- ence symptoms of a mental health problem with 50% of these occurring by age 14 years and 75% by age 24 years [4]. Common internalising and externalising presentations include anxi- ety, depression and conduct or behaviour problems with high rates of comorbidities among CYP [1, 5]. Notably, some studies highlight disparities in prevalence of mental health problems among CYP identifying as specific minority ethnic groups and those from low and middle income and developing countries [6]. The higher prevalence rates among these groups have mainly been attributed to poverty and social disadvantages but less is known about other risk and protective factors [7, 8]. Although previous reviews suggest some similarities in risk factors for mental health problems in low and middle income and developing countries and those found in high income countries [8, 9], other studies suggest that the factors associated with mental health may be complex and bi-directional and further influenced by culture [10, 11]. Therefore, experts consistently call for more research to provide a deeper understanding of regional differences [12]. The existing literature identified a wide range of demographic, personal, familial, school, social and interpersonal characteristics as key factors associated with CYP’s mental health [13, 14] and subsequent service utilisation [15]. More specifically these factors include age, gender, ethnicity, family composition, urbanisation, family and friend support, social isolation, peer victimization, physical/sexual abuse or emotional neglect and parent psychopathology [16, 17]. In more severe cases additional factors include substance use, comorbid disorders and intellectual disabilities [18, 19]. As for marginalised groups, based on ethnicity or sexuality, experiences of discrimination were also identified as having a negative association with mental health [9, 20]. Conversely, improved self-esteem and optimism have been associated with posi- tive mental health and resilient outcomes [13, 21]. While acknowledging the efforts of researchers in the previous reviews, the evidence from some regions like the English-speaking Caribbean is still under-represented. The English-speaking Caribbean is made up of about 18 countries or territories, of which the majority are classed as low and middle income or developing status [22–24]. Population statistics suggest that this region consists mainly of families of African, mixed-race, Indian or indigenous origins [25, 26]. Conclusion Individual, relationship, community and societal factors may influence CYP’s mental health outcomes in the English-speaking Caribbean. Knowledge of these factors is useful to inform early identification and early interventions. More research is needed to explore inconsistent findings and understudied areas. evidence for associations between sexuality, screen time and policies/procedures and CYP’s mental health. At least 40% of the evidence contributing to each of the factors was judged as high quality. evidence for associations between sexuality, screen time and policies/procedures and CYP’s mental health. At least 40% of the evidence contributing to each of the factors was judged as high quality. Abbreviations: CYP, Children and young people; YP, Young people. Abbreviations: CYP, Children and young people; YP, Young people. Abbreviations: CYP, Children and young people; YP, Young people. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region Literature search strategy The first author (SL) conducted the initial literature search in January 2021 and updated the search in January 2022 using academic databases (CINAHL, Cochrane Library, EMBASE, MEDLINE, PsycINFO, LILACS, and Web of Science). We also used grey literature sources (OpenGrey, ResearchGate and the first 10 pages of Google) to track any recent publications that were not yet indexed in the academic databases. Search terms and key words included “children OR adolescent OR young people” AND “mental health OR well-being” AND “West Indies OR Caribbean”. Definitions of mental health and wellbeing were guided by diagnostic manuals (e.g., DSM-5 and ICD 11) and the frame of reference used in the individual studies. The search strategy was developed and piloted through an iterative process with a research librarian. Further details of the literature search have been published as part of the initial scop- ing review [27]. Methods The review process was guided by recommendations from the Joanna Briggs Institute [34], Cochrane Campbell Collaborations [35] and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA checklist, [36]–S1 File). The study protocol was regis- tered with the International Prospective Register of Systematic Reviews (PROSPERO, CRD42021283161). Introduction Studies conducted in the English-speaking Caribbean region also reported high rates of mental health problems and a limited number of evidence-based inter- ventions [27]. A recent report also highlighted a scarcity of appropriate mental health policies and funding [6]. Due to the complexity of mental health and the great need for services, not all PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 2 / 23 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region CYP locally or regionally are able to receive professional mental health services [28]. Therefore, there is an obvious demand for a better understanding of the factors that influence CYP’s men- tal health to develop interventions to identify, prevent and manage mental health problems. Two previous reviews were conducted in 2009 and 2012 and explored individual as well as micro-and-macro-system factors influencing adolescents’ (10–19-year-olds) mental health in the English-speaking Caribbean region [29, 30]. Between the two reviews the authors identi- fied gender, age, family, home environment, school, religion and engagement in risk behav- iours as important factors associated with adolescent’s mental health. Since those studies were conducted there have been an exponential increase in research as well as a growing interest and need for CYP mental health support [27, 31]. There have also been increased attention and advocacy to focus on a wider age range up to 24 years to capture the key transition periods from childhood to adulthood [32]. Therefore, based on recommendations and consensus from academic, practice and lived experience experts, an updated systematic review was deemed appropriate [27, 33]. Subsequently, the aims of the current study were threefold. First, to update the existing reviews to investigate any new or emerging factors that could potentially influence the mental health of CYP in the English-speaking Caribbean region. Second, to build on previous reviews by extending the inclusion criteria to include studies that examined the mental health of CYP below age 10 and up to age 24 years. Third, to improve on the limitations of the previous reviews by conducting quality appraisals of the reviewed articles using established critical appraisal tools. Quality appraisal The methodological quality of the included studies was assessed using six corresponding checklists from the Joanna Briggs Institute [37]. The checklists assessed methodological quality of the included studies using statements like “Was the sample frame appropriate to address the target population?” and “Was the sample size adequate?”. Fifty-four (65%) of the included studies were independently appraised by three reviewers (JP, SL, BP) resulting in 80% interra- ter reliability. The remaining studies were appraised by the primary author (SL) and verified by a second reviewer (BP or JP). When necessary, discussions were held to achieve consensus. Items on each checklist were judged as “yes”, “no”, “unclear” or “not applicable”. Similar to other reviews [38, 39], the risk of bias of the contributing results were classified into 1) low risk (high quality evidence), if the studies reached more than 70% of “yes” scores; 2) moderate risk (medium quality evidence), if the studies reached between 50% and 69% of “yes” scores; and 3) high risk (low quality evidence), if the studies reached less than 49% of “yes” scores. Notably some reports presented separate findings from the same dataset. Based on the nature of this review each report was appraised individually, but when necessary, we verified information from the primary study. As this review did not include a meta-analysis the risk of double counting and the related problems were low [40]. Data extraction At least two reviewers independently extracted data on publication date, primary author, country of data collection, psychosocial problem explored, study design, sample size, demo- graphics of the sample, outcome measures and recruitment settings. Regarding factor associa- tions, two reviewers (JD and YD) extracted the description of the effect measure between the factors and the outcome, but in cases where no effect measures were present a description of the association was extracted from the text. We also extracted data for negligible and inconsis- tent associations to provide a non-biased overview of the literature. The information was then verified by a third reviewer (SL or MO) and discrepancies (<10%) discussed at team meetings to reach consensus. The data management was conducted using Microsoft Excel. Screening and eligibility criteria Four reviewers (SL, JP, BP, CT) were involved in the two-stage independent screening process. First, titles and abstracts were screened followed by full texts while applying the following crite- ria. Meetings and email communications were used to resolve disagreements and reach consensus. Articles were included if: 3 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region • Primarily focused on CYP (0–24 years) • Primarily focused on CYP (0–24 years) • Explored mental health symptoms or diagnoses or any psychosocial problem • Conducted in the English-speaking Caribbean region • Detailed peer-reviewed primary research published in English Articles were excluded if: • The sample (or over 50%) reported a mean age above 24 years • The sample (or over 50%) reported a mean age above 24 years • The main outcomes were physical health, substance abuse, neurodevelopmental and intellec- tual disabilities • The main outcomes were physical health, substance abuse, neurodevelopmental and intellec- tual disabilities • Focused on Caribbean migrants or CYP living in non-English-speaking Caribbean countries • Focused on Caribbean migrants or CYP living in non-English-speaking Caribbean countries Results 9684 records were retrieved from the database and grey literature searching. 7901 records remained after duplicates were removed. After title and abstract screening, 311 publications were subjected to full-text screening, of which 83 were eligible for inclusion in this review (Fig 2). Characteristics of the reviewed studies The reviewed articles were published between 1976 and 2020 [44–126]. Most of the studies focused on depressive symptoms, behaviour/conduct problems and suicidality. Most of the studies were conducted in Jamaica and Trinidad and Tobago and in education settings. More than half of the reviewed studies were representative in terms of sex (i.e., 50 to 60% males and females) but most focused on adolescents 12 to 17 years. Sample sizes were considerably large with most studies recruiting at least 50 participants. Table 1 provides an overview of the body of evidence and the S2 File provides further details of the reviewed studies. Data analysis First, the study and population characteristics were charted to provide an overall description of the body of evidence. Second, narrative synthesis, supported by thematic and content analy- sis, as outlined by Popay et al. [41] was conducted. To achieve this, we developed a preliminary synthesis using tables and charts, explored relationships and patterns in the data and then 4 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region assessed robustness of the synthesis product based on the size, consistency and strength of evi- dence. Third, factors were then organised into conceptually coherent themes based on the social-ecological model [42, 43]. The social-ecological model was selected as it is a commonly used framework to help understand the complex interplay between individual, relationship, community and societal factors that influence health outcomes (Fig 1). Fig 1. Social ecological model. https://doi.org/10.1371/journal.pone.0282666.g001 Fig 1. Social ecological model. https://doi.org/10.1371/journal.pone.0282666.g001 https://doi.org/10.1371/journal.pone.0282666.g001 assessed robustness of the synthesis product based on the size, consistency and strength of evi- dence. Third, factors were then organised into conceptually coherent themes based on the social-ecological model [42, 43]. The social-ecological model was selected as it is a commonly used framework to help understand the complex interplay between individual, relationship, community and societal factors that influence health outcomes (Fig 1). assessed robustness of the synthesis product based on the size, consistency and strength of evi- dence. Third, factors were then organised into conceptually coherent themes based on the social-ecological model [42, 43]. The social-ecological model was selected as it is a commonly used framework to help understand the complex interplay between individual, relationship, community and societal factors that influence health outcomes (Fig 1). Factors associated with children and young people’s mental health problems Twenty-one factors associated with CYP’s mental health were identified and mapped onto the four levels of the social-ecological model. Of the 21 factors, 12 were individual demographic, 5 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region Fig 2. PRISMA flow chart depicting the screening process. https://doi.org/10.1371/journal.pone.0282666.g002 Fig 2. PRISMA flow chart depicting the screening process. https://doi.org/10.1371/journal.pone.0282666.g002 psychosocial or behavioural factors that included sex/gender, age, comorbidity, academic level, race/ethnicity, sexuality, experience of adverse events, positive affect, health risk behaviours, cop- ing strategies, religion/prayer and screen time. The five relationship factors included parent to parent, parent to child, peer to peer and child to sibling relationships and parent history. The three community factors included school/employment, geography and social status. The only societal factor identified related to existing policies/procedures. Table 2 provides an overview of the factors associated with CYP’s mental health in the English-speaking Caribbean. The blocks are shaded according to the number of studies; darker shading indicates more evidence (number of reports in brackets). The colours represent whether the supporting evidence for each factor associ- ation was consistent (green), mixed or inconsistent (yellow) or limited (grey). Factors were judged as having limited or insufficient evidence if they were explored in less than five reports. psychosocial or behavioural factors that included sex/gender, age, comorbidity, academic level, race/ethnicity, sexuality, experience of adverse events, positive affect, health risk behaviours, cop- ing strategies, religion/prayer and screen time. The five relationship factors included parent to parent, parent to child, peer to peer and child to sibling relationships and parent history. The three community factors included school/employment, geography and social status. The only societal factor identified related to existing policies/procedures. Table 2 provides an overview of the factors associated with CYP’s mental health in the English-speaking Caribbean. The blocks are shaded according to the number of studies; darker shading indicates more evidence (number of reports in brackets). The colours represent whether the supporting evidence for each factor associ- ation was consistent (green), mixed or inconsistent (yellow) or limited (grey). Factors were judged as having limited or insufficient evidence if they were explored in less than five reports. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE PLOS ONE Table 2. Factors associated with children and young people’s mental health in the English-speaking Caribbean. INDIVIDUAL RELATIONSHIP COMMUNITY SOCIETY DEMOGRAPHIC PSYCHOSOCIAL BEHAVIOURAL Sex/Gender (43) Adverse events (10) Health risks (14) Parent-parent (18) School/Employment (20) Policies/Procedures (2) Age (21) Positive affect (7) Coping strategies (5) Parent-child (16) Geography (12) Comorbidity (15) Religion/Prayer (5) Peer-peer (14) Social status (10) Academic level (8) Screen time (1) Parent history (12) Race/Ethnicity (8) Child-sibling (5) Sexuality (1) Note: Darker shading indicates more evidence (number of reports in brackets). Green represents factors with consistent evidence, yellow represents mixed or inconsistent evidence and grey represents limited evidence. Table 2. Factors associated with children and young people’s mental health in the English-speaking Caribbean. INDIVIDUAL RELATIONSHIP COMMUNITY SOCIETY DEMOGRAPHIC PSYCHOSOCIAL BEHAVIOURAL Sex/Gender (43) Adverse events (10) Health risks (14) Parent-parent (18) School/Employment (20) Policies/Procedures (2) Age (21) Positive affect (7) Coping strategies (5) Parent-child (16) Geography (12) Comorbidity (15) Religion/Prayer (5) Peer-peer (14) Social status (10) Academic level (8) Screen time (1) Parent history (12) Race/Ethnicity (8) Child-sibling (5) Sexuality (1) Note: Darker shading indicates more evidence (number of reports in brackets). Green represents factors with consistent evidence, yellow represents mixed or inconsistent evidence and grey represents limited evidence. Table 2. Factors associated with children and young people’s mental health in the English-speaking Caribbean. Note: Darker shading indicates more evidence (number of reports in brackets). Green represents factors with consistent evidence, yellow represents mixed or inconsistent evidence and grey represents limited evidence. https://doi.org/10.1371/journal.pone.0282666.g003 Quality assessment of the reviewed studies 58 studies (70%) were judged to have low risk of bias, 13 studies (16%) were judged to have moderate risk of bias, and 11 studies (14%) were judged to have high risk of bias. 6 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region Table 1. Overview of the reviewed articles. Characteristics of the reviewed articles Number of articles % of articles Year of publication 1976–1989 4 4.82 1990–1999 6 7.23 2000–2009 30 36.14 2010–2020 43 51.81 Country Jamaica 44 53.01 Trinidad & Tobago 16 19.28 Barbados 5 6.02 Guyana 4 4.82 Bermuda 2 2.41 St Vincent & the Grenadines 1 1.20 The Bahamas 1 1.20 St Kitts & Nevis 1 1.20 St Lucia 1 1.20 Multiple 8 9.64 Presenting problem Depressive symptoms 25 30.12 Behavioural and conduct problems 17 20.48 Suicidality 15 18.07 Disordered eating & image issues 6 7.23 Anxiety 1 1.20 Multiple 19 22.89 Recruitment settings Education 66 79.52 Healthcare 12 14.46 Community 5 6.024 Sex of participants Majority males (>60%) 9 10.84 Majority females (>60%) 24 28.92 50 to 60% males and females 46 55.42 Not clearly stated 4 4.82 ~Age of participants (years) Under 12 9 10.84 12 to 17 46 55.42 18 to 24 28 33.73 Sample size Small (<50 participants) 12 14.46 Medium (50–300 participants) 25 30.12 Large (>300 participants) 46 55.42 https://doi.org/10.1371/journal.pone.0282666.t001 Table 1. Overview of the reviewed articles. Correspondingly, more studies were of high or medium quality and fewer studies were judged as low quality. The quality assessment details for each study can be found in the S3 File. The quality of the body of evidence informing each factor was organised and presented in Fig 2. Each of the factors comprised of at least 40% of high-quality studies. 7 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Factors associated with children and young people’s mental health in the English-speaking Caribbean region Individual demographic factors 1. Sex/gender. There were mixed (or inconsistent) findings on the association between sex/gender and CYP’s mental health. Data from 43 studies informed this association (see Table 2). Of these 31 (72%) were judged as high-quality evidence (see Fig 3). In a large proportion of the studies (20 out of 43 or 46.5%) females were more likely than males to report depressive symptoms [44–52], disordered eating/body image issues [53–56], psychiatric disorders [57] and other internalising problems [58]. Although females were more likely to have suicide ideation and non-fatal suicide attempts [59–67], males were more likely to be at risk of completing suicide [59, 68]. Similarly, although older females were more likely than males to express significantly higher indirect aggression [69], younger males displayed more verbal and physical aggression [70]. Younger females were also more likely than males to Fig 3. Summary of the quality of evidence for each factor. https://doi.org/10.1371/journal.pone.0282666.g003 Fig 3. Summary of the quality of evidence for each factor. https://doi.org/10.1371/journal.pone.0282666.g003 8 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region be placed in group homes for behaviour/conduct problems [71], while there were more males than females with mental disabilities in schools [72] and university programmes [73]. Both males and females reported having specific types of phobias [74]. In other studies sex/gender was not associated with depressive symptoms [75–78], suicidal plan or risk [79], stress [80], disordered eating/body image issues [81], aggressive behaviour/conduct problems [82] and other internalising and externalising problems [83–86]. 2. Age. There were mixed findings on the association between age and CYP’s mental health. Data from 21 studies informed this association. Of these 14 (67%) were judged as high- quality evidence. In some studies (6 out of 14 or 42.8%), behaviour/conduct problems and depressive symp- toms were more common among older adolescents than primary school-aged children [72] or younger adolescents [50, 51, 76, 82, 87]. However, self-report depressive symptoms did not always increase with age [77]. For example, students who were younger or older than expected for their grade level reported higher depression scores than students who were at the expected age [46]. Younger adolescents were more likely to have suicidal thoughts [62, 63, 67] but more suicide attempt cases were reported among older adolescents and YP (16–20 years) [65]. Individual demographic factors In terms of behaviour/conduct problems, younger children and adolescents displayed more behaviour/conduct problems than older adolescents [58, 83, 88], but older boys were more likely to be committed for more serious offending behaviours like robbery [71]. Specific pho- bias and anxiety varied with age; with older students expressing fears of nuclear war and school failure while younger students expressed fears of diseases [74]. In other studies age was not associated with clinical profiles [85], behaviour/conduct problems [89], depressive symptoms [78] or suicidal ideation [66]. 3. Comorbidity. There were mixed findings on the association between having a disability and CYP’s mental health. Data from 15 studies informed this association. Of these 12 (80%) were judged as high-quality evidence. Most studies (14 out of 15 or 93.3%) suggested that the presence of mental health symptoms was common when YP experienced other chronic conditions or disabilities. For example, studies suggested that having learning difficulties was associated with more depressive symp- toms [47], behaviour/conduct problems and anxiety [90]. Studies also suggested that having chronic and acute mental and physical conditions, including self-harm practices, were associ- ated with disordered eating attitudes [54, 81], depressive symptoms [91], behaviour/conduct problems [85, 89], suicide ideation [60, 63, 66, 67, 92, 93] and other psychiatric disorders [57]. Notably, one study suggested that mental disorders was not frequently found in YP with behaviour/conduct problems [94]. 4. Academic level. There were consistent findings on the association between level of edu- cation or academic performance and CYP’s mental health. Data from 8 studies informed this association. Of these 6 (75%) were judged as high-quality evidence. Studies suggested that YP with higher levels of education or better academic performance experienced lower depressive symptoms [48, 95], were less likely to plan suicide [64] and were at reduced risk of anxiety [90]. In terms of behaviour/conduct problems, inadequate education or poorer academic performance was associated with increased behaviour/conduct problems [89, 94], but higher grade levels were also sometimes significantly associated with more fre- quent behaviour/conduct problems [87]. Among older students, being in the final year of uni- versity or those with academic issues were more likely than other students to experience burnout, higher stress levels and other mental disorders [95, 96]. 5. Race/Ethnicity. There were mixed findings on the association between race/ethnicity and CYP’s mental health problems. Data from 8 studies informed this association. Of these 7 (88%) were judged as high-quality evidence. 5. Race/Ethnicity. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Individual demographic factors There were mixed findings on the association between race/ethnicity and CYP’s mental health problems. Data from 8 studies informed this association. Of these 7 (88%) were judged as high-quality evidence. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 9 / 23 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region Mixed-race YP (i.e., African and Indian ethnic origins) and Afro-Trinidadians were more likely than Indo-Trinidadians to report higher levels of suicidality [64], and in Guyana, callers to a suicide hotline were more likely to be Indo-Guyanese [59]. Regarding disordered eating/ body image issues, one study suggested Indo-Trinidadians were more likely than Afro-Trini- dadians and mixed-race Trinidadians to report body dissatisfaction/eating issues [97] contra- dicting another study which suggested that Afro-Trinidadian adolescent females reported significantly higher scores on body dissatisfaction and binge eating practices [98]. Relatedly, a desire for lighter skin complexion was also associated with disordered eating/body image issues [54]. Regarding behaviour/conduct problems, more Afro-Trinidadians than other eth- nicities were represented at group homes [71]. In other studies race/ethnicity was not signifi- cantly associated with suicidality [61] or depressive symptoms [44]. 6. Sexuality. One high quality study suggested that YP with conflict surrounding their sexual orientation were more likely to experience emotional and social distress [59]. Individual psychosocial factors 7. Positive affect. There were mixed findings on the association between positive affect and CYP’s mental health. Data from 7 studies informed this association. Of these 6 (86%) were judged as high-quality evidence. In most studies (5 out of 7 or 71.4%), increased self-esteem, life satisfaction, resilience and positive emotions (e.g., happiness) was associated with lower levels of depression, anxiety, stress, aggressive behaviours and increased psychological wellbeing [99–103]. As for disor- dered eating, this association was mediated by gender, indicating that females with higher self- esteem were at reduced risk of disordered eating but this association was not significant for males [54]. However, in one study there was no significant association between self-esteem and depressive symptoms [50]. 8. Adverse events. There were consistent findings on the association between negative affect or adverse experiences and CYP’s mental health. Data from 10 studies informed this association. Of these 7 (70%) were judged as high-quality evidence. All of these studies (10 out of 10, 100%) suggested that YP who were lonely, unhappy, trau- matised from experiences of abuse (e.g., sexual, verbal and physical) or had thoughts of harm- ing self or others or decreased life expectancy or lower future ambitions, or negative opinions and thoughts were more likely to report higher levels of distress [104], depressive symptoms [50, 86], suicidal ideation and attempts [62, 63, 66, 67, 105] and behaviour/conduct problems [66, 94, 106]. Individual behavioural factors One high quality study suggested that there was no significant difference between the amount of television watched and aggressive or prosocial behaviours in CYP [89]. Individual behavioural factors 9. Health risk behaviours. There were mixed findings on the association between health risk behaviours and CYP’s mental health. Data from 14 studies informed this association. Of these 13 (93%) were judged as high-quality evidence. Most studies (13 out 14 or 92.9%) suggested that YP who admitted to alcohol or drug (mis) use (e.g., cannabis) and unsafe sexual practices were at increased risk of disordered eating/ body image issues [54, 81], psychotic and depressive symptoms [45, 77, 107, 108] and behav- iour/conduct problems [87, 109]. However, there were mixed findings for suicide ideation indicating that alcohol or drug (mis)use was associated with suicide ideation [60, 62, 63, 67, 89] but the association was not always significant [79]. 10. Coping strategies. There were consistent findings on the association between self- care strategies and CYP’s mental health. Data from 5 studies informed this association. Of these 2 (40%) were judged as high-quality evidence. 10 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region Avoidant, emotional and support coping were used by YP to manage problem behaviours [86, 110] and relaxation techniques like reading, deep breathing and sleeping were used to manage stress [111]. These techniques alongside being in control of their daily schedules were associated with reduced rates of burnout and depressive symptoms in older YP [95]. Effective social adjustment skills were also associated with reduced psychiatric distress [57]. 11. Religion/Prayer. There were mixed findings on the association between religion or prayer and CYP’s mental health. Data from 5 studies informed this association. All studies were judged as high-quality evidence. All of the studies (5 out of 5 or 100%) suggested that YP who identified as having a religion (e.g., Catholic, Seventh-day Adventists and Pentecostal) or those who attended a place of wor- ship were less likely than YP with no religious affiliation to have suicide ideation/attempts [61, 64] or behaviour/conduct problems [89] or burnout and depressive symptoms [44, 95]. How- ever, one study suggested that YP identifying as non-Christian religions were at increased risk of suicide ideation/attempts [64]. Notwithstanding statistical significance the same study found that YP who reported praying with their families were less likely to experience suicide ideation/attempt [64]. 12. Screen time. Relationship factors 13. Parent to parent. There were mixed findings on the association between parent-to- parent relationship (e.g., parental conflict or separation) and CYP’s mental health. Data from 18 studies informed this association. Of these 12 (67%) were judged as high-quality evidence. 18 studies informed this association. Of these 12 (67%) were judged as high quality evidence. In most of the studies (14 out of 18 or 77.8%) CYP living with unmarried parents, parents in conflict, or reconstructed families (e.g., living with relatives) were more likely than other groups to report symptoms of mental health problems. For example, CYP living with unmar- ried parents or reconstructed families were more likely than other groups to report disordered eating/body image issues [53, 54, 81], depressive symptoms [44, 50, 112] or suicide ideation [61, 86]. Similarly, YP not living with their both parents or experiencing other sources of fam- ily conflict were at increased risk of disordered eating/body image issues [54, 77], depressive symptoms [45, 96], stress [57, 59] and behaviour/conduct problems [113]. In terms of behav- iour/conduct problems, some evidence suggested the absence of a parent or separation did not have a significant impact if the separation occurred within the first five years of the child’s life, while separation later in the young person’s life was associated with psychological distress [94, 112]. Reasons for parental separation, for example, witnessing inter-adult verbal aggression or domestic violence also predicted behaviour/conduct problems [89], depressive symptoms, sui- cide ideation and psychological distress in YP [82]. In another study, family conflict was not significantly associated with YP’s mental health [114]. g y 14. Parent to child. There were mixed findings on the association between parent to child relationships and CYP’s mental health. Data from 16 studies informed this association. Of these 14 (88%) were judged as high-quality evidence. YP who reported being afraid of their parents or had unhealthy attachments to their moth- ers were more likely than others to report depressive symptoms [45], but YP with stronger attachments to their mothers were also likely to display offending behaviours like owning a gun [114]. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Relationship factors Authoritarian or neglectful parenting styles and using physical punishment or excess monitoring also increased the risk of YP experiencing psychological maladjustment [115], behaviour/conduct problems [89], depressive symptoms [77, 116], or suicide ideation [70], but an agreement between YP and parents on the style of parenting reduced the risk of PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 11 / 23 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region poor psychosocial outcomes [69, 70]. The reverse occurred in other studies with parental mon- itoring of free time being associated with lower odds of mental health problems [66, 117]. However, one study reported that the severity of parental punishment alone had little effect on the variations in YPs psychological adjustments [115]. One study also found no significant association between corporal punishment and psychosocial outcomes, but a significant nega- tive association between parent-child verbal punishment and psychological outcomes [82]. Conversely, YP who described their parents as understanding and YP who received regular emotional or social support from their family were at reduced risk of burnout, depressive symptoms or suicide ideation [62, 63, 99, 105] and overall psychological distress [101]. Yet, one study found that some YP who had parental understanding were still more likely than other YP to experience suicidality [67]. 15. Peer to peer. There were consistent findings on the association between peer relation- ships and CYP’s mental health. Data from 14 studies informed this association. Of these 10 (71%) were judged as high-quality evidence. YP who experienced peer pressure were more likely to feel overwhelmed and in need of emotional support [59]. Similarly, YP who were victims of bullying were also more likely to experience suicide ideation [60, 63, 84] and excessive worry [84]. However, YP with close friendships, good interpersonal skills and in receipt of social support were less likely to experi- ence suicidal thoughts [60, 62, 63, 93] and reduced psychological wellbeing [99, 101]. In terms of behaviour/conduct problems, YP with friends who were in trouble with the law were more likely than other YP to display delinquent behaviours [71]. Studies also suggested that YP who were not in committed relationships or YP who experi- enced conflict in their romantic relationships were more likely than other YP to experience suicidal thoughts [59, 65], psychological distress [57, 100] or stress and anxiety [96]. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Community factors 18. School/Employment. There were mixed findings on the association between school/ university environment, employment and CYP’s mental health. Data from 20 studies informed this association. Of these 12 (60%) were judged as high-quality evidence. YP attending non-traditional and non-prestigious high schools were more likely than other students to report depressive symptoms [44, 75]. However, no significant association was found between the type of school or sense of belonging and behaviour/conduct problems [87] and suicidal ideation [61] in other studies. Studies also suggested an association between miss- ing school or classes and suicidality [62, 63] and behaviour/conduct behaviours [120]. Rela- tionships with teachers, sometimes impacted by school punishments (e.g., beaten by hand) was inversely associated with behaviour/conduct problems [87, 89, 110, 121]. Among older YP (e.g., university students) the amount of material to be studied, exams/grades, campus facilities and quality of teaching contributed to stress levels [80, 100]; while combining employment and studies resulted in lower occurrences of depressive symptoms [47] or psychiatric disorders [57]. Specifically, students attending nursing programmes experienced moderately high levels of stress in clinical environments [111]. Notably, in one study students were also more likely than non-students to report suicidality [68]. As for the school environment, one study sug- gested that schools offering mental health interventions (e.g., whole school approaches) were not significantly beneficial to YP’s mental health [121], but other studies suggested some improvements in behaviour problems like the use of profanity [122, 123]. Another study highlighted that a universal intervention in a school was useful in supporting children with behaviour and conduct problems but not for prosocial and emotional problems [124]. How- ever, another multi-modal intervention implemented in schools made significant improve- ment in school social and behaviour adjustments, particularly for boys [102]. 19. Geography. There were mixed findings on the association between geographic loca- tion and CYP’s mental health problems. Data from 12 studies informed this association. Of these 8 (67%) were judged as high-quality evidence. YP from rural areas were more likely than other YP to display externalising problems [83] and suicidality [68]. In other studies, YP from urban communities or violent prone areas were more likely that other YP to report suicidal behaviours [79], depressive symptoms [50], disor- dered eating [56] and offending behaviours [94]. Relationship factors Among older YP, married students reported significantly lower depressive symptoms than students in visiting relationships [47]. 16. Parent history. There were mixed findings on the association between parent history or background and CYP’s mental health. Data from 12 studies informed this association. Of these 9 (75%) were judged as high-quality evidence. In 50% of these studies (6 out of 12 studies) YP with family members with mental health problems, legal issues or substance (mis)use were at increased risk of disordered eating/body image issues [81], depressive symptoms [44], behaviour/conduct problems [71, 89, 114] and suicidality [61]. This also meant that when parents exhibited tolerant attitudes towards drug use and gun ownership YP were more likely to display offending behaviour/conduct problems [113, 114]. One study noted however that mental disorders and criminal activity was infre- quently found among parents of YP with delinquent behaviours [94] but poor family manage- ment and lack of specific practices (e.g., structured mealtimes) was associated with emotional distress in YP [114, 118]. As for parent education level, YP whose mothers had post-secondary education were at reduced risk of depressive symptoms [46–48]. However, one study suggested that there was no significant association between parent education and behaviour/conduct problems in YP [89]. 17. Child to sibling. There were consistent findings on the association between relation- ships among siblings and CYP’s mental health. Data from 5 studies informed this association. Of these 2 (40%) were judged as high-quality evidence. As for other family relationships, YP with male siblings or multiple siblings or siblings with a history of antisocial behaviours were significantly more likely than other YP to display spe- cific behaviour/conduct problems like gun ownership [94, 113, 114] or be at increased risk of disordered eating/body image issues [81]. YP with a chronically ill sibling also reported greater distress and poorer social adjustments than other YP [119]. 12 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Summary of findings Our systematic review and analysis of 83 reports identified 21 factors with varied evidence of associations with CYP’s mental health across four levels (i.e., individual, relationship, commu- nity and society). These factors include age, sex/gender, race/ethnicity, academic level, comor- bidities, sexuality, positive affect, adverse events, health risk behaviours, coping strategies, religion/prayer, screen time, parent to parent, parent to child, peer to peer and child to sibling relationships, parent history, social status, school/employment, geography and policies/ procedures. Community factors On a country level, one study suggested that YP from Jamaica reported significantly higher depressive symptoms than YP from St Kitts and Nevis or St Vincent and the Grenadines [48, 116], while another study suggested that YP from the Bahamas had higher levels of depressive symptoms than YP from Jamaica [125]. Commu- nity opportunities and rewards for prosocial involvement did not have statistically significant association with behaviour/conduct problems [109] but socially organised communities or higher quality neighbourhoods (e.g., lower crime rates) were more likely to contribute to reduced behaviour/conduct problems [89] and depressive symptoms [109, 125, 126]. Having a sense of belonging to a particular neighbourhood was also associated with lower levels of depressive symptoms [125, 126]. 20. Social status. There were mixed findings on the association between social status and CYP’s mental health. Data from 10 studies informed this association. Of these 6 (60%) were judged as high-quality evidence. In most of the studies (6 out of 10 studies, 60%) YP with parents who were unemployed or underemployed or had manual jobs (e.g., plumbing) or those who belonged to poorer house- holds were at increased risk of disordered eating [81], depressive symptoms [51] or behaviour/ conduct problems [58, 71, 89, 94]. This also meant that for females the lack of domestic ameni- ties (e.g., water) or YP who were undernourished had increased psychological distress [54, 76]. 13 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region For example, even YP from high economic status families who were unable to have time for family meals or those with financial difficulties resulting in lack of food security were more likely to report psychological distress [63, 111]. Societal factors 21. Policies or procedures. There were mixed findings on the association between poli- cies/procedures and CYP’s mental health. Data from 2 studies informed this association. Both studies were judged as high-quality evidence. Although laws and norms disfavouring drug use and firearms did not have a significant effect on offending behaviours, the risk of apprehension was significantly associated with less frequent gang membership [109]. Other institutional policies, procedures, and regional norms also sometimes contributed to contention and disappointment among CYP [101]. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 Comparison to previous reviews In line with previous reviews, our study confirms the association of individual, relationship, community and societal factors with the mental health of CYP [13–21, 29]. Unlike previous reviews there were mixed finding for some of the key factors like parent-to-parent relationship and social status. This further adds to the body of evidence suggesting that mental health is complex and not all CYP may be affected in the same way by the various factors [10, 11]. Simi- lar to other reviews sexuality and screen time were explored in very few studies and resulted in limited evidence on which to draw stronger conclusions. In addition to the factors identified in the international literature, we found religion/prayer and policies/procedures to be associ- ated with mental health outcomes in CYP in the English-speaking Caribbean. We also built on the two regional reviews by identifying child to sibling relationships, cop- ing strategies, screen time, sexuality and policies/procedures as emerging factors that have not been previously explored. One possible explanation for any of the differences could be meth- odological. For example, our review included a wider age range (3–24 years) compared to other reviews, allowing us to capture issues around romantic relationships and sexual experi- ences under individual and relationship factors as possible sources of distress for CYP. These factors appeared to be missed or understudied in some of the international literature around CYP under age 18. Our findings further highlight the importance of exploring these factors, especially in regions like the English-speaking Caribbean where higher rates of teenage preg- nancies and risky sexual behaviours are common [30, 127]. The second possible explanation could be cultural. Notwithstanding the mixed findings, CYP’s identification with religion/prayer was seen as potential to be a protective factor against poorer mental health outcomes. The mixed findings could suggest that a deeper understanding of the differences between spiritual views and religious affiliations among CYP would be PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 14 / 23 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region needed before moving forward. This is especially important as some CYP and their families seek out mental health support from religious leaders [128]. Lastly, similar to other reviews, the studies included in our review were of varying quality. Comparison to previous reviews However, in line with recommendations for a narrative synthesis, we also explored the body of evidence contributing to each factor allowing us to make specific recommendations with greater confidence [41]. Despite urgent calls for mental health support for CYP in the Carib- bean [31], the amount of research that investigated some of the influencing factors (e.g., screen time and sexuality) appears to be small relative to other regions [20, 129]. Implications for practice This review identified several factors that have the potential to overlap or influence other fac- tors. Knowledge of these factors can help professionals identify CYP at risk of developing psy- chosocial problems or at risk of experiencing worsening mental health symptoms. Our findings also suggest a need for early interventions that can support CYP at different stages and in different settings. There are also opportunities to promote protective factors like helpful coping strategies and positive affect. By focusing on factors across the various levels there is also potential to achieve population impact. For example, universal interventions focusing on schools and families or those targeting specific groups of CYP based on sex/gender or religious affiliations may be beneficial. Notably, emerging evidence from the Caribbean suggest an interest for innovative delivery methods for CYP to receive support [131]. Therefore, these could include mobile apps and online resources or a combination of different approaches. Implications for future research Owing to the mixed findings on some of the associating factors (e.g., parent to child relation- ships) and the dearth of evidence on others (e.g., sexuality), we suggest that further research is needed to better our understanding as to why and in what context some CYP are at greater risk of developing mental health problems. Further cross-disciplinary research focusing on possible interactions among these factors would also be useful since the evidence reviewed did not always provide a consistent narrative of the risk profiles. The absence of some key factors (e.g., impact of natural disasters), presenting problems (e.g., psychosis), and countries (e.g., Belize) also reinforces recent appeals for more research in this area [27]. Despite the gradual increase in research activity in the last 40 years, there was also a lack of diversity in research methods used in the included studies and variation in the quality of the evidence, suggesting a need for a wider range of high-quality rigorous research activity. Implications for policy To our knowledge, this review of 83 articles is the largest and most up to date review of the evi- dence on the factors that contribute to psychological distress among CYP in the English-speaking Caribbean which comprise of majority low-and-middle-income and developing countries. There- fore, policy makers can use the findings as a guide during key decision-making periods. Also, based on the findings in this review the impact of policies/procedures on CYP is not yet clear. However, it appears that in some instances policies can be a source of distress for CYP; so, it is rec- ommended that CYP are involved in the development of policies that are relevant to them [130]. S1 File. PRISMA checklist. (DOCX) S2 File. Characteristics of the included studies. (PDF) S2 File. Characteristics of the included studies. (PDF) S3 File. Quality appraisal. (XLSX) S3 File. Quality appraisal. (XLSX) Strengths and limitations The main strength of this review is the conceptual organisation of factors associated with CYP’s mental health which contributes to the theoretical framework for identifying CYP who 15 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 PLOS ONE Factors associated with children and young people’s mental health in the English-speaking Caribbean region might be most at risk of mental health problems. The other strengths of this study include our comprehensive search strategy applied to academic databases and grey literature sources and the inclusion of peer reviewed articles. This study also benefited from the participation of at least two reviewers during screening and study selection, quality appraisal, data extraction and the narrative synthesis which could strengthen the reliability of our findings. However, the review is not devoid of limitations. Despite our best attempts to identify all relevant studies some information could have been missed. It is also important to note that this review pooled data from a number of studies, each with its own limitations. The decision to only include peer-reviewed material could also contribute to publication bias. Another limitation is the underrepresentation of specific age groups (e.g., <12 years), psychosocial problems (e.g., eat- ing disorders), geographic locations (e.g., Grenada) and limited exploration of some common associating factors (e.g., sexuality). However, where appropriate the authors were explicit in reporting specific information; but in keeping with guidelines for the narrative synthesis this was not always possible. Therefore, caution is advised if attempts are made to generalize our findings. Acknowledgments The authors thank the librarian who guided the academic database searches. We would also like to thank Miles Weekes for his support at the time of preparing the manuscript for submis- sion. We also acknowledge the Research Internship for Minority Ethnic Students programme at Edge Hill University which provided a stipend for two of the authors (JT and YD) during the data analysis period. Conclusions It is well established that CYP from regions consisting of low and middle income and develop- ing countries are at increased risk of experiencing mental health problems. This review adds to the international evidence by providing insights on 21 factors associated with CYP’s mental health in one such region. Of these, the role of religion/prayer had not been fully explored in the international reviews. Sexuality, screentime and the impact of policies/procedures are understudied and other factors like gender issues and wider societal problems (e.g., Covid-19) are not yet explored. Our findings support the relevance of considering individual, relation- ship, community and societal aspects in early identification and early interventions aimed at CYP’s mental health and wellbeing. Project administration: Shaun Liverpool. Project administration: Shaun Liverpool. Project administration: Shaun Liverpool. Resources: Shaun Liverpool, Yasmin Draoui, Judea Tucker, Brent Pereira, Jamal Prescod, Michael Owen, Catherine Trotman. Software: Shaun Liverpool. Supervision: Shaun Liverpool, Michael Owen. Supervision: Shaun Liverpool, Michael Owen. Validation: Shaun Liverpool, Brent Pereira, Michael Owen, Catherine Trotman Visualization: Shaun Liverpool. Writing – original draft: Shaun Liverpool, Yasmin Draoui, Judea Tucker. Writing – review & editing: Shaun Liverpool, Brent Pereira, Jamal Prescod, Michael Owen, Catherine Trotman. Formal analysis: Shaun Liverpool, Yasmin Draoui, Judea Tucker, Brent Pereira. Formal analysis: Shaun Liverpool, Yasmin Draoui, Judea Tucker, Brent Pereira. Investigation: Shaun Liverpool, Yasmin Draoui, Judea Tucker, Brent Pereira, Jamal Prescod, Catherine Trotman. Methodology: Shaun Liverpool. Methodology: Shaun Liverpool. Author Contributions Conceptualization: Shaun Liverpool. 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Function-Based Intervention for an Adolescent With Emotional and Behavioral Disorders in Bermuda: Moving Across Culture. PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023 References BEHAVIORAL DISOR- DERS. 2007; 33: 23–32. https://doi.org/10.1177/019874290703300102 123. Baker-Henningham H, Scott S, Jones K, Walker Kelvyn, ORCID: http://orcid.org/0000-0001-8398- 2190, Susan A I—ORCID: http://orcid.org/ Jones, H. B, et al. Reducing child conduct problems and promoting social skills in a middle-income country: Cluster randomised controlled trial. British Journal of Psychiatry. 2012; 201: 101–108. http://dx.doi.org/10.1192/bjp.bp.111.096834. 124. H. B, S.P. W, C. P, Baker-Henningham H, Walker SP, Powell C, et al. Preventing behaviour problems through a universal intervention in Jamaican basic schools: a pilot study. West Indian Med J. 2009; 58: 460–464. PMID: 20441066 125. Lowe GA, Lipps G, Gibson RC, Halliday S, Morris A, Clarke N, et al. Neighbourhood factors and depression among adolescents in four Caribbean countries. PloS one. 2014; 9: e95538. https://doi. org/10.1371/journal.pone.0095538 PMID: 24760035 126. [Anonymous]. The association of perceived neighbourhood factors, social class and other demo- graphic factors with depressive symptoms among Grade Six elementary school children in Jamaica. West Indian Med J. 2017; 66: 24. 127. Pradhan R, Wynter K, Fisher J. Factors associated with pregnancy among adolescents in low-income and lower middle-income countries: a systematic review. Journal of epidemiology and community health (1979). 2015; 69: 918–924. https://doi.org/10.1136/jech-2014-205128 PMID: 26034047 128. Rathod S, Pinninti N, Irfan M, Gorczynski P, Rathod P, Gega L, et al. Mental Health Service Provision in Low- and Middle-Income Countries. Health Services Insights. 2017; 2017: 1178632917694350. https://doi.org/10.1177/1178632917694350 PMID: 28469456 129. Stiglic N, Viner RM. Effects of screentime on the health and well-being of children and adolescents: a systematic review of reviews. BMJ Open. 2019; 9: e023191. https://doi.org/10.1136/bmjopen-2018- 023191 PMID: 30606703 130. [Anonymous]. The Impact of Children and Young People’s Participation on Policy Making. Scottish Government People and Society. 2018. 131. Maloney CA, Abel WD, McLeod HJ. Jamaican adolescents’ receptiveness to digital mental health ser- vices: A cross-sectional survey from rural and urban communities. Internet Interventions. 2020; 21: 100325. https://doi.org/10.1016/j.invent.2020.100325 PMID: 32455121 23 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666 March 8, 2023
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Transmission of Hepatitis E Virus from Rabbits to Cynomolgus Macaques
Emerging infectious diseases
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Transmission of Hepatitis E Virus from Rabbits to Cynomolgus Macaques Peng Liu,1 Qiu-Ning Bu,1 Ling Wang, Jian Han, Ren-Jie Du, Ya-Xin Lei, Yu-Qing Ouyang, Jie Li, Yong-Hong Zhu, Feng-Min Lu, and Hui Zhuang The zoonotic nature of HEV was first confirmed in 1997 with the identification of HEV isolates in swine in the United States, which were most closely related to an isolate of HEV from a person in the United States, and this isolate could experimentally infect nonhuman primates (5,6). Zoonotic transmission of HEV was further substantiated with the demonstration of HEV infection in persons after they ate undercooked infected meat from wild boars and wild deer (7,8). Antibodies against HEV have been detected in numerous animal species, including dogs, cats, sheep, goats, horses, cattle, bison, and rats; and HEV strains have been genetically identified from domestic and wild pigs, chickens, deer, mongooses, and rabbits (4,9). The recent discoveries of HEV-like viruses in rats and fish have further broadened understanding of the host range and diversity of HEV (10–12).i The recent discovery of hepatitis E virus (HEV) strains in rabbits in the People’s Republic of China and the United States revealed that rabbits are another noteworthy reservoir of HEV. However, whether HEV from rabbits can infect humans is unclear. To study the zoonotic potential for and pathogenesis of rabbit HEV, we infected 2 cynomolgus macaques and 2 rabbits with an HEV strain from rabbits in China. Typical hepatitis developed in both monkeys; they exhibited elevated liver enzymes, viremia, virus shedding in fecal specimens, and seroconversion. Comparison of the complete genome sequence of HEV passed in the macaques with that of the inoculum showed 99.8% nucleotide identity. Rabbit HEV RNA (positive- and negative-stranded) was detectable in various tissues from the experimentally infected rabbits, indicating that extrahepatic replication may be common. Thus, HEV is transmissible from rabbits to cynomolgus macaques, which suggests that rabbits may be a new source of human HEV infection. y ( ) The first strain of rabbit HEV was isolated from Rex Rabbits on 2 rabbit farms in Gansu, People’s Republic of China (13). Additional studies indicated that rabbit HEV was prevalent among various breeds of farmed rabbits throughout much of China, and the prevalence of antibodies against HEV was 57.0% in Lanzhou and 54.6% in Beijing (13–15). Rabbit HEV has also been isolated from rabbits in Virginia, USA, which showed a high prevalence of antibodies against HEV (36%) and HEV RNA (16.5%) (16). Phylogenetic analyses revealed that rabbit HEV was most closely related to genotype 3 HEV, which has been confirmed to infect humans. 1These authors contributed equally to this article. Author affiliation: Peking University Health Science Center, Beijing, People’s Republic of China DOI: http://dx.doi.org/10/3201/eid1904.120827 Experimental Inoculation of Nonhuman Primates To determine whether rabbit HEV strains are transmissible to nonhuman primates, we inoculated intravenously 2 cynomolgus monkeys, housed separately, with 2 mL of the rabbit HEV inoculum. After inoculation, serial serum and fecal samples were collected 2×/week for 16 weeks. Serum samples were tested for ALT levels and for IgM and IgG against HEV. All samples were also assayed for HEV RNA by RT-nPCR (15). Materials and Methods (2 rabbits per group) and inoculated intravenously with either 1 mL of PBS (negative control) or 1 mL of rabbit HEV inoculum. Serum and fecal specimens were collected weekly after inoculation. Serum samples were tested for ALT activity and HEV RNA. Fecal specimens were also assayed for HEV RNA. If serum and fecal specimens became simultaneously positive for HEV RNA, a complete necropsy was performed of each rabbit. Bile and various different types of tissues and organs, including liver, kidney, small intestine, spleen, stomach, heart, brain, bladder, and lung, were collected and stored at -80°C. To prevent cross-contamination during necropsy, we used individually wrapped, sterile disposable materials and new sterile scalpel blades for each sample. Transmission of Hepatitis E Virus from Rabbits to Cynomolgus Macaques Peng Liu,1 Qiu-Ning Bu,1 Ling Wang, Jian Han, Ren-Jie Du, Ya-Xin Lei, Yu-Qing Ouyang, Jie Li, Yong-Hong Zhu, Feng-Min Lu, and Hui Zhuang Furthermore, a recent study indicated that rabbit HEV is antigenically related to the other known animal strains of HEV and is experimentally transmissible to swine (17). However, to our knowledge, no study had determined the zoonotic potential of rabbit HEV. Therefore, in this study, we endeavored to ascertain whether rabbit HEV can cross species barriers and infect nonhuman primates and to further clarify the pathogenesis and replication of rabbit HEV in its natural host. H epatitis E virus (HEV) is the causative agent of acute hepatitis E, which is endemic to many developing countries and occurs sporadically in some industrialized countries. HEV is a small nonenveloped virus with a pos- itive-sense single-stranded RNA genome of ≈7.2 kb; it is currently classified as the sole member of the genus Hep- evirus, family Hepeviridae (1). Thus far, at least 4 geno- types, which comprise a single serotype, of HEV have been identified in mammals: genotypes 1 and 2 are restricted to strains that infect humans, and genotypes 3 and 4 are zoo- notic (2). More recently, a putative fifth HEV genotype was identified in wild boars in Japan (3). HEV from chickens, which is phylogenetically distinct from HEV from mam- mals, is likely to be classified as a new genus within the family Hepeviridae (4). H Author affiliation: Peking University Health Science Center, Beijing, People’s Republic of China DOI: http://dx.doi.org/10/3201/eid1904.120827 1These authors contributed equally to this article. 559 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 RESEARCH Virus Inocula The rabbit HEV strain (CHN-BJ-R14) used in this study was originally recovered from the feces of a farmed Rex Rabbit in the suburbs of Beijing in 2011. The fecal sample was diluted in phosphate-buffered saline (PBS) (pH 7.4) containing 1% bovine serum albumin to make a 10% (wt/vol) suspension. The clarified suspension was subsequently filtered through 0.45-μm and 0.22-μm filters. Titers of the rabbit HEV inoculum were determined by a semiquantitative nested reverse transcription PCR (RT- nPCR) (18), and the titer was 104 genome equivalents (GE) per milliliter (mL). Approximately 100 mg of each tissue and organ was homogenized in 1 mL of sterile PBS (pH 7.4) to make 10% (wt/vol) suspensions and clarified by centrifugation at 4,500 g for 10 min at 4°C. Thereafter, 100 µL of the clarified supernatants was used for total viral RNA extraction, and positive-stranded and negative- stranded HEV RNA were detected by RT-nPCR as described below. Determination of ALT Levels All serum samples were tested immediately for ALT levels with a Hitachi Automatic Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan), by using chemical reagents purchased from Roche (Basel, Switzerland), according to the manufacturer’s instructions. Biochemical evidence of hepatitis was recorded when the serum ALT level exceeded the baseline ALT level by >2- fold, as defined by a peak ALT value that was equal to or greater than double the prechallenge values (19,20). ELISA to Detect Antibodies against HEV The serum specimens collected from monkeys were tested for IgM and IgG against HEV by using an ELISA based on the virus E2 protein (amino acids 394–606 of HEV open reading frame [ORF] 2) (20), according to the manufacturer’s instructions (Wantai, Beijing, China). The serum samples collected from rabbits were also examined for antibodies by using the same assay. Signal-to-cutoff values were calculated, and values >1 were considered positive. Preinoculation baseline serum specimens were used as negative controls for each monkey. Animals Two juvenile male cynomolgus monkeys (Macaca fascicularis), weighing 2.0–2.5 kg, designated as Cy1 and Cy2, were obtained from the Beijing Xierxing Institute of Biologic Resources (Beijing, China) for the cross-species infection study. For the rabbit infection study, four 7-week- old specific-pathogen free (SPF) New Zealand white rabbits, weighing 750–1,000 g, were obtained from the Department of Laboratory Animal Science of Peking University Health Science Center. Preinoculation serum and feces specimens were collected once a week for 3 weeks, and all animals were tested for alanine aminotransferase (ALT) to establish a baseline, and were confirmed as negative for antibodies against HEV by an ELISA and negative for HEV RNA by RT-nPCR. The animal experiments were approved by the Committee of Laboratory Animal Welfare and Ethics, Peking University Health Science Center. The regulations of the review committee of Laboratory Animal Welfare and Ethics and the protocol for the review on Laboratory Animal Welfare and Ethics, Peking University Health Science Center, were followed. Cross-Species Transmission of Rabbit HEV to Nonhuman Primates In both of the macaques inoculated with rabbit HEV, hepatitis developed, as determined on the basis of ALT elevation, viremia, fecal shedding of viruses, and seroconversion (Figure). Dramatic elevations in serum ALT were observed 5 and 10 wpi for both monkeys, with a peak value of 135 U/L at 9 wpi for monkey Cy1 and 97 U/L at 5.5 wpi for monkey Cy2. Before inoculation, both monkeys were seronegative for HEV and became seropositive for antibodies against HEV at 6–7 wpi. IgM against HEV was detectable from 7 to 12 wpi for Cy1 and from 6 to 8 wpi for Cy2. The rise in IgM against HEV was followed closely by a strong response of IgG against HEV for Cy1, whereas both responses occurred at about the same time for Cy2. The IgG level against HEV remained markedly elevated at the end of the 16-week experiment. Results The PCR protocol used in this study could detect as few as 10 GE copies of HEV plasmid DNA. Negative and positive controls were included in each assay to exclude the possibility of contamination and failure of amplification. A recombinant plasmid containing HEV ORF1 and ORF2 fragments at a concentration of 102 copies per mL and serum or fecal specimens or tissues from naive rabbits were used as positive and negative controls, respectively. Samples showing a band of the expected size on a 1.5% (w/v) agarose gel were considered positive, and the positive products were directly sequenced. Transmission of HEV from Rabbits to Macaques Transmission of HEV from Rabbits to Macaques RNA was reverse transcribed at 42°C for 60 min with SuperScript II reverse transcription (Invitrogen) and the external reverse primer P4 or S4 in a reaction mixture of 20 mL. Then, nested PCRs were carried out to amplify the partial fragments of ORF1 (129–373 nt) and ORF2 (5,983– 6,349 nt) of the HEV genome by using the 2 sets of specific external and internal primer pairs listed in online Technical Appendix Table 1 (wwwnc.cdc.gov/EID/article/19/4/12- 0827-Techapp1.pdf). The PCR parameters for both sets of primers and both rounds of PCR were the same, with an initial incubation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 40 s, with a final incubation at 72°C for 10 min. entire viral genome. The nested PCR was done as described (21). The nucleotide sequences at the 5′ and 3′ termini of the genome were determined by using a rapid amplification of cDNA ends (RACE) kit (Invitrogen), according to the manufacturer’s instructions. Sequence Analyses The expected PCR products amplified from the inoculum and monkey fecal sample at 3 weeks wpi were purified and ligated into a pGEM-T vector (Promega). At least 3 positive clones for each region of the viral genome were sequenced commercially in both directions by using an automated DNA sequencer (ABI model 3730 sequencer; Applied Biosystems, Foster City, CA, USA). Tissues with detectable positive-stranded HEV RNA were then assayed for negative-sense HEV RNA by RT- nPCR with the same 2 sets of universal primers (online Technical Appendix Table 1). The extracted RNA was subjected to cDNA synthesis with the external forward primer P1 or S1. Then parental RNAs were degraded by RNaseH, and this was followed by nested PCR. The amplification conditions for negative-stranded HEV RNA detection were essentially the same as those used in the detection of positive-sense HEV RNA. Nucleotide sequences were assembled and analyzed with the MEGA 4.0 and ALIGNX software (Vector NTI package version 9.0; Invitrogen). ORFs were identified by using the EMBOSS software (version 5.0.0; emboss. sourceforge.net). The full-length genomic sequences of CHN-BJ-R14 and rHEV-Cy1 reported in this study have been deposited in GenBank under accession nos. JX109834 and JX121233, respectively. Experimental Infection of Rabbits RNA was extracted from 100 μL of serum, bile, tissue suspension, or 10% fecal suspension by using TRIzol reagent (Invitrogen, Burlington, ON, Canada), and purified RNA was resuspended in 11 μL of RNase-free water. To detect positive-stranded HEV RNA, 11 μL of purified To clarify the extrahepatic replication sites of HEV, rabbits were experimentally infected with rabbit HEV as described (19). In brief, 4 SPF rabbits, which were housed in separate cages, were divided randomly into 2 groups Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 560 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 Amplification of the Full-Length Genome of Rabbit HEV Alanine aminotransferase (ALT) levels are plotted as U/L. The baseline ALT levels were 33 U/L and 38 U/L for Cy1 and Cy2, respectively. The titers of HEV IgM and IgG are plotted as ELISA signal-to-cutoff (S/CO) values. Presence and absence of HEV RNA in serum or feces are indicated by + and – signs, respectively. Sequence Analyses of Rabbit HEV during Cross-Species Transmission Amplification of the Full-Length Genome of Rabbit HEV To compare the complete genome sequence of the HEV passed in the macaques to that of the inoculum, the fecal sample (rHEV-Cy1) of 1 monkey at 3 weeks’ postinoculation (wpi) and the inoculum (CHN-BJ-R14) were sequenced to determine the full-length genome as reported (21). Briefly, total RNA was extracted from 120 μL of the rabbit HEV inoculum and a 10% monkey fecal suspension in PBS by using the Total RNA Isolation System (Promega, Madison, WI, USA). cDNA was synthesized from 12 μL of purified RNA by using 1 μL (200 U) of Moloney murine leukemia virus reverse transcription (Promega) and 2 μL (10 pmol/L) of OligodT primer. With 6 sets of specific external and internal primer pairs (online Technical Appendix Table 2), a set of nested PCRs were performed by using the first-strand cDNA to amplify the Serum and fecal samples taken before inoculation from both monkeys were negative for HEV RNA. Viremia and fecal shedding of viruses were detected in both monkeys after intravenous inoculation. Fecal excretion of rabbit HEV, indicative of replication, was first detected at 1 wpi and persisted for 5–9 weeks. HEV viremia was first detected at 5.5 wpi for Cy1 and at 2 wpi for Cy2 and lasted for 2.5–3.5 weeks. The partial sequences of the PCR products from both monkeys shared 99%–100% nucleotide identity with the original inoculum. 561 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 RESEARCH Figure. Cross-species trans- mission of rabbit hepatitis E virus (HEV) to 2 cynomolgus macaques (Cy1 and Cy2). Alanine aminotransferase (ALT) levels are plotted as U/L. The baseline ALT levels were 33 U/L and 38 U/L for Cy1 and Cy2, respectively. The titers of HEV IgM and IgG are plotted as ELISA signal-to-cutoff (S/CO) values. Presence and absence of HEV RNA in serum or feces are indicated by + and – signs, respectively. Figure. Cross-species trans- mission of rabbit hepatitis E virus (HEV) to 2 cynomolgus macaques (Cy1 and Cy2). Alanine aminotransferase (ALT) levels are plotted as U/L. The baseline ALT levels were 33 U/L and 38 U/L for Cy1 and Cy2, respectively. The titers of HEV IgM and IgG are plotted as ELISA signal-to-cutoff (S/CO) values. Presence and absence of HEV RNA in serum or feces are indicated by + and – signs, respectively. Figure. Cross-species trans- mission of rabbit hepatitis E virus (HEV) to 2 cynomolgus macaques (Cy1 and Cy2). Extrahepatic Replication of HEV in Experimentally Infected Rabbits food safety (22,23). The recent discovery of rabbit strains of HEV in China (13) and the United States (16) showed that farmed rabbits are another key reservoir of HEV. In our previous study, phylogenetic analysis of the genome of rabbit HEV suggested the potential for cross-species transmission of rabbit HEV (21). A recent study also demonstrated that rabbit HEV can cross species barriers and infect SPF pigs (17). In the study described here, we showed that under experimental conditions, rabbit HEV is transmissible to cynomolgus macaques, which can serve as surrogates for humans. This finding suggests that rabbit HEV may be a new source of human HEV infection. Both control rabbits remained negative for HEV RNA throughout the study. Viremia and fecal shedding of HEV were detected in rabbits inoculated with the rabbit HEV inoculum. Both rabbits were necropsied, at 5.5 wpi and 12 wpi, respectively, when ALT elevation was observed, and HEV RNA was detected simultaneously in serum and feces. Bile and 9 different types of tissues and organs were collected and tested for positive-stranded HEV RNA. Positive-stranded HEV RNA was detected in bile and in 5 of the tissues—liver, kidney, small intestine, spleen, and stomach. Detection of positive-stranded HEV RNA from various tissues and organs did not indicate that the virus was replicating in these tissues because contamination of the tissue samples by virus circulating in the blood could not be ruled out. To further identify the replicating sites of HEV, we screened for negative-stranded RNA, which is an intermediate product during HEV replication, in all tissues that were positive for the positive-stranded HEV RNA. Negative-stranded RNA was also detectable in the 5 types of tissues. The positive products were sequenced and found to be identical to the original inoculum. In both cynomolgus monkeys infected in this study with 104 GEs of rabbit HEV, typical acute hepatitis E developed. The patterns of HEV infection in cynomolgus monkeys infected with rabbit HEV were similar to those of animals inoculated with HEV strains of genotypes 1–4, that is, characterized by fecal excretion of virus, followed by viremia and liver enzyme elevation and finally by seroconversion (24–27). Although the same viral doses were inoculated into both monkeys, the overall course of disease varied somewhat, findings in accord with those of previous studies (28). Sequence Analyses of Rabbit HEV during Cross-Species Transmission the inoculum (CHN-BJ-R14) revealed 18 nt mutations over the entire genome, resulting in 9 nonsynonymous amino acid changes. ORF1 harbored 16 of the 18 nt mutations; 11 were in the helicase domain and in the RNA-dependent RNA polymerase domain (Table). To analyze mutations in the rabbit HEV genome that appeared during a single passage between the 2 different host species, we sequenced rabbit HEV strains recovered from the inoculum (CHN-BJ-R14) and from experimentally infected cynomolgus monkeys (rHEV-Cy1) over the entire genome. The CHN-BJ-R14 and rHEV-Cy1 isolates had the same genomic length of 7,284 nt, excluding the 3′ poly (A) tail, and contained 3 ORFs—ORF1, ORF2, and ORF3— which encoded proteins of 1,722 aa (nt 26–5194), 660 aa (nt 5232–7214), and 113 aa (nt 5221–5562), respectively. The 5′ untranslated region (UTR) and 3′ UTR comprise 25 nt and 71 nt, respectively. Sequence analyses showed that CHN- BJ-R14 and rHEV-Cy1 shared 99.8% nucleotide identity with each other. Comparison of the complete genome sequence of rabbit HEV passed in the macaques (rHEV-Cy1) with that of Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) analysis showed that CHN-BJ-R14 and rHEV- Cy1 were most closely related to genotype 3 HEV with a maximum nucleotide identity of 81%, with the exception of 3 other rabbit HEV strains isolated in Gansu (13) and Beijing (21). However, several unique features possessed only by rabbit HEVs, but not genotype 3 or other HEV strains, were observed in the 2 rabbit HEV isolates of this study. These features, discovered in a previous study (21), were characterized by an insertion of 31 aa in ORF1 (929–959 aa) and a unique A residue at nt 13 (sites based on CHN-BJ-R14) in the 5′ UTR (data not shown). 562 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 Transmission of HEV from Rabbits to Macaques Extrahepatic Replication of HEV in Experimentally Infected Rabbits In an earlier study, cross-species infection of pigs infected with rabbit HEV showed a delayed onset and short duration of viremia and fecal virus shedding and an absence of seroconversion (17), which differed from findings observed in infected monkeys of this study. The differences might suggest that pigs are less susceptible than nonhuman primates to rabbit HEV. However, because the inocula in both the current study and in other studies (17,19) Discussioni Since the first animal strain of HEV, swine HEV, was identified from a pig in the United States in 1997 (5), the increasing identification of HEV infection among a wide range of animals, including pigs, chickens, wild boar, and deer (4), has raised public health concern for zoonoses and Table. Comparison of the complete genome sequence of rabbit HEV passed in macaques with that of the inoculum* Nucleotide position† Genomic region Nucleotide Amino acid CHN-BJ-R14‡ rHEV-Cy1§ Position† Substitution 614 ORF1-MeT C T 197 Silent 957 ORF1-Y T C 311 Thr to Ile 1667 ORF1-PCP T C 548 Silent 1875 ORF1 T C 617 Pro to Leu 2706 ORF1-X G A 894 Asp to Gly 3553 ORF1-Hel A T 1176 Silent 3571 ORF1-Hel C T 1182 Silent 3859 ORF1-RdRp C A 1278 Silent 3889 ORF1-RdRp C T 1288 Silent 3972 ORF1-RdRp G A 1316 Glu to Gly 4215 ORF1-RdRp C T 1397 Leu to Pro 4285 ORF1-RdRp A G 1420 Silent 4414 ORF1-RdRp T C 1463 Silent 4427 ORF1-RdRp C T 1468 Tyr to His 4882 ORF1-RdRp T C 1619 Silent 5028 ORF1-RdRp T C 1668 Ala to Val 5531 ORF2 C T 100 Silent ORF3 C T 104 Ala to Val 5713 ORF2 T A 161 Ile to Asn *HEV, hepatitis E virus; ORF, open reading frame; Thr, Threonine; Ile, Isoleucine; Pro, proline; Leu, leucine; Asp, aspartic acid; Gly, glycine; Glu, glutamic acid; Tyr, tyrosine; His, histidine; Ala, alanine; Val, valine; Asn, asparagine. †Nucleotide or amino acid position according to the rabbit HEV CHN-BJ-R14 strain. ‡CHN-BJ-R14, HEV isolate recovered from the rabbit HEV inoculum in this study. §rHEV-Cy1, HEV isolate recovered from the fecal sample of 1 monkey at 3 wpi in this study. ¶Putative domains in ORF1. MeT, methyltransferase domain; Y, Y domain; PCP, papain-like cysteine protease domain; X, X or macro domain; Hel, helicase domain; RdRp, RNA-dependent RNA polymerase domain. Table. Comparison of the complete genome sequence of rabbit HEV passed in macaques with that of the in Nucleotide Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013 56 563 RESEARCH have not yet been titrated for infectivity and because HEV infections are virus dose dependent (18), additional studies should be performed to determine the infectivity titer of rabbit HEV and to demonstrate whether the rate of inducing hepatitis increases with virus dose of infection. Acknowledgments We are grateful to Malcolm A. McCrae for proofreading the revised manuscript. This work was partially supported by the National Science Foundation of China (grant no. 81271827). Dr Peng is a PhD student at Department of Microbiology, School of Basic Medical Sciences, Peking University. His primary research interests are the molecular epidemiology and pathogenesis of HEV. Discussioni HEV found in this study and the other reports of extrahepatic manifestations of HEV infection in humans (34), clinicians should consider the possibility of HEV infection in patients with nonhepatic diseases, especially patients with acute pancreatitis, neurologic syndromes, thrombocytopenia, hemolysis, and autoimmune manifestations. In the current study, although comparison of the full- length sequences of rHEV-Cy1 and CHN-BJ-R14 showed 99.8% nucleotide identity, 18 nt changes, resulting in 9 nonsynonymous amino acid substitutions, were found in the genome of HEV. These results suggest that adaptation of rabbit HEV to growth in cynomolgus monkeys may be associated with a certain number of mutations. Eleven of the 16 mutations fell within ORF1, accompanied by 4 nonsynonymous substitutions, mapped to the helicase region and the RNA-dependent RNA polymerase region, which are essential for efficient replication of the genomes of HEV (29). Moreover, although most mutations are expected to be in the third codon position, of the 16 substitutions in ORF1, 7 occur at the first codon position and 3 at the second codon position. These facts may indicate that positive selection is operating in the infection of the cynomologus monkeys with the rabbit HEV inoculum. A recent study revealed that high-throughput sequencing of isolates from bile and feces from 2 pigs experimentally infected with human HEV of genotype 3f shared the same full-length consensus sequence as in the human sample, although a limited spectrum of mutations were observed during the interspecies transmission (30). The genomic sequences in this study were determined by sequencing several randomly selected positive clones, which is much less extensive than high-throughput sequencing; consequently, additional studies will be needed to verify whether the sequence changes that occurred after cross-species transmission of rabbit HEV to cynomolgus monkeys are adaptive mutations or result from the quasispecies structure of HEV. In conclusion, the successful infection of cynomolgus macaques with rabbit HEV suggests that humans might be at risk for infection with rabbit HEV. Further, rabbit HEV was detectable in multiple rabbit tissues and organs, indicating extrahepatic replication may be a common feature of rabbit HEV. These findings raise additional concern for zoonotic transmission of HEV infection among persons who have occupational exposure to rabbits or persons who eat undercooked rabbit meat. 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Infect Genet Evol. 2011;11:2020–5. http:// dx.doi.org/10.1016/j.meegid.2011.09.006 Address for correspondence: Ling Wang, Department of Microbiology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, China; email: lingwang@bjmu.edu.cn Emerging Infectious Diseases • www cdc gov/eid • Vol 19 No 4 April 2013 565 565 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 19, No. 4, April 2013