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https://openalex.org/W2909937546
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https://elpub.vir.nw.ru/jour/article/download/169/119
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Russian
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AGROBIOLOGICAL CHARACTERISTICS OF THE MALTING SPRING BARLEY CULTIVAR ‘OMSKY 100
|
Trudy po prikladnoj botanike, genetike i selekcii
| 2,017
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cc-by
| 7,785
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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
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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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IOP conference series. Materials science and engineering
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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
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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
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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
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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,
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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
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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
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Iverson, L., Fels, S., and Fisher, B. Towards
understanding IT security professionals and their
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[5] CA Corporation. How can a comprehensive identity
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Gagne, A., and Beznosov, K. Human, Organizational,
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[7] Heckle, R., Lutters, W. G., and Gurzick, D. Network
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[8] Jaferian, P., Botta, D., Raja, F., Hawkey, K., and
Beznosov, K. Guidelines for Designing IT Security
Management Tools. In CHIMIT . ACM, 2008,
7:1–7:10.
[9] Kandogan, E. and Haber, E. M. Security
administration tools and practices. In L. F. Cranor
and S. Garfinkel, eds., Security and Usability:
Designing Secure Systems that People Can Use,
O’Reilly Media, Inc., 2005. 357–378.
[10] Knapp, K. J., Marshall, T. E., Rainer, R. K., and
Ford, F. N. Managerial dimensions in information
security: A theoretical model of organizational
effectiveness. https://www.isc2.org/
download/auburn study2005.pdf (2005).
[11] Microsoft Corporation. Microsoft identity and access
management series: Fundamental concepts (2006).
[12] Post, G. V. and Kagan, A. Evaluating information
security tradeoffs: Restricting access can interfere
with user tasks. Computers & Security, 26 , 3 (2007),
229 – 237.
[13] Stake, R. E. The Art of Case Study Research. Sage
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[14] Vaidya, J., Atluri, V., and Guo, Q. The role mining
problem: Finding a minimal descriptive set of roles. In
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2007, 175–184.
[15] Werlinger, R., Hawkey, K., and Beznosov, K. 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
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[17] Wright, J. Final progress reports.
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|
en
|
Comment on acp-2020-1270
| null | 2,021
|
cc-by
| 12,943
|
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
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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).
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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).
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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
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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
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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
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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
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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.
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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. This research has been supported by the
National Research Foundation of Korea (grant nos. NRF2020R1A4A1018818 and NRF-2021M1A5A1065425 (KOPRIPN21011)).
Review statement. This paper was edited by Aurélien Dommergue
and reviewed by two anonymous referees.
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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
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2 3 218 00 2012
d A
06 2012 [ ]
,
p
p
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(
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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https://openalex.org/W4386038831
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http://waocp.com/journal/index.php/apjcb/article/download/918/2172
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English
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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
| 2,022
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cc-by
| 6,910
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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
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SR, Spanos WJ. Palliation of pelvic and head and neck cancer
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Prabhash K. Palliative chemotherapy in head and neck
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an update on 93 randomised trials and 17,346 patients. Radiotherapy and Oncology: Journal of the European
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RB, Jones CU, et al. Radiotherapy plus cetuximab for
squamous-cell carcinoma of the head and neck. The New
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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
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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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Zenodo (CERN European Organization for Nuclear Research)
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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
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SEAMEO Regional Centre for Special Educational Needs Proceedings of the International Conference on Special Education
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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
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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
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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
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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
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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
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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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https://openalex.org/W3033506244
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https://www.hal.inserm.fr/inserm-02881214/file/s41598-020-66016-7.pdf
|
English
| null |
miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy
|
Scientific reports
| 2,020
|
cc-by
| 14,498
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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
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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
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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
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The authors declare no competing interests. p
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1765–1786 (2013). 73. Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. J. R. Soc. 57, 289–300 (1995).t 74. Lê, S., Josse, J. & Husson, F. FactoMineR: An R Package for Multivariate Analysis. J. Stat. Softw. 25, 1–18 (2008). 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
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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. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway
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Research Square (Research Square)
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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
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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
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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
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https://openalex.org/W4383058277
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https://link.springer.com/content/pdf/10.1007/s11538-023-01170-3.pdf
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English
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Chemical Systems with Limit Cycles
|
Bulletin of mathematical biology
| 2,023
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cc-by
| 16,866
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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. References Boros B, Hofbauer J (2021) Oscillations in planar deficiency-one mass-action systems. J Dyn Differ Equ. https://doi.org/10.1007/s10884-021-10051-z 123 Page 29 of 29
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P, Craciun G (2018) Mathematical analysis of chemical reaction systems. Isr J Chem 58:1–10 u P, Craciun G (2018) Mathematical analysis of chemical reaction systems. Isr J Chem 58:1–10 Yu P, Craciun G (2018) Mathematical analysis of chemical reaction systems. Isr J Chem 58:1–10 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps
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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.
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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
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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. 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). 12.
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ZnII and CdII Metal Ions: Synthesis, Potentiometric Studies, and Crystal Structures. Inorg. Chem. 2016, mbrosi, G.; Formica, M.; Fusi, V.; Giorgi, L.; Macedi, E.; Micheloni, M.; Paoli, P.; Rossi, P. A Biphenol-Based
nII and CdII Metal Ions: Synthesis, Potentiometric Studies, and Crystal Structures. Inorg. Chem. 2016, 55, 7676 g
p
nd CdII Metal Ions: Synthesis, Potentiometric Studies, and Crystal Structures. Inorg. Chem. 2016, 55, 7676–76 45. Ambrosi, G.; Battelli, C.; Formica, M.; Fusi, V.; Giorgi, L.; Macedi, E.; Micheloni, M.; Pontellini, R.; Prodi, L. Two polyaminopheno-
lic fluorescent chemosensors for H + and Zn(II). Spectroscopic behaviour of free ligands and of their dinuclear Zn(II) complexes. New J. Chem. 2009, 33, 171–180. [CrossRef] 46. Ambrosi, G.; Formica, M.; Fusi, V.; Giorgi, L.; Micheloni, M. Polynuclear metal complexes of ligands c
Coord. Chem. Rev. 2008, 252, 1121–1152. [CrossRef] ca, M.; Fusi, V.; Giorgi, L.; Micheloni, M. Polynuclear metal complexes of ligands containing phenolic units
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Distance in Dinuclear Complexes. New Ligand with a 2,2‘-Biphenol Fragment as Key Unit: Synthesis, Coordination Behavior,
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Maggi, F.; la Cecilia, D.; Tang, F.H.M.; McBratney, A. The global environmental hazard of glyphosate use. Sci. Total Environ. 2020,
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66. Aragoni, M.C.; Arca, M.; Bencini, A.; Caltagirone, C.; Conti, L.; Garau, A.; Valtancoli, B.; Isaia, F.; Lippolis, V.; Palomba, F.; et al. Zn2+/Cd2+ optical discrimination by fluorescent acridine-based bis -macrocylic receptors. Supramol. Chem. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. 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] 2017, 29,
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HYPERQUAD suite of programs. Talanta 1996, 43, 1739–1753. [CrossRef] 67. Gran, G. Determination of the equivalence point in potentiometric titrations. Part II. Analyst 1952, 77, 661 671. [CrossRef]
68. Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of equilibrium constants with the
HYPERQUAD suite of programs. Talanta 1996, 43, 1739–1753. [CrossRef] 68. Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in solution. Determination of equilibrium constants with the
HYPERQUAD suite of programs. Talanta 1996, 43, 1739–1753. [CrossRef] 69. Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Hyperquad simulation and speciation (HySS): A utility
program for the investigation of equilibria involving soluble and partially soluble species. Coord. Chem. Rev. 1999, 184,
311–318. [CrossRef] 69. Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Hyperquad simulation and speciation (HySS): A utility
program for the investigation of equilibria involving soluble and partially soluble species. Coord. Chem. Rev. 1999, 184,
311–318. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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Goldene Konfirmation des Jahrgangs 1940/41
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Backnanger Jahrbuch
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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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English
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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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E3S web of conferences
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cc-by
| 4,771
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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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https://openalex.org/W4299693788
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https://zenodo.org/records/167761/files/02_IJRG16_SE10_02.pdf
|
English
| null |
ATTITUDE OF PLUS TWO STUDENTS TOWARDS PUBLIC EXAMINATION IN RELATION TO PARENTAL PRESSURE
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,016
|
cc-by
| 1,979
|
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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https://openalex.org/W4254106807
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https://www.qeios.com/read/4HJWLK/pdf
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English
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Postneoadjuvant Therapy Stage I Esophageal Adenocarcinoma AJCC v8
|
Definitions
| 2,020
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cc-by
| 90
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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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https://openalex.org/W2789840097
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https://europepmc.org/articles/pmc5871819?pdf=render
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English
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Lung Topology Characteristics in patients with Chronic Obstructive Pulmonary Disease
|
Scientific reports
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| 11,038
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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.). The data are not publicly available due to them containing information that
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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. References Adcock, A., Carlsson, G. & Rubin, D. Classification of hepatic lesions using the matching metric. Comput. Vis. Image Und. 36–42 (2014). 30. Bendich, P., Marron, J. S., Miller, E., Pieloch, A. & Skwerer, S. Persistent homology analysis of brain artery trees. Ann. Appl. Stat. 10,
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with conventional methods. European Journal of Radiology 84(5), 963–974 (2015). 33. Kirby, M. et al. A Novel Method of Estimating Small Airway Disease Using Inspiratory-to-Expiratory Computed Tomography. Respiration 94(4), 336–345 (2017). p
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disease progression. Nature Medicine 18(11), 1711–1715 (2012). SCIENtIFIC REPOrTS | (2018) 8:5341 | DOI:10.1038/s41598-018-23424-0 11 www.nature.com/scientificreports/ 35. Perea, J. A. Persistent homology of toroidal sliding window embeddings, presented at 2016 IEEE International Conferen
Acoustics, Speech and Signal Processing (ICASSP), (unpublished) (2016). p
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39. Sieren, J. P. SPIROMICS Protocol for Multicenter Quantitative Computed Tomography to Phenotype the Lungs. Am Journal Crit
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J t l TDA
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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
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https://openalex.org/W1998062667
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https://europepmc.org/articles/pmc5956421?pdf=render
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English
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Quantified Hypoxia and Anoxia in Lakes and Reservoirs
|
The scientific world journal/TheScientificWorldjournal
| 2,004
|
cc-by
| 7,501
|
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.
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Gordon, J.A. (1993) Dissolved oxygen in streams and reservoirs. Water Environ. Res. 65, 571–573.
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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
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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
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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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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”
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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;
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Macmillan, 2017. ELIAS, Ana Sofia; GILL, Rosalind; SCHARFF, Christina. Aesthetic Labour: Rethinking Beauty Politics in
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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
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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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The Effect of Traditional Cupping on Pain and Mechanical Thresholds in Patients with Chronic Nonspecific Neck Pain: A Randomised Controlled Pilot Study
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Evidence-based complementary and alternative medicine
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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
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Confounding adjustment performance of ordinal analysis methods in stroke studies
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medRxiv (Cold Spring Harbor Laboratory)
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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
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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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Scientific reports
| 2,017
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cc-by
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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
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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
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users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
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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
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*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. Since all experiments were performed on
embryonic mice, whole litters, which included both male and female mice, were used for
experiments. We did not detect any sex-based differences in our phenotype. The ages used in
each experiment are included in the relevant text, figures, and figure legends. Experiments
were performed with the observer blind to genotype, though the ensuing image analyses
were not due to the obvious nature of the phenotypes. All animal experiments were approved
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•
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
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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
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https://www.frontiersin.org/articles/10.3389/fmicb.2019.00590/pdf
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English
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Metatranscriptomic Signatures Associated With Phytoplankton Regime Shift From Diatom Dominance to a Dinoflagellate Bloom
|
Frontiers in microbiology
| 2,019
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cc-by
| 11,272
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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. 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
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(CC BY). The use, distribution or reproduction in other forums is permitted, provided
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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
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a Prorocentrum donghaiense bloom in the coastal waters of the East China
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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
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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
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https://openalex.org/W2570057063
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http://jurnalalqalam.or.id/index.php/Alqalam/article/download/341/256
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Indonesian
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IMPLEMENTASINILAI-NILAIPENDIDIKAN DALAM SURAH ALI 'IMRAN PADA MTsN WATAMPONE
|
Al Qalam - Balai Penelitian Lektur Keagamaan Ujung Pandang/Al-Qalam
| 2,014
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cc-by-sa
| 7,223
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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,
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Abiy al-Husain. 1999. Mu'jam Maqyis al-Lugah. Bairut: Dar
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terimplementasi
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Madrasah
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Negeri
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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
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Kamus Arab Indonesia. Yogyakarta:
Pondok
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Islam. Solo: Kurnia Kalam Semesta.
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https://openalex.org/W2753999658
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https://kar.kent.ac.uk/83566/1/Garner_Incentive_Value_And_Spatial_Certainty_2020.pdf
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English
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Distinct selection mechanisms for when predictions and rewards guide visual selective attention
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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
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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
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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
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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-
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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
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tation, distribution and reproduction in any medium or format, as long as
you give appropriate credit to the original author(s) and the source, pro-
vide 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
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are available online. See: https://github.com/kel-github/attention-value-
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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)
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hydraulic conductance within Prunus root systems. Tree 16(7):479–487 Volpe V, Marani M, Albertson J, Katul G (2013) Root controls on water redistribution and carbon uptake
in the soil plant system under current and future climate. Adv Water Resour 60:110–120 Wasson AP, Richards RA, Chatrath R, Misra SC, Prasad SV, Rebetzke GJ, Kirkegaard JA, Christopher J,
Watt M (2012) Traits and selection strategies to improve root systems and water uptake in water-limited
wheat crops. J Exp Bot 63(9):3485–3498 Wheeler TD, Stroock AD (2008) The transpiration of water at negative pressures in a synthetic tree. Nature
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tradeoffs between water uptake and root length utilization. J Plant Growth Regul 21(4):315–323 12 123
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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. IEEE Wireless Communications. 22,122-128(2015) 2. G. F. Silva, A. Donaire, A. McFadyen, J. J. Ford: String stable integral design for
vehicle platoons with disturbance. Automatica. 127,109542(2021) 3. S. Darbha, S. Konduri, P. R. Pagilla: Vehicle platooning with constant spacing
strategies and multiple vehicle look ahead information. IET Intell. Trans. Syst. 14,589-600(2020) 4. J. I. Ge, G. Orosz: Dynamics of connected vehicle system with delayed acceleration
feedback. Transp. Res. C, Emerg. Technol. 46,46-64(2014) 5. F. Lin, M. Fardad, M. R. Jovanovic: Optimal control of vehicular formations with
nearest neighbor interactions. IEEE Trans. Autom. Control. 57,2203-2218(2012) 6. Y. G. Bian, Y. Zheng, W. Ren, et al.: Reducing time headway for platooning of
connected vehicles via V2V communication. Transp. Res. C,102,87-105(2019) 7. B. Besselink, K. H. Johansson: String stability and a delay-based spacing policy for
vehicle platoons subject to disturbances. IEEE Trans. Autom. 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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Phenotypic Parameters Prediction of Lettuce based on Computer Vision and Regression
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cc-by
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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.
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https://openalex.org/W2324343575
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https://zenodo.org/records/2414695/files/article.pdf
|
English
| null |
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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https://openalex.org/W2082413425
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English
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FURTHER OBSERVATIONS ON THE PNEUMOTHORAX TREATMENT OF PHTHISIS.
|
Lancet
| 1,912
|
public-domain
| 4,839
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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. RÉSUMÉ. 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 hemihyperæsthesia 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 Médicale, 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 AXILLÆ; 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, dyspnœa,
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 Générales de Médecine 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 Münchener Medizinische Wochenschrift, Feb. 28th, 1911. g
6 Brit. Med. Jour., Oct. 12th, 1912.
7 Medical Press and Circular, Nov. 13th, 1912. 4 Münchener 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 AXILLÆ; 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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https://openalex.org/W2950690143
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https://europepmc.org/articles/pmc6631453?pdf=render
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English
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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
|
Molecules/Molecules online/Molecules annual
| 2,019
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cc-by
| 10,164
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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,
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loading compounds are not available. © 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/). © 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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https://doi.org/10.5771/0506-7286-1985-2-233_1, am 14.07.2024, 03:19:32
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https://doi.org/10.5771/0506-7286-1985-2-233_1, am 14.07.2024, 03:19:32
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https://openalex.org/W2053422221
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https://www.mdpi.com/1420-3049/13/2/319/pdf?version=1403112475
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English
| null |
Nitroalkanes as Central Reagents in the Synthesis of Spiroketals
|
Molecules/Molecules online/Molecules annual
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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
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https://openalex.org/W2508396876
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English
| null |
Extrasolar planets and brown dwarfs around AF-type stars
|
Astronomy & astrophysics
| 2,017
|
cc-by
| 27,692
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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
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Astrophysics Astronomy
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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
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103
HD43318
10
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103
HD82328
10
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Minimal mass (Mjup)
HD91312
10
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103
HD143584
10
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102
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103
HD148048
10
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103
Minimal mass (Mjup)
HD185395
10
1
100
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102
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103
HD196524
10
1
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101
102
Separation (au)
10
1
100
101
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103
HD199254
10
1
100
101
102
Separation (au)
10
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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
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101
102
103
HD82328
HD148048 10
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102
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10
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103
HD199254 10
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HD196524
HD212754 101
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5
5 Separation (au) 10
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2
3
HD210715
10
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102
Separation (au)
10
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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
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10
-1
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< 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
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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
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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
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546, A69 Appendix A: SOPHIE sample The main physical properties and the main characteristics of the spectroscopic observables for each of the targets of our
SOPHIE sample are detailed in Table A.1. The main physical properties and the main characteristics of the spectroscopic observables for each of the targets of our
SOPHIE sample are detailed in Table A.1. p y
p
p
p
p
g
SOPHIE sample are detailed in Table A.1. Table A.1. 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
|
https://openalex.org/W2923107797
|
https://europepmc.org/articles/pmc6476923?pdf=render
|
English
| null |
Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury
|
Frontiers in pharmacology
| 2,019
|
cc-by
| 8,541
|
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. Therefore, collectively these findings indicate REFERENCES doi: 10.1016/j.nbd.2009.10.023 Lever, C., Burton, S., and O’Keefe, J. (2006). Rearing on hind legs, environmental
novelty, and the hippocampal formation. Rev. Neurosci. 17, 111–134. doi: 10.1515/revneuro.2006.17.1-2.111 D’Aniello, A., Luongo, L., Romano, R., Iannotta, M., Marabese, I., Boccella, S., et al. (2017). d-Aspartic acid ameliorates painful and neuropsychiatric changes and
reduces beta-amyloid abeta1-42 peptide in a long lasting model of neuropathic
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J.,
Xiong,
X.,
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Y. (2019). Isoliquiritigenin
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translational challenges and emerging therapeutic strategies. Trends Pharmacol. Sci. 31, 596–604. doi: 10.1016/j.tips.2010.09.005 De Gregorio, D., McLaughlin, R. J., Posa, L., Ochoa-Sanchez, R., Enns, J., Lopez-
Canul, M., et al. (2018). Cannabidiol modulates serotonergic transmission and
prevents allodynia and anxiety-like behavior in a model of neuropathic pain. Pain 160, 136–150. doi: 10.1097/j.pain.0000000000001386 Luongo, L., de Novellis, V., Gatta, L., Palazzo, E., Vita, D., Guida, F., et al. (2013). Role of metabotropic glutamate receptor 1 in the basolateral amygdala-
driven prefrontal cortical deactivation in inflammatory pain in the rat. Neuropharmacology 66, 317–329. doi: 10.1016/j.neuropharm.2012.05.047 Feliciano, D. P., Sahbaie, P., Shi, X., Klukinov, M., Clark, J. D., and
Yeomans, D. C. (2014). Nociceptive sensitization and BDNF up-regulation
in a rat model of traumatic brain injury. Neurosci. Lett. 583, 55–59. doi: 10.1016/j.neulet.2014.09.030 Mayeux, J., Katz, P., Edwards, S., Middleton, J. W., and Molina, P. E. (2017). Inhibition of endocannabinoid degradation improves outcomes from mild
traumatic brain injury: a mechanistic role for synaptic hyperexcitability. J. Neurotrauma 34, 436–443. doi: 10.1089/neu.2016.4452 Fernández-Ruiz, J., Sagredo, O., Pazos, M. R., García, C., Pertwee, R.,
Mechoulam, R., et al. (2013). Cannabidiol for neurodegenerative disorders:
important new clinical applications for this phytocannabinoid? Br. J. Clin. Pharmacol. 75, 323–333. doi: 10.1111/j.1365-2125.2012.04341.x Palazzo, E., Romano, R., Luongo, L., Boccella, S., De Gregorio, D., Giordano,
M. E., et al. (2015). MMPIP, an mGluR7-selective negative allosteric modulator,
alleviates pain and normalizes affective and cognitive behavior in neuropathic
mice. Pain 156, 1060–1073. doi: 10.1097/j.pain.0000000000000150 Giordano, C., Cristino, L., Luongo, L., Siniscalco, D., Petrosino, S., Piscitelli, F.,
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cortex of neuropathic mice: impact on glial caspases and pain perception. Cereb. Cortex 22, 2495–2518. doi: 10.1093/cercor/bhr328 Paxinos, G., and Franklin, K. B. (2004). The Mouse Brain in Stereotaxic Coordinates. Houston, TX: Gulf professional publishing. REFERENCES Guida, F., Palazzo, E., Luongo, L., Marabese, I., de Novellis, V., Maione, S.,
et al. (2017b). Supraspinal metabotropic glutamate receptors: an endogenous
substrate for alleviating chronic pain and related affective disorders. mGLU
Receptors 31, 15–31. Apps, R., and Strata, P. (2015). Neuronal circuits for fear and anxiety-the missing
link. Nat. Rev. Neurosci. 16:642. doi: 10.1038/nrn4028 Guida, F., Luongo, L., Marmo, F., Romano, R., Iannotta, M., Napolitano, F.,
et al. (2015). Palmitoylethanolamide reduces pain-related behaviors and
restores glutamatergic synapses homeostasis in the medial prefrontal cortex of
neuropathic mice. Mol. 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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https://openalex.org/W3121560790
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https://www.scielo.br/j/ambiagua/a/CZRLgKJZJ9jMCGDHTvFSjvP/?lang=en&format=pdf
|
English
| null |
Simulation of nitrate and potassium concentrations in soil solution using parametric models and Hydrus-2D
|
Revista Ambiente & Água
| 2,021
|
cc-by
| 9,962
|
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
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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
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Indian journal of science and technology
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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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https://openalex.org/W2536582757
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http://www.scielo.br/pdf/pab/v51n9/0100-204X-pab-51-09-1371.pdf
|
Portuguese
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Mapping soil carbon, particle-size fractions, and water retention in tropical dry forest in Brazil
|
Pesquisa Agropecuária Brasileira
| 2,016
|
cc-by
| 11,233
|
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 ,
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,
, 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 ,
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, p
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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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Design as an interactive boundary object
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Journal of organization design
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cc-by
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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://creativeco
mmons.org/licenses/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
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In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients
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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. Additionally, the culture
exhibited relatively high levels of biofilm production, which
might be helpful in the protection against viral invasion. Discussion The authors acknowledge COST (European Co-
operation in the field of Scientific and Technical Research) action
BM1003, BMicrobial cell surface determinants of virulence as targets
for new therapeutics in Cystic Fibrosis.^ TO and KDW were cofinanced
by the European Union as part of the European Social Fund. Conflict of interest
The authors declare that they have no competing
interests. Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
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Adjuvant bevacizumab for melanoma patients at high risk of recurrence: survival analysis of the AVAST-M trial
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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-
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thelial growth factor in metastatic melanoma. Br J Cancer 1997; 76(7):
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VEGF in melanoma patients: a pilot study. Anticancer Res 2004; 24(6):
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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-
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2011; 344: 129–148. 17. Thomas NE, Edmiston SN, Alexander A et al. Association between NRAS
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359–368. 25. Li Y-L, Zhao H, Ren X-B. Relationship of VEGF/VEGFR with immune
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Predictive factors of endometriosis progression into ovarian cancer
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© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. 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/journal.pone.0076808. 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
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inducible factor-1alpha and nitric oxide synthase isoforms in ovarian
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W2892374527.txt
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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
| 2,018
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cc-by
| 7,695
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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
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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
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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
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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
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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
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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.
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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.
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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
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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
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provided the original author(s) and the copyright owner(s) are credited and that the
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https://openalex.org/W2567293515
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https://jurnal.unimed.ac.id/2012/index.php/jpf/article/download/Rizki%20Maulida%20dan%20Mariati%20P.%20Simanjuntak/pdf
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Indonesian
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PENGEMBANGAN BAHAN AJAR FISIKA SMA BERBASIS INVESTIGASI PADA MATERI FLUIDA DINAMIS UNTUK MENINGKATKAN HASIL BELAJAR SISWA
|
Jurnal Pendidikan Fisika
| 2,015
|
cc-by
| 3,458
|
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
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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
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(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
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(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
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( )
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,
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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
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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
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Mayberg S, Green P: Assessing the Organizational Social Con-
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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-
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20. Jerrell JM, Hargreaves WA: The Operating Philosophy of Com-
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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
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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
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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"
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CANCER: Childhood Leukemia and Proximity to Nuclear Power Plants
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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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PloS one
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cc-by
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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):
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Link to published version (if available):
10.1371/journal.pone.0190393
Link to publication record on the Bristol Research Port
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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 10ðS2 S1Þ=400
ð1Þ
E2 ¼
1
1 þ 10ðS1 S2Þ=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):
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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
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(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-
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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-
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tion processing systems. 2006; 19: 569–576. 21. Elliott LL. Reliability of judgments of figural complexity. Journal of Experimental Psychology. 1958; 56
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Conference on Mobile Computing, Applications, and Services 2011 Oct 24 (pp. 20–31). Springer Berlin
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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
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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
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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
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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
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3: An unexpected regulator of cell lineage determination in skin. Genes
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catenin signalling in adult mouse epidermis is suffi cient to induce new
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profi ling adult hair follicle stem cells. Nat Biotechnol 22: 411–417. 61. Amoh Y, Li L, Katsuoka K, Penman S, Hoffman RM (2005) Multipotent
nestin-positive, keratin-negative hair-follicle bulge stem cells can form
neurons. Proc Natl Acad Sci U S A 102: 5530–5534. neurons. Proc Natl Acad Sci U S A 102: 5530–5534. 53. Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM (2000) Involvement of
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56. Nishimura EK, Jordan SA, Oshima H, Yoshida H, Osawa M, et al. (2002) PLoS Biology | www.plosbiology.org 1877 November 2005 | Volume 3 | Issue 11 | e372
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Bio-activities and phytochemical investigation of <i>Cnestis palala</i> (Lour.) Merr.
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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
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g
p
,
p
g (
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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 SephadexLH-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 SephadexLH-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 SephadexLH-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
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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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https://cp.copernicus.org/preprints/cp-2020-58/cp-2020-58.pdf
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Testing Hypotheses About Glacial Dynamics and the Stage 11 Paradox Using a Statistical Model of Paleo-Climate
| null | 2,020
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cc-by
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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
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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
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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
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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
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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
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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
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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
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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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https://openalex.org/W4205629705
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http://real.mtak.hu/159548/1/1788-6120-article-p3.pdf
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Hungarian
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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
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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,
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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
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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
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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
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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:
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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
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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
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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
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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-
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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: )
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Fadlan: the Volga Route from Baghdad to Bolgar). Moscow: “Izdatel'skii dom Mardzhani” Publ. (in
Russian). )
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(Antiquities of Biliar) II. Kazan: “Zaman” Publ. (in Russian). )
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Steppes) 25. Kazan: “Fen” Publ. (in Russian). pp
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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
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Urals: in 8 volumes) 5. Chelyabinsk: South Ural State University (in Russian). )
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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
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IX-XIII vv. (Swords and Sabers of 9th–13th Centuries). Series: Svod Arkheologicheskikh Istochniko
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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
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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
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evaluations and estimating optimal model complexity for Maxent ecological niche models. Methods in Ecology and Evolution, 5, 1198–1205. lla, R., Galante, P.J., Soley-Guardia, M., Boria, R.A., Kass, J.M., Uriarte, M. & Muscarella, R., Galante, P.J., Soley-Guardia, M., Boria, R.A., Kass, J.M., Uriarte, M. &
Anderson, R.P. (2014) ENMeval: An R package for conducting spatially independent Owens, H.L., Campbell, L.P., Dornak, L.L., Saupe, E.E., Barve, N., Soberón, J., Ingenloff, K.,
Lira-Noriega, A., Hensz, C.M., Myers, C.E. & Peterson, A.T. (2013) Constraints on
interpretation of ecological niche models by limited environmental ranges on calibration
areas. Ecological Modelling, 263, 10–18. Park, D.S. & Potter, D. (2015a) A reciprocal test of Darwin’s naturalization hypothesis in two
mediterranean-climate regions. Global Ecology and Biogeography, 24, 1049–1058. Park, D.S. & Potter, D. (2015b) Why close relatives make bad neighbours: phylogenetic
conservatism in niche preferences and dispersal disproves Darwin’s naturalization
hypothesis in the thistle tribe. Molecular Ecology, 24, 3181–3193. Peterson, A.T., Papeş, M. & Soberón, J. (2008) Rethinking receiver operating characteristic
analysis applications in ecological niche modeling. Ecological Modelling, 213, 63–72. Peterson, A.T. & Soberón, J. (2012) Species distribution modeling and ecological niche 20 modeling: getting the concepts right. Natureza & Conservação, 10, 102–107. Proosdij, A.S.J., Sosef, M.S.M., Wieringa, J.J. & Raes, N. (2016) Minimum required number of
specimen records to develop accurate species distribution models. Ecography, 39, 542–552. Qiao, H., Feng, X., Escobar, L.E., Peterson, A.T., Soberón, J., Zhu, G. & Papeş, M. (2018) An
evaluation of transferability of ecological niche models. Ecography, 42, 521-534. Saupe, E.E., Barve, V., Myers, C.E., Soberón, J., Barve, N., Hensz, C.M., Peterson, A.T., Owens,
H.L. & Lira-Noriega, A. (2012) Variation in niche and distribution model performance: The
need for a priori assessment of key causal factors. Ecological Modelling, 237-238, 11–22. Soberon, J. & Townsend Peterson, A. (2005) Interpretation of Models of Fundamental Ecological Niches and Species’ Distributional Areas. Biodiversity Informatics, 2. 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
|
https://openalex.org/W4386482945
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https://www.nature.com/articles/s41386-023-01727-9.pdf
|
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G protein-biased LPAR1 agonism of prototypic antidepressants: Implication in the identification of novel therapeutic target for depression
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Neuropsychopharmacology
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cc-by
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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
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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. 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
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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
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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
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Enhancing the Spin Hall Effect of Cylindrically Polarized Beams
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Micromachines
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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. Express 2015, 23, 1767–1774. [CrossRef] [PubMed] ,
,
g,
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p
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18. Jia, Y.; Liu, Y.; Zhang, W.; Wang, J.; Wang, Y.; Gong, S.; Liao, G. Ultra-wideband metasurface with linear-to-circular polarization 18. Jia, Y.; Liu, Y.; Zhang, W.; Wang, J.; Wang, Y.; Gong, S.; Liao, G. Ultra-wideband metasurface with linea
conversion of an electromagnetic wave. Opt. Mater. Express 2018, 8, 597–604. [CrossRef] g
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24. References Barnett, S.M.; Allen, L. Orbital angular momentum and nonparaxial light beams. Opt. Commun. 1994, 110, 670–678. [CrossRef] 36. Zhou, J.; Zhang, W.; Liu, Y.; Ke, Y.; Liu, Y.; Luo, H.; Wen, S. Spin-dependent manipulating of vector beams
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38. Bayat, J.; Hajizadeh, F.; Khazaei, A.M.; Rasouli, S. Gear-like rotatable optical trapping with radial carpet beams. Sci. Rep. 2020,
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et al. Transmission of optical communication signals through ring core fiber using perfect vortex beams. Opt. Express 2023,
31, 40113–40123. [CrossRef]ii 41. Porfirev, A.P.; Khonina, S.N.; Ivliev, N.A.; Fomchenkov, S.A.; Porfirev, D.P.; Karpeev, S.V. Polarization-Sensitive Patterning of
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author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). References Cheng, H.; Chena, S.; Yu, P.; Li, J.; Xie, B.; Li, Z.; Tian, J. Dynamically tunable broadband mid-infrared cross polarization converter
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32. Gori, F.; Guattari, G.; Padovani, C. Bessel-Gauss beams. Opt. Commun. 1987, 64, 491–495. [CrossRef] 33. Karimi, E.; Zito, G.; Piccirillo, B.; Marrucci, L.; Santamato, E. Hypergeometric-Gaussian beams. Opt. [CrossRef] 34. Hebri, D.; Rasouli, S. Combined half-integer Bessel-like beams: A set of solutions of the wave equation. Phys. Rev. A 2018,
98, 043826. [CrossRef] [
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35. Barnett, S.M.; Allen, L. Orbital angular momentum and nonparaxial light beams. Opt. Commun. 1994, 110, 670–678. [CrossRef]
36. Zhou, J.; Zhang, W.; Liu, Y.; Ke, Y.; Liu, Y.; Luo, H.; Wen, S. Spin-dependent manipulating of vector beams by tailoring polarization. Sci. Rep. 2016, 6, 34276. [CrossRef] [PubMed] 35. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content. References MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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https://zenodo.org/records/2185742/files/article.pdf
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Ausfallssymptome nach läsion des linken gyrus angularis in einem falle von Schädel-und gehirnverletzung
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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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https://europepmc.org/articles/pmc5382201?pdf=render
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A Clinical Perspective of Anti-Fibrotic Therapies for Cardiovascular Disease
|
Frontiers in pharmacology
| 2,017
|
cc-by
| 7,361
|
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
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conducted in the absence of any commercial or financial relationships that could
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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
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Art. no. 123619. [6] O. Castrejon-Campos, L. Aye, a
mixes more robust: An integrativ
clean energy transitions,’’ Energy
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top–down and bottom–up approa
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2022. [Online]. Available: http://pv
[9] Atlantic Shores Buoys, Atlantic S
Offshore Wind LLC, Brooklyn, N
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[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
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Jun. 2022,
Art. no. 123619. (15) [6] O. Castrejon-Campos, L. Aye, and F. K. P. Hui, ‘‘Making policy
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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
|
https://openalex.org/W2275876161
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https://centaur.reading.ac.uk/69936/1/Dyspraxia_2017.pdf
|
English
| null |
Dyspraxia and autistic traits in adults with and without autism spectrum conditions
|
Molecular autism
| 2,016
|
cc-by
| 6,473
|
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
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. 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
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Psychiatry Clin Neurosci. 1944;117:76–136. 6. Gowen E, Hamilton A. Motor abilities in autism: a review using a
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the development of autism spectrum conditions. Frontiers Neurology:
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control. J Autism Dev Disord. 1996;26:99–107. 12. Miyahara M, Tsujii M, Hori M, Nakanishi K, Kageyama H, Sugiyama T. Brief
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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
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Неред А.С., Мусаев Э.Р. Малоинвазив-
ные хирургические технологии при по-
ражениях позвоночника в онкогемато-
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2013;6(2):177–94. [Valiyev A.K.,
Sokolovskiy A.V., Nered A.S.,
Musaev E.R. Minimally invasive surgical
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холей позвоночника у пациентов, по-
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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-
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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. Giant cell
tumor of the sacrum and spinе: series of 23
cases and review of the literature. Iowa
Orthop J 2010;30:69–75. 34. Dalbayrak S., Yaman O., Ozer A.F. Minimally invasive approaches
in metastatic spinal tumor surgery. Turk Neurosurg 2015;25(3);357–61. DOI: 10.5137/1019-5149.JTN.8990-13.1. 22. Tomita K., Kawahara N., Kobayashi T. et al. Surgical strategy for spinal
metastases. Spine (Phila Pa 1976)
2001;26(3):298–306. DOI: 10.1097/00007632-200102010-
00016. 35. Barzilai O., Boriani S., Fisher C.G. et al. Essential concepts for the management
of metastatic spine disease: what the
surgeon should know and practice. Global
Spine J 2019;9(1 Suppl):98S–107S. DOI: 10.1177/2192568219830323. 46. Charest-Morin R., Dirks M.S., Patel S. et al. Ewing sarcoma of the spine
prognostic variables for survival and local
control in surgically treated patients. Spine
(Phila Pa 1976) 2018;43(9):622–9. DOI: 10.1097/BRS.0000000000002386. 23. Boriani S. En bloc resection in the spine:
a procedure of surgical oncology. J Spine Surg 2018;4(3):668–76. DOI: 10.21037/jss.2018.09.02. 36. Goetz M.P., Callstrom M.R., Charbo-
neau J.W. et al. Percutaneous image-
guided radiofrequency ablation of painful
metastases involving bone: a multicenter
study. J Clin Oncol 2004;22(2):300–6. DOI: 10.1200/JCO.2004.03.097. 47. Овчинников В.А., Угляница К.Н., Вол-
ков В.Н. Современные методы лучево-
го лечения онкологических больных. Журнал ГрГМУ 2010;1(29):93–7. [Ovchinnikov V.A., Uglyanitsa K.N.,
Volkov V.N. Modern metods
of radiotherapy of oncological patients. Zhurnal GrGMU = Journal of the
Grodno State Medical University
2010;1(29):93–7. (In Russ.)]. 47. Овчинников В.А., Угляница К.Н., Вол-
ков В.Н. Современные методы лучево-
го лечения онкологических больных. Журнал ГрГМУ 2010;1(29):93–7. [Ovchinnikov V.A., Uglyanitsa K.N.,
Volkov V.N. Modern metods
of radiotherapy of oncological patients. Zhurnal GrGMU = Journal of the
Grodno State Medical University
2010;1(29):93–7. (In Russ.)]. 24. Boriani S., Weinstein J.N., Biagini R. Primary bone tumors of the spine. Terminology and surgical staging. Spine
(Phila Pa 1976) 1997;22(9):1036–44. DOI: 10.1097/00007632-199705010-
00020. 37. НЕЙРОХИРУРГИЯ
Том 24 Volume 24 Russian Journal of Neurosurgery НЕЙРОХИРУРГИЯ
Том 24 Volume 24 Обзор литературы solitary spinal metastasis. Paraplegia
1994;32(1):36–46. DOI: 10.1038/sc.1994.7. on surgical management of myeloma bone
disease. Orthop Surg 2016;8(3):263–9. DOI: 10.1111/os.12267. ния агрессивных гемангиом позвон-
ков. Хирургия позвоночника
2018;15(1):79–90. [Klimov V.S.,
Kosimshoev M.A., Evsyukov A.V. et al. Results of differentiated surgical treatment
of aggressive vertebral hemangiomas. Hirurgia Pozvonocnika = Spine Surgery
2018;15(1):79–90. (In Russ.)]. DOI: http://dx.doi.org/10.14531/
ss2018.1.79-90. 40. Guzik G. Oncological and functional
results of the surgical treatment of ver
tebral metastases in patients with multiple
myeloma. BMC Surg 2017;17(1):92. DOI: 10.1186/s12893-017-0288-9. 28. Howell E.P., Williamson T., Karikari I. et al. Total en bloc resection of primary
and metastatic spine tumors. Ann Transl
Med 2019;7(10):226. DOI:10.21037/atm.2019.01.25. 41. Peng X., Wan Y., Chen Y. et al. Primary
non-Hodgkin’s lymphoma of the spine
with neurologic compression treated
by radiotherapy and chemotherapy alone
or combined with surgical decompression. Oncol Rep 2009;21(5):1269–75. DOI: 10.3892/or_00000350. 29. Ohashi M., Hirano T., Watanabe K. et al. En bloc spondylectomy for spinal
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at a minimum of 2 years after surgery. Asian Spine J 2019;13(2):296–304. DOI:10.31616/asj.2018.0145. 16. Vasudeva V.S., Chi J.H., Groff M.W. Surgical treatment of aggressive vertebral
hemangiomas. Neurosurg Focus
2016;41(2):E7. DOI: 10.3171/2016.5.FOCUS16169. 30. Deng Z., Xu B., Jin J. et al. Strategies
for management of spinal metastases:
a comprehensive review. Cancer
Translational Medicine 2015;1(3):94–100. DOI: 10.4103/2395-3977.159536. 42. Harris E., Butler J.S., Cassidy N. Aggressive plasmablastic lymphoma
of the thoracic spine presenting as acute
spinal cord compression in a case
of asymptomatic undiagnosed human
immunodeficiency virus infection. Spine J 2014;14(7):e1–5. DOI: 10.1016/j.spinee.2013.12.018. 17. Parker J., Soltani S., Boissiere L. et al. Spinal aneurysmal bone cysts (ABCs):
optimal management. Orthop
Res Rev 2019;11:159–66. DOI: 10.2147/ORR.S211834. 31. Conti A., Acker G., Kluge A. et al. Decision making in patients with meta
static spine. The role of minimally invasive
treatment modalities. Front Oncol
2019;9:915. DOI: 10.3389/
fonc.2019.00915. 18. Tsagozis P., Brosjo O. Current strategies
for the treatment of aneurysmal bone
cysts. Orthop Rev (Pavia) 2015;7(4):6182. DOI: 10.4081/or.2015.6182. 43. Denaro L., Berton A., Ciuffreda M. et al. Surgical management of chordoma:
a systematic review. J Spinal Cord
Med 2020;43(6):797–812. DOI: 10.1080/10790268.2018.1483593. 19. Aiba H., Kobayashi1 M., Waguri-
Nagaya Y. et al. Treatment of aneurysmal
bone cysts using endoscopic curettage. BMC Musculoskelet Disord
2018;19(1):268. DOI: 10.1186/s12891-018-2176-6. 32. Spinazze S., Caraceni A., Schrijvers D. et al. Epidural spinal cord compression. Critical Reviews in Oncology/Hematology
2005;56(3):397–406. DOI: 10.1016/j.critrevonc.2005.04.005. 44. Л И Т Е Р А Т У Р А / r e f e r e n c e s Surgical treatment
of primary spinal tumors: tactics and
results. Hirurgia pozvonocnika = Spine 14. Laredo J.D., Reizine D., Bard M.,
Merland J.J. Vertebral hemangiomas:
radiologic evaluation. Radiology
1986;161(1):183–9. DOI: 10.1148/radiology.161.1.3763864. 5. Mesfin A., McCarthy E.F., Kebaish
K.M. Surgical treatment of aneurysmal
bone cysts of the spine. Iowa Orthop
J 2012;32:40–5. 15. Климов В.С., Косимшоев М.А.,
Евсюков А.В и др. Результаты диффе-
ренцированного хирургического лече- 102 2’ 2022 НЕЙРОХИРУРГИЯ
Том 24 Volume 24 Song H.-M., Gu Y.-F., Li Y.-D. et al. Interventional tumor removal: a new
technique for malignant spinal tumor and
malignant vertebral compression fractures
without epidural involvement. Acta Radiol
2014;55(8):976–84. DOI: 10.1177/0284185113508761. 25. Tokuhashi Y., Matsuzaki H., Oda H. et al. A revised scoring system for preoperative
evaluation of metastatic spine tumor
prognosis. Spine (Phila Pa 1976)
2005;30(19):2186–91. DOI: 10.1097/01. brs.0000180401.06919.a5. 48. Трофимова О.П., Ткачев С.И.,
Юрьева Т.В. Прошлое и настоящее
лучевой терапии в онкологии. Клини-
ческая онкогематология 2013;6(4):
355–64. [Trofimova O.P., Tkachev S.I.,
Yuryeva T.V. Past and present of radio
therapy in management of malignancies. Klinicheskaya onkogematologiya =
Clinical oncohematology 2013;6(4):
355–64. (In Russ.)]. /
38. Gu Y.-F., Tian Q.-H., Li Y.-D. et al. Percutaneous vertebroplasty and inter
ventional tumor removal for malignant
vertebral compression fractures and/or
spinal metastatic tumor with epidural
involvement: a prospective pilot study. J Pain Res 2017;10:211–8. DOI: 10.2147/JPR.S122211. 26. Pennington Z., Ahmed A.K., Molina C.A. et al. Minimally invasive versus
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с первичными злокачественными опу-
холями кости Ассоциации онкологов
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et al. Direct decompressive surgical
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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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State Capture Deconstructed: Risk Measurement in Vulnerable Economic Sectors in Europe
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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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.
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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-
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1
f 20. Stockholm county council. Behovsindex 2011-2013 [Care need index 2011-
2013]. http://dok.slso.sll.se/CES/FHG/Jamlik_halsa/Rapporter/Behovsindex-
2011-2013.pdf. 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
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jämförande studie mellan tre landsting före och efter vårdvalets införande. [PHC Choice Reform and equitable care in primary health care. A
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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
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(In Swedish)
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ROLE OF URIC ACID IN CARDIAC CHANGES IN OVERWEIGHT AND OBESE CHILDREN.
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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:
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Hair growth-promoting effect of Geranium sibiricum extract in human dermal papilla cells and C57BL/6 mice
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BMC complementary and alternative medicine
| 2,017
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cc-by
| 8,126
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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
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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. Choi SJ, Cho AR, Jo SJ, Hwang ST, Kim KH, Kwon OS. Effects of
glucocorticoid on human dermal papilla cells in vitro. J Steroid Biochem
Mol Biol. 2013;135:24–9. 22. He J, Kang T, Yin H, Zheng Y, Zhang S. The content determination of gallic
acid of Geranium sibiricum collected in three Provinces in Northeast. Chin
Arch Tradit Chin Med. 2007;25:822–3. 22. He J, Kang T, Yin H, Zheng Y, Zhang S. The content determination of gallic
acid of Geranium sibiricum collected in three Provinces in Northeast. Chin
Arch Tradit Chin Med. 2007;25:822–3.
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https://www.frontiersin.org/articles/10.3389/fped.2023.1142196/pdf
|
English
| null |
Corrigendum: Different frequency bands in various regions of the brain play different roles in the onset and wake-sleep stages of infantile spasms
|
Frontiers in pediatrics
| 2,023
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cc-by
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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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|
English
| null |
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
| 2,021
|
cc-by
| 3,185
|
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
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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:
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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
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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
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)
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. 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
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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-
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March 4, 2016 Citrullinated Proteoglycan in Human Cartilage TTG. Purification and storage of highly purified cartilage PG: TTG. All quality controls for
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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. BMC Public Health, 18, Article 740. https://doi.org/10.1186/s12889-018-
5666-4 General rights General rights
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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
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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
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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
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June 7, 2016 11 / 13 MRSA in Hospitals in NYC in 2014 12. PLOS ONE | DOI:10.1371/journal.pone.0156924
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sette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: “SCCmec IV multiplex.”
J Antimicrob Chemother. 2007; 60: 42–48. PMID: 17468509 27. Okuma K, Iwakawa K, Turnidge JD, Grubb WB, Bell JM, O'Brien FG, et al. Dissemination of new methi-
cillin-resistant Staphylococcus aureus clones in the community. J Clin Microbiol. 2002; 40: 4289–4294. PMID: 12409412 28. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, et al. Involvement of Panton-Val-
entine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin
Infect Dis. 1999; 29: 1128–1132. PMID: 10524952 29. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, et al. Complete genome sequence of
USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet. 2006; 367: 731–739. PMID: 16517273 30. Diep BA, Stone GG, Basuino L, Graber CJ, Miller A, Etages des S-A, et al. The arginine catabolic
mobile element and staphylococcal chromosomal cassette mec linkage: convergence of virulence and
resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus. J Infect Dis. 2008; 197:
1523–1530. doi: 10.1086/587907 PMID: 18700257 31. Otto M. Community-associated MRSA: what makes them special? Int J Med Microbiol. 2013; 303: 324–
330. doi: 10.1016/j.ijmm.2013.02.007 PMID: 23517691 32. Thurlow LR, Joshi GS, Richardson AR. Virulence strategies of the dominant USA300 lineage of com-
munity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). FEMS Immunol Med
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lence survey of health care-associated infections. N Eng J Med. 2014; 370: 1198–1208. 34. Wilson J, Guy R, Elgohari S, Sheridan E, Davies J, Lamagni T, et al. Trends in sources of meticillin-
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lence of EMRSA-15 in Portuguese public buses: a worrisome finding. PLoS One. 2011; 6: e17630. doi:
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20206987 40. David MZ, Cadilla A, Boyle-Vavra S, Daum RS. Replacement of HA-MRSA by CA-MRSA Infections at
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10.1093/cid/cir034 PMID: 21217178 42. Chadha P, Mariano N, LaBombardi V. In Vitro Activities of Mupirocin, Tigecycline, Ceftaroline, Vanco-
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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
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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
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Influenza A infection interferes with the development of asthma
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Respiratory research
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BioMed Central
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BioMed Central PublisherInfo
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BioMed Central
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London
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BioMed Central PublisherInfo
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BioMed Central
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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
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:
1
ArticleLastPage
:
1
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:
RegistrationDate : 2001–9–19
Received
: 2000–9–29
Accepted
: 2001–9–19
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: 2001–9–19
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:
Biomed Central Ltd2001
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ArticleContext
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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
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Biomed Central Ltd2001
ArticleGrants
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ArticleContext
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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.
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http://www.sciedupress.com/journal/index.php/ijfr/article/download/8709/5240
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An Analysis on the Behaviour of Corporate Social Responsibility towards Profitability of Islamic Banks: Asean and Europe
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International journal of financial research
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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
்ቁ2ሺ݇ܶሻ (6)
Schwarz Information Criterion (SIC): െ2 ቀ
ଵ
்ቁ݇ሺ
୪୭ሺTሻ
T
ሻ (7)
Hannan-Quinn Information Criterion (HQ): െ2 ቀ
ଵ
்ቁ2݇ܮܱܩሺ
୪୭ሺTሻ
T
ሻ (8) Akaike information crieterion (AIC):
െ2 ቀ
ଵ
்ቁ2ሺ݇ܶሻ (6) (6) Schwarz Information Criterion (SIC): െ2 ቀ
ଵ
்ቁ݇ሺ
୪୭ሺTሻ
T
ሻ (7)
Hannan-Quinn Information Criterion (HQ): െ2 ቀ
ଵ
்ቁ2݇ܮܱܩሺ
୪୭ሺTሻ
T
ሻ (8) Schwarz Information Criterion (SIC): െ2 ቀ
ଵ
்ቁ݇ሺ
୪୭ሺTሻ
T
ሻ (7) (7) ்
T
Hannan-Quinn Information Criterion (HQ): െ2 ቀ
ଵ
்ቁ2݇ܮܱܩሺ
୪୭ሺTሻ
T
ሻ (8) Hannan-Quinn Information Criterion (HQ): െ2 ቀ
ଵ
்ቁ2݇ܮܱܩሺ
୪୭ሺTሻ
T
ሻ (8) (8) Subject to: 1 = the value of lag likelihood is the same as െ
்
ଶ൬1 logሺ2ߨሻ݈݃ ቀ
ఌ"ఌᇱ
்ቁ൰ ; ߝ"ߝ where it is sum of squared 1 = the value of lag likelihood is the same as െ
்
ଶ൬1 logሺ2ߨሻ݈݃ 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 yY୲ିଵβ୧∑
∆Y୲ି୧ାଵ
୮
୧ୀଵ
ߝ௧ ∆ܻ௧ൌ ߙ yY୲ିଵβ୧∑
∆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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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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https://www.degruyter.com/document/doi/10.1515/opphil-2020-0004/pdf
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Objects, Relations, Potential and Change
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Open Philosophy
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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
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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
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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
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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
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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
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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 )
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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
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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
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& H
lth
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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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https://openalex.org/W2021616451
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https://zenodo.org/records/2348934/files/article.pdf
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English
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IV. <i>On surface concentration, and the formation of liquid films</i>
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The London, Edinburgh and Dublin philosophical magazine and journal of science
| 1,907
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public-domain
| 7,164
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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
|
https://openalex.org/W2035023923
|
https://hal.archives-ouvertes.fr/hal-03246862/file/1-s2.0-S0166685109002114-main.pdf
|
English
| null |
Quantitative assessment of DNA replication to monitor microgametogenesis in Plasmodium berghei
|
Molecular and biochemical parasitology
| 2,009
|
cc-by
| 5,245
|
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
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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
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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://openalex.org/W2039422427
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https://journals.iucr.org/e/issues/2011/03/00/hb5794/hb5794.pdf
|
Khmer
| null |
(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
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cc-by
| 5,369
|
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
| 2,021
|
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 toand . 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 ˆdecreases 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 ˆdecreases with increasing n for all the methods of estimation. 2. The bias of ˆdecreases with increasing n for all the methods of estimation. Comparison of estimation methods for unit-Gamma distribution 780 3. The bias of ˆdecreases with increasing n for all the methods of estimation. 3. The bias of ˆdecreases with increasing n for all the methods of estimation. 4. The bias of ˆgenerally 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. Hence, we can argue that the MPS
estimators, AD2, ADR and ML estimators are among the best performing estimators for unit-
Gamma distribution. From both simulation study and real data examples, we observe that
performance of AD2L estimators is the worst among all methods of estimation. Acknowledgements The authors would like to thank to the Referees and the Editor for several helpful comments
which had improved the earlier versions of the manuscript. Sanku Dey, Andre F. B. Menezes and Josmar Mazucheli 799 [12]
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Para una crítica inmanente del lberalismo. Theodor Adorno y la cuestión del individuo en Mínima Moralia
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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.
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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
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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
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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
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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
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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
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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.
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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”.
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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
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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
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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).
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https://openalex.org/W4307553301
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https://www.frontiersin.org/articles/10.3389/fonc.2022.1010122/pdf
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English
| null |
Meta analysis of indocyanine green fluorescence in patients undergoing laparoscopic colorectal cancer surgery
|
Frontiers in oncology
| 2,022
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cc-by
| 7,126
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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
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after laparoscopic colorectal cancer surgery. Conflict of interest This study is supported by the National Natural Science
Foundation of China (No. 81860854, 82260936) and the
National Natural Science Foundation of China’s Post Grant
Fund (No. 2018YFC170610512). 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. Author contributions JD performed the search and drafted the manuscript. WH and
YL performed the data extraction. TY and KX analyzed the data. XL
and TX designed the study and amended the original draft. YL,
WH, TY and KX equally involved and equally contributed into the
study conduction. All authors contributed to the article and
approved the submitted version. Conclusively, ICG is dependable and effective in
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Quantifying a Sustainable Management Space for Human Use of Coastal Groundwater under Multiple Change Pressures
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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
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Water. doi:10.1111/j.1745-6584.2008.00535.x Werner AD, Alcoe DW, Ordens CM, et al. (2011) Current practice and future challenges in coastal aquifer. Flux-
Based and Trigger-Level Approaches with Application to an Australian Case Study. Water Resour Manage,
Management. doi:10.1007/s11269-011-9777-2 g
Werner AD, Ward JD, Morgan LK, et al. (2012) Vulnerability indicators of sea water intrusion. Ground Water 50:
48–58. doi:10.1111/j.1745-6584.2011.00817.x Zuurbier KG, Raat KJ, Klaasjan J, et al. (2016) How subsurface water technologies (SWT) can provide robust,
effective, and cost-efficient solutions for freshwater Management in Coastal Zones. Water Resour Manag. doi:10.1007/s11269-016-1294-x Zuurbier KG, Raat KJ, Klaasjan J, et al. (2016) How subsurface water technologies (SWT) can provide robust,
effective, and cost-efficient solutions for freshwater Management in Coastal Zones. Water Resour Manag. doi:10.1007/s11269-016-1294-x
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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
//
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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
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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
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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
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syndrome. Obes. Rev. 2015, 16, 914–941. [CrossRef] 7. Bub, A.; Malpuech-Brugère, C.; Orfila, C.; Amat, J.; Arianna, A.; Blot, A.; Di Nunzio, M.; Holmes, M.;
Kertész, Z.; Marshall, L.; et al. A Dietary Intervention of Bioactive Enriched Foods Aimed at Adults at
Risk of Metabolic Syndrome: Protocol and Results from PATHWAY-27 Pilot Study. Nutrients 2019, 11, 1814. [CrossRef] 7. Bub, A.; Malpuech-Brugère, C.; Orfila, C.; Amat, J.; Arianna, A.; Blot, A.; Di Nunzio, M.; Holmes, M.;
Kertész, Z.; Marshall, L.; et al. A Dietary Intervention of Bioactive Enriched Foods Aimed at Adults at
Risk of Metabolic Syndrome: Protocol and Results from PATHWAY-27 Pilot Study. Nutrients 2019, 11, 1814. [CrossRef] 8. Gibney, M.J.; Walsh, M.; Brennan, L.; Roche, H.-M.; German, B.; Van Ommen, B. 5. Concluding Remarks In the score plot, each dot represents
a different serum sample, and each colour represents a different group: dark green dots= AC; cyan dots= OBG;
orange dots= DHA; yellow dots= DHA+AC; purple dots= DHA+OBG; Figure S2: Score plot of M-PLS analysis
(T0 vs. T1) of the spectral region (0.92–0.71 ppm) containing the signal of lipoprotein methyl group (-CH3)
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; 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. (Hungary), and Desarrollos Panaderos
Levantinos SLL (Spain). flicts of Interest: The authors declare that they have no conflict of interest. Conflicts of Interest: The authors declare that they have no conflict of interest. Conflicts of Interest: The authors declare that they have no conflict of interest. References Metabolomics in human
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50. Backes, J.; Anzalone, D.; Hilleman, D.; Catini, J. The clinical relevance of omega-3 fatty acids in the
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Substituição do Milho pela Farinha de Varredura (Manihot esculenta, Crantz) na Ração de Bezerros Holandeses.: 1. Desempenho e Parâmetros Sangüíneos
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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. Means in the same collums for each parameter, followed by different letters, differ (P<.01) by Tukey test. 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 Médias nas mesmas colunas para cada parâmetro, seguidas de letras diferentes diferem (P<0,01) pelo teste Tukey. Means in the same collums for each parameter, followed by different letters, differ (P<.01) by Tukey test. Conclusões REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE
ZOOTECNIA, 37, 2000. Viçosa. Anais... Viçosa: Sociedade
Brasileira de Zootecnia, 2000b, p.378. A substituição do milho pela farinha de varredura
reduziu o consumo de matéria seca, proteína bruta e
fibra em detergente neutro, mas não o consumo de
amido. O ganho de peso foi reduzido com a inclusão
de farinha de varredura e a conversão alimentar não
foi alterada. Os níveis substituição não influenciaram
os níveis de hematócrito, uréia e glicose sangüíneos. CAMPOS, O.F.; LIZIEIRE, R.S. Produção do bezerrão. Coro-
nel Pacheco: EMBRAPA/CNPGL, 2000. 12p. CEREDA, M.P. Caracterização dos resíduos da industrialização
da mandioca. In: CEREDA, M.P. (Ed.) Resíduos da industri-
alização da mandioca. Botucatu: Paulicéia, 1994. p.11-50. CHARLES, T.P.; CAMPOS, O.F.; LIZIEIRE, R.S. Uso do teste de
coagulação pelo glutaraldeído como indicador do nível de
imunoglobulinas no soro de bezerros recém nascidos. Revista da
Sociedade Brasileira de Zootecnia, v.23, n.1 p.65-72, 1994. g
Sociedade Brasileira de Zootecnia, v.23, n.1 p.65-72, 1994. A farinha de varredura pode ser utilizada em
substituição total ao milho na alimentação de bezer-
ros holandeses do desaleitamento até 180 dias de
idade, com ganhos de pesos satisfatórios. De BEM, I.A.B. A mandioca como componente de rações
comerciais. In: CONGRESSO LATINO-AMERICANO DE
RAÍZES TROPICAIS, 1., 1996, São Pedro. Anais... São
Pedro: 1996. p.75-77. R. Bras. Zootec., v.31, n.1, p.192-204, 2002 204 PEREIRA, J.R.A.; ROSSI Jr., P. Manual prático de avaliação
nutricional de alimentos. Piracicaba: Fundação de Estudos
Agrários "Luiz de Queiroz", 1995. 25p. DIAZ-CASTANEDA, M.; BRISSON, G.J. Blood responses of
calves fed milk substitutes containing hydrolyzed fish protein
and lime-treated corn flour. Journal of Dairy Science,
72:2095-2106, 1989. POORE, M.H.; ECK, T.P.; SWINGLE, R.S. et al. Total starch
and relative starch availability of feed grains. In: BIENNIAL
CONFERENCE ON RUMEN FUNCTION, 1989, Chicago. Abstract... Chicago: 1989. (Abstract, 35) GRANT, R.; STOCK, R.; MADER, T. Feeding and managing
Holstein steers. 1993. Disponível na Internet
http://ianrwww.unl.edu/pubs/dairy/g1177.htm KEARL, L.C. Nutrients requirements of ruminants in
developing countries. 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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English
| null |
Topological frustration of artificial spin ice
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Nature communications
| 2,017
|
cc-by
| 8,530
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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
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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
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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
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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
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31. Ravlic´, R., Bode, M., Kubetzka, A. & Wiesendanger, R. Correlation of
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32. Chin, G. Y. in Deformation of Ceramic Materials (Springer, 1975). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
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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,
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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
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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
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Li Y (2020) Engineering isolated Mn-N2C2 atomic interface sites for efficient bifunctional oxygen
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(10): 1655-1661. http://dx.doi.org/10.1016/s0013-4686(99)00389-8 55. Meng K, Liu Q, Huang Y, Wang Y (2015) Facile synthesis of nitrogen and fluorine Co-doped carbon
materials as efficient electrocatalysts for oxygen reduction reactions in air-cathode microbial fuel
cells. J. Mater. Chem. A 3 (13): 6873-6877. http://dx.doi.org/10.1039/c4ta06500j 55. Meng K, Liu Q, Huang Y, Wang Y (2015) Facile synthesis of nitrogen and fluorine Co-doped carbon
materials as efficient electrocatalysts for oxygen reduction reactions in air-cathode microbial fuel
cells. J. Mater. Chem. A 3 (13): 6873-6877. http://dx.doi.org/10.1039/c4ta06500j References Cheng W, Lu X F, Luan D, Lou X W D (2020) Nimn-based bimetal-organic framework nanosheets
supported on multi-channel carbon fibers for efficient oxygen electrocatalysis. Angew. Chem. Int. Ed. Engl. 59 (41): 18234-18239. http://dx.doi.org/10.1002/anie.202008129 48. Li K, Xu S, Liu X, Li H, Zhan S, Ma S, Huang Y, Liu S, Zhuang X (2022) The organic contaminants
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flexible all-solid-state nico-Zn batteries with high capacity based on advanced ion-buffering
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021-00375-1 50. Zhang J-Y, Yan Y, Mei B, Qi R, He T, Wang Z, Fang W, Zaman S, Su Y, Ding S, Xia B Y (2021) Local
spin-state tuning of cobalt–iron selenide nanoframes for the boosted oxygen evolution. Energy
Environ. Sci. 14 (1): 365-373. http://dx.doi.org/10.1039/d0ee03500a 51. Li R-Q, Wan X-Y, Chen B-L, Cao R-Y, Ji Q-H, Deng J, Qu K-G, Wang X-B, Zhu Y-C (2021) Hierarchical
Ni3N/Ni0.2Mo0.8N heterostructure nanorods arrays as efficient electrocatalysts for overall water and
urea electrolysis. Chem. Eng. J. 409: 128240. http://dx.doi.org/10.1016/j.cej.2020.128240 Page 14/20 Page 14/20 52. Li W, Li F, Yang H, Wu X, Zhang P, Shan Y, Sun L (2019) A bio-inspired coordination polymer as
outstanding water oxidation catalyst via second coordination sphere engineering. Nat. Commun. 10
(1): 5074. http://dx.doi.org/10.1038/s41467-019-13052-1 52. Li W, Li F, Yang H, Wu X, Zhang P, Shan Y, Sun L (2019) A bio-inspired coordination polymer as
outstanding water oxidation catalyst via second coordination sphere engineering. Nat. Commun. 10
(1): 5074. http://dx.doi.org/10.1038/s41467-019-13052-1 53. Hu B, Zhu X, An X, Wang C, Wang X, He J, Zhao Y (2020) Separation of metal-N4 units in metal-
organic framework for preparation of M-Nx/C catalyst with dense metal sites. Inorg. Chem. 59 (23):
17134-17142. http://dx.doi.org/10.1021/acs.inorgchem.0c02420 53. Hu B, Zhu X, An X, Wang C, Wang X, He J, Zhao Y (2020) Separation of metal-N4 units in metal-
organic framework for preparation of M-Nx/C catalyst with dense metal sites. Inorg. 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. Page 19/20 Page 19/20 SupportingInformation.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupportingInformation.docx Page 20/20
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Explore the interaction between root metabolism and rhizosphere microbiota during the growth of Angelica sinensis
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Frontiers in plant science
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cc-by
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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. E., and Schulze-Lefert, P. (2013). Structure and functions of the bacterial microbiota of plants. Annu. Rev. Plant Biol. 64, 807–838. doi: 10.1146/annurev-arplant-050312-120106 An, Z., Guo, F., Chen, Y., Bai, G., and Chen, Z. (2020). Rhizosphere bacterial and
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specific patterns that are developmentally programmed and correlate with soil
microbial functions. PloS One 8, e55731. doi: 10.1371/journal.pone.0055731 Badri, D. V., Quintana, N., El, K. E., Kim, H. K., Choi, Y. H., Sugiyama, A., et al. (2009). An ABC transporter mutation alters root exudation of phytochemicals that
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FASTQ preprocessor. Bioinformatics 34, i884–i890. doi: 10.1093/bioinformatics/
bty560 Bourceret, A., Guan, R., Dorau, K., Mansfeldt, T., Omidbakhshfard, A.,
Medeiros, D. B., et al. (2022). Maize field study reveals covaried microbiota and
metabolic changes in roots over plant growth. MBio 13, e258421. doi: 10.1128/
mbio.02584-21 Cotton, T. E. A., Pétriacq, P., Cameron, D. D., Meselmani, M. A.,
Schwarzenbacher, R., et al. (2019). Metabolic regulation of the maize rhizobiome
by benzoxazinoids. ISME J. 13, 1647–1658. doi: 10.1038/s41396-019-0375-2 Bulgarelli, D., Garrido-Oter, R., Munch, P. C., Weiman, A., Droge, J., Pan, Y.,
et al. (2015). SUPPLEMENTARY FIGURE 2 OPLS-DA for the comparison of metabolomic profiles between M and G. Correlation between the differential secondary metabolites and
differential microbial taxa in rhizosphere of A. sinensis. (*, P <=0.05; **,
P <= 0.01; ***, P <=0.001.). 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. Frontiers in Plant Science 13 frontiersin.org Chen et al. 10.3389/fpls.2022.1005711 Acknowledgments Thanks to Guangzhou Genedenovo Biotechnology Co., Ltd. for helping with the sequencing and bioinformatics analysis. We
also thank Zeng-Xiang Guo for assisting with the seedling
samples, technical support for field management, and
permission to conduct research at Min County Angelica
sinensis institute. SUPPLEMENTARY FIGURE 1 SUPPLEMENTARY FIGURE 1 Species accumulation curve of bacteria. Correlation between the medicinal components and differential microbial
taxa in rhizosphere of A. sinensis. (*, P <=0.05; **, P <= 0.01; ***,
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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. Data curation: Shaun Liverpool, Yasmin Draoui, Judea Tucker, Brent Pereira, Jamal Prescod,
Catherine Trotman. 16 / 23 PLOS ONE | https://doi.org/10.1371/journal.pone.0282666
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Transmission of Hepatitis E Virus from Rabbits to Cynomolgus Macaques
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Emerging infectious diseases
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cc-by
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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. Future studies
should be conducted to investigate rabbit HEV infection in
human populations and assess whether close contact with
rabbits is a risk factor for HEV infection. References 1. Emerson SU, Anderson D, Arankalle A, Meng XJ, Purdy M,
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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
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